The standard method of isolating neural stem cells in vitro is with the  neurosphere  culture system, the method originally used to identify NSCs. After some proliferation, the cells are either induced to differentiate by withdrawing the  mitogens  or by exposing the cells to another factor that induces some of the cells to develop into different lineages. [ 18 ]  Cellular fates are analysed by staining with antibodies directed against antigens specific for astrocytes, oligodendrocytes, and neurons. In some cases, cells are plated at low density and monitored to determine if a single cell can give rise to the three phenotypes. [ 19 ]   Immunomagnetic  cell separation strategies using antibodies directed against cell surface markers present on stem cells, progenitors and mature CNS cells have been applied to the study of NSCs. Other non-immunological methods have been used to identify populations of cells from normal and tumorigenic CNS tissues, which demonstrate some of the in vitro properties of stem cells, including high  aldehyde dehydrogenase  (ALDH) enzyme activity. ALDH cells from embryonic rat and mouse CNS have been isolated and shown to have the ability to generate neurospheres, neurons, astrocytes and oligodendrocytes in vitro, as well as neurons in vivo when transplanted into the adult mouse cerebral cortex. [ 18 ]  Once a stem cell divides asymmetrically, the more mature progenitor is born and migrates to regions of differentiation. As the progenitor migrates, it matures further until it reaches a site where it stops and either becomes quiescent or fully differentiates into a functioning cell. The major obstacle to identifying and discovering markers that define a stem cell is that the most primitive cells are probably in a  quiescent  state and do not express many unique antigens. Thus, as with other fields like  haematopoiesis , a combination of positive and negative markers will be required to better define the central nervous system stem cell. [ 19 ]
Nonetheless, changes in the expression levels of specific molecules can be used to indicate the presence of neural stem cells in studies focused on further differentiation toward specific neural lineages. Usual markers used for neural stem cells include  Nestin  and  SOX2 . Although Nestin it is expressed predominantly in stem cells of the central nervous system (CNS), its expression is absent from nearly all mature CNS cells, thus it is an efficient marker for neural stem cells. [ 20 ]  During  neurogenesis , Sox2 is expressed throughout developing cells in the neural tube as well as in proliferating CNS progenitors, hence is thought to be centrally important for neural stem cell proliferation and differentiation. [ 20 ]  In addition to intracellular molecules, products are available to study proteins which are expressed at the cell surface, including  ABCG2 ,  FGF R4 , and  Frizzled-9 .


The differentiation of neural stem cells is controlled, in a context-dependent manner, by intrinsic factors and extracellular signalling molecules that act as positive or negative regulators that can be used as markers. [ 21 ]


A neural progenitor cell is distinct from a neural stem cell since it is incapable of continuous self-renewal and usually has the capacity to give rise to only one class of differentiated progeny. They are tripotent cells which can give rise to neurons, astrocytes and oligodendrocytes. An oligodendroglial progenitor cell, for example, gives rise to oligodendrocytes until its mitotic capacity is exhausted. [ 17 ]
Some neural progenitor markers are capable of tracking cells as they undergo expansion and differentiation from rosettes to neurons. The  neural rosette  is the developmental signature of neuroprogenitors in cultures of differentiating embryonic stem cells; rosettes are radial arrangements of columnar cells that express many of the proteins expressed in neuroepithelial cells in the neural tube. It has been shown that cells within rosettes express multiple cell markers, including among others  Nestin ,  NCAM  and  Musashi-1 , a RNA-binding protein that is expressed in proliferating neural stems cells. [ 22 ]
 Neuroepithelial progenitors  (NEP) are responsible for neurogenesis in the neural tube and also give rise to two other types of neural progenitor cell,  radial glia  and basal progenitors. Radial glia are the dominant progenitor cell type in the developing brain whereas basal progenitors are specifically located at the  subventricular zone  (SVZ) in the developing  telencephalon . Although functional studies of radial glia are increasing, it is difficult to distinguish them from neuroprogenitors and astrocytes. Like neuroprogenitors, radial glia express intermediate filament proteins nestin as well as the transcription factor  PAX6  that is expressed in some neuroprogenitors in the ventral half of the neural tube. Radial glia also express proteins characteristic of astrocytes, including the widely used glial fibrillary acidic protein ( GFAP ), among others. Cytological markers that might be unique to radial glia include modified forms of nestin identified by the RC1 and RC2 antibodies that recognize the murine antigens. [ 23 ]


Markers can detect neurons in different stages of development from nuclear, cytoplasmic, membrane or perisynaptic products present in neurons. It is also possible to label specifically  cholinergic ,  dopaminergic ,  serotonergic ,  GABAergic  or  glutamatergic  neurons. [ 24 ]  Pan neuron markers have multiple targets (somatic, nuclear, dendritic, spine and axonal proteins) and consequently label across all parts of the neuron. It is used to study neuronal morphology, although there are specific markers that label particular regions of the neuron. [ 25 ]


Doublecortin  (DCX) is a microtubule-associated protein that is widely expressed in the soma and leading processes of migrating neurons and in the axons of differentiation neurons. Its expression is downregulated with maturation  [ 26 ]


Neuron-specific  Class III β-tubulin  (TuJ1) is present in newly generated immature postmitotic neurons and differentiated neurons and in some mitotically active neuronal precursors. [ 11 ]


Microtubule-associated protein 2  (MAP-2) is a cytoskeletal protein. Its expression is weak in neuronal precursors but it increases during neuron development process. In general, its expression is confined to neurons and reactive astrocytes. [ 15 ]


Neuron specific enolase  (NSE), also called as gamma-enolase or enolase 2, is a cytosolic protein that is expressed in mature neurons. NSE levels increase along the neuron development reaching higher level in later stages. It can be expressed in glial cells during oligodendrocyte differentiation with the same levels that have been found in neuron culture, but is repressed when cells become mature. In pathological conditions was also reported that glial neoplasms and reactive glial cells expressed this marker. [ 15 ]


Calretinin  is widely distributed in different neuronal populations of vertebrate retina, being a valuable marker for immature postmitotic neurons. [ 11 ]


Neuronal Nuclei antigen ( NeuN ) or  Fox-3  is a nuclear protein present in postmitotic cell, at the point of differentiation into mature cells. [ 27 ]  It can be used to detect almost all neuronal cell types except Purkinje cells, olfactory bulb mitral cells, retinal photoreceptor and dopaminergic neurons in the  substantia nigra . [ 15 ]


Calbindin is expressed by cerebellar Purkinje cells and granule cells of the hippocampus. [ 11 ]  The reorganization and migration of calbindin-stained Purkinje neurons in rat cerebellum after peripheral nerve injury suggests that calbindin may be a marker for immature post-mitotic neurons, similar to calretinin. [ 28 ]


Tyrosine hydroxylase  (TH) is an enzyme involved in the synthesis of  dopamine  and  norepinephrine . Generally, it is used as a marker for dopaminergic neurons, but it can also be found in some forebrain neurons which make  norepinephrine  (which is the product of dopamine and the enzyme dopamine β-hydroxylase). [ 29 ]


Choline Acetyltransferase  (ChAT) is expressed in cholinergic neurons of both the CNS and PNS. In the CNS, ChAT is expressed in motor neurons and pre-ganglionic autonomic neurons of the spinal cord, a subset of neurons in the  neostriatum  and in the  basal forebrain . On the other hand, in PNS it is present in a small group of sympathetic neurons and in all parasympathetic neurons. [ 30 ]


GABA  is a mature neuronal marker expressed in GABAergic interneurons (inhibitor neurons which are generally interneurons in the brain). GAD65/67 are two enzymes involved in GABA synthesis by GABAergic interneurons. [ 29 ]


Amyloid precursor protein  (APP), the central protein in Alzheimer's Disease, is expressed differently during neuron differentiation. The expression of APP isoforms 695 and 714 is up-regulated in well-differentiated post-mitotic neuronal cells, while APP isoform 770 is down-regulated. These dynamics provide valuable insights into the differentiation state of neurons, aiding in the identification and study of neuronal development and maturation stages. [ 31 ]


Neuronal lineage markers can be used in clinical research to identify diseased cells and/or in repair process. Since selective degeneration of functional neurons is associated with the pathogenesis of  neurodegenerative disorders , such as degeneration of midbrain dopaminergic neurons in  Parkinson's disease , forebrain cholinergic neurons in  Alzheimer's disease  and cortical GABAergic neurons in  schizophrenia , markers of neuronal cell phenotype are of particular interest because of their utility in understanding pathology of clinical disease. [ 32 ]  There are two key markers in these studies:  choline acetyltransferase  and  tyrosine hydroxylase .  Choline acetyltransferase  (ChAT) is an enzyme responsible for catalyzing the synthesis of acetylcholine, and is expressed in the majority of cholinergic neurons. Hence, ChAT immunoreactivity is used to detect cognitive decline in several neurodegenerative disorders. [ 15 ]
In motor regions, sensory cortex and in the basal forebrain these immunolabeling has been applied to evaluate disruptions in cholinergic neurons of the ChAT fiber network and also for overall morphology. [ 33 ]
The Tyrosine hydroxylase (TH) immunolabeling has been very useful for Parkinson's disease investigation. It is used to determine the quantity of dopaminergic cell loss in Parkinson's patients.


ABCG2 ;
 NeuroD1 ;
 ASCL1/Mash1 ;
 Noggin ;
 Beta-catenin ;
 Notch-1 ;
 Notch-2 ;
 Brg1 ;
 Nrf2 ;
 N-Cadherin ;
 Nucleostemin ;
 Calcitonin R ;
 Numb ;
 CD15 / Lewis X ;
 Otx2 ;
 CDCP1 ;
 Pax3 ;
 COUP-TF I/NR2F1 ;
 Pax6 ;
 CXCR4 ;
 PDGF R alpha ;
 FABP7 / B-FABP ;
 PKC zeta ;
 FABP 8 /M-FABP;
 Prominin-2 ;
 FGFR2 ;
 ROR2 ;
 FGFR4 ;
 RUNX1/CBFA2 ;
 FoxD3 ;
 RXR alpha/NR2B1 ;
 Frizzled-9 ;
 sFRP-2 ;
 GATA-2 ;
 SLAIN 1 ;
 GCNF / NR6A1 ;
 SOX1 ;
 GFAP ;
 SOX2 ;
 Glut1 ;
 SOX9 ;
 HOXB1 ;
 SOX11 ;
 ID2 ;
 SOX21 ;
 Meteorin ;
 SSEA-1 ;
 MSX1 ;
 TRAF-4 ;
 Musashi-1 ;
 Vimentin ;
 Musashi-2 ;
 ZIC1 ;
 Nestin


A2B5 ;
 AP-2 Alpha ;
 ATPase Na+/K+ transporting alpha 1 ;
 Activin RIIA ;
 Brg1 ;
 CD168 / RHAMM ;
 CD4 ;
 Doublecortin / DCX ;
 Frizzled 4 / CD344 ;
 GAP43 ;
 Jagged1 ;
 Laminin ;
 MSX1 /HOX7;
 Mash1 ;
 Musashi-1 ;
 Nestin ;
 Netrin-1 ;
 Netrin-4 ;
 Neuritin ;
 NeuroD1 ;
 Neurofilament alpha-internexin/NF66 ;
 Notch1 ;
 Notch2 ;
 Notch3 ;
 Nucleostemin ;
 Otx2 ;
 PAX3 ;
 S100B ;
 SOX2 ;
 Semaphorin 3C ;
 Semaphorin 6A ;
 Semaphorin 6B ;
 Semaphorin 7A ;
 TROY / TNFRSF19 ;
 Tubulin βII ;
 Tuj 1 ;
 Vimentin


ATOH1 / MATH1 ;
 ASH1 / MASH1 ;
 HES5 ;
 HuC /Hu;
 HuD ;
 Internexin α ;
 L1 neural adhesion molecule ;
 MAP1B /MAP5;
MAP2A;
MAP2B;
Nerve Growth Factor Rec/ NGFR ;
 Nestin ;
 NeuroD ;
Neurofilament L 68 kDa;
 Neuron Specific Enolase /NSE;
 NeuN ;
 Nkx-2.2 /NK-2;
 Noggin ;
 Pax-6 ;
 PSA-NCAM ;
 Tbr1 ;
 Tbr2 ;
 Tubulin βIII ;
 TUC-4 ;
 Tyrosine hydroxylase /TH


Calbindin;
Calretinin;
Collapsin Response Mediated Protein 1 / CRMP1 ;
Collapsin Response Mediated Protein 2 /CRMP2;
Collapsin Response Mediated Protein 5 /CRMP5;
 Contactin-1 ;
Cysteine-rich motor neuron 1/ CRIM1 ;
c-Ret phosphor Serine 696;
 Doublecortin / DCX ;
 Ephrin A2 ;
Ephrin A4;
 Ephrin A5 ;
Ephrin B1;
Ephrin B2;
 GAP-43 ;
HuC;
 HuD ;
 Internexin alpha ;
 Laminin-1 ;
LINGO-1;
 MAP1B /MAP5;
Mical-3;
NAP-22;
 NGFR ;
 Nestin ;
 Netrin-1 ;
 Neuropilin ;
 Plexin-A1 ;
RanBPM;
Semaphorin 3A;
Semaphorin 3F;
 Semaphorin 4D ;
Slit2;
Slit3;
 Staufen ;
 Tbr 1 ;
Tbr 2;
 Trk A ;
Tubulin βIII;
TUC-4


NeuN ;
NF-L;
NF-M;
 GAD ;
 TH ;
 PSD-95 ;
 Synaptophysin ;
 VAMP ;
ZENON;
Up-regulated  Amyloid-beta precursor protein  isoform 695 & isoform 714; Down-regulated  Amyloid-beta precursor protein  isoform 770;
 Neuron Specific Enolase ;
 MAP2 ;
 MAPT ;


ChAT / choline acetyltransferase ;
Chox10;
 En1 ;
Even-skipped/Eve;
 Evx1 ;
Evx2;
 Fibroblast growth factor-1 / FGF1 ;
HB9;
Isl1;
Isl2;
Lim3;
Nkx6;
p75 neurotrophin receptor;
REG2;
Sim1;
SMI32;
Zfh1


4.1G;
 Acetylcholinesterase ;
Ack1;
AMPA Receptor Binding Protein/ABP;
ARG3.1;
Arp2;
E-Cadherin;
N-Cadherin;
 Calcyon ;
 Catenin  alpha and beta;
 Caveolin ;
CHAPSYN-110/PSD93;
 Chromogranin A ;
Clathrin light chain;
 Cofilin ;
Complexin 1/ CPLX1 /Synaphin 2;
Contactin-1;
 CRIPT ;
Cysteine String Protein/CSP;
 Dynamin 1 ;
Dymanin 2;
Flotillin-1;
Fodrin;
 GRASP ;
 GRIP1 ;
Homer;
Mint-1;
 Munc-18 ;
NSF;
 PICK1 ;
 PSD-95 ;
RAB4;
Rabphillin 3A;
SAD A;
SAD B;
SAP-102;
SHANK1a;
 SNAP-25 ;
Snapin;
 Spinophilin /Neurabin-1;
 Stargazin ;
Striatin;
SYG-1;
Synaptic Vesicle Protein 2A;
Synaptic Vesicle Protein 2B;
 Synapsin 1 ;
 Synaptobrevin /VAMP;
Synaptojanin 1;
 Synaptophysin ;
 Synaptotagmin ;
synGAP;
Synphilin-1;
Syntaxin 1;
Syntaxin 2;
Syntaxin 3;
Syntaxin 4;
Synuclein alpha;
VAMP-2;
Vesicular Acetylcholine Transporter/VAChT;
 Vesicular GABA transporter /VGAT/VIAAT;
Vesicular Glutamate Transporter 1, 2, 3/VGLUT;
 Vesicular monoamine transporter 1 ,  2


Acetylcholine/ACh ;
 Acetylcholinesterase ;
 Choline Acetyltransferase/ChAT ;
 Choline transporter ;
 Vesicular Acetylcholine Transporter/VAChT


Adrenaline ;
 Dopamine ;
 Dopamine Beta Hydroxylase/DBH ;
 Dopamine Transporter/DAT ;
 L-DOPA ;
Nitric Oxide-Dopamine;
 Norepinephrine ;
 Norepinephrine Transporter/NET ;
Parkin;
 Tyrosine Hydroxylase/TH ;
 TorsinA


DL-5-Hydroxytryptophan;
 Serotonin ;
 Serotonin Transporter/SERT ;
 Tryptophan Hydroxylase


DARPP-32 ;
 GABA ;
 GABA Transporters 1 ;
GABA Transporters 2;
GABA Transporters 3;
 Glutamate Decarboxylase/GAD ;
 Vesicular GABA transporter/VGAT/VIAAT


Glutamate ;
 Glutamate Transporter ;
 Glutamine ;
 Glutamine Synthetase ;
 Vesicular Glutamate Transporter 1 ;
Vesicular Glutamate Transporter 2;
Vesicular Glutamate Transporter 3


A  neutral network  is a set of  genes  all related by  point mutations  that have equivalent function or  fitness . [ 1 ]  Each node represents a gene sequence and each line represents the mutation connecting two sequences. Neutral networks can be thought of as high, flat plateaus in a  fitness landscape . During  neutral evolution , genes can randomly move through neutral networks and traverse regions of  sequence space  which may have consequences for  robustness  and  evolvability .


Neutral networks exist in  fitness landscapes  since proteins are  robust  to mutations. This leads to extended networks of genes of equivalent function, linked by  neutral mutations . [ 2 ] [ 3 ]  Proteins are resistant to mutations because many sequences can fold into highly similar  structural folds . [ 4 ]  A protein adopts a limited ensemble of native conformations because those conformers have lower energy than unfolded and mis-folded states (ΔΔG of folding). [ 5 ] [ 6 ]  This is achieved by a distributed, internal network of cooperative interactions ( hydrophobic ,  polar  and  covalent ). [ 7 ]  Protein structural robustness results from few single mutations being sufficiently disruptive to compromise function. Proteins have also evolved to avoid  aggregation [ 8 ]  as partially folded proteins can combine to form large, repeating, insoluble  protein fibrils  and masses. [ 9 ]  There is evidence that proteins show negative design features to reduce the exposure of aggregation-prone  beta-sheet  motifs in their structures. [ 10 ]
Additionally, there is some evidence that the  genetic code  itself may be optimised such that most point mutations lead to similar amino acids ( conservative ). [ 11 ] [ 12 ]  Together these factors create a  distribution of fitness effects  of mutations that contains a high proportion of neutral and nearly-neutral mutations. [ 13 ]


Neutral networks are a subset of the sequences in  sequence space  that have equivalent function, and so form a wide, flat  plateau  in a  fitness landscape .  Neutral evolution  can therefore be visualised as a population diffusing from one set of sequence nodes, through the neutral network, to another cluster of sequence nodes. Since the majority of evolution is thought to be neutral, [ 14 ] [ 15 ]  a large proportion of gene change is the movement though expansive neutral networks.


The more neutral neighbours a sequence has, the more  robust to mutations  it is since mutations are more likely to simply neutrally convert it into an equally functional sequence. [ 1 ]  Indeed, if there are large differences between the number of neutral neighbours of different sequences within a neutral network, the population is predicted to evolve towards these robust sequences. This is sometimes called circum-neutrality and represents the movement of populations away from cliffs in the  fitness landscape . [ 16 ]


In addition to in silico models, [ 17 ]  these processes are beginning to be confirmed by  experimental evolution  of  cytochrome P450s [ 18 ]  and  B-lactamase . [ 19 ]


Interest in the interplay between  genetic drift  and selection has been around since the 1930s when the shifting-balance theory proposed that in some situations, genetic drift could facilitate later adaptive evolution. [ 20 ]  Although the specifics of the theory were largely discredited, [ 21 ]  it drew attention to the possibility that drift could generate cryptic variation that, though neutral to current function, may affect selection for new functions ( evolvability ). [ 22 ]


By definition, all genes in a neutral network have equivalent function, however some may exhibit  promiscuous activities  which could serve as starting points for adaptive evolution towards new functions. [ 23 ] [ 24 ]  In terms of  sequence space , current theories predict that if the neutral networks for two different activities overlap, a neutrally evolving population may diffuse to regions of the neutral network of the first activity that allow it to access the second. [ 25 ]  This would only be the case when the distance between activities is smaller than the distance that a neutrally evolving population can cover. The degree of interpenetration of the two networks will determine how common cryptic variation for the promiscuous activity is in sequence space. [ 26 ]


The fact that neutral mutations were probably widespread was proposed by Freese and Yoshida in 1965. [ 27 ]   Motoo Kimura  later crystallized a theory of neutral evolution in 1968 [ 28 ]  with King and Jukes independently proposing a similar theory (1969). [ 29 ]   Kimura computed the rate of nucleotide substitutions in a population (i.e. the average time for one base pair replacement to occur within a genome)  and found it to be ~1.8 years. Such a high rate would not be tolerated by any mammalian population according to  Haldane 's formula. He thus concluded that, in mammals, neutral (or nearly neutral) nucleotide substitution mutations of  DNA   must dominate. He computed that such mutations were occurring at the rate of roughly 0-5 per year per gamete.


In later years, a new paradigm emerged, that placed  RNA  as a precursor molecule to  DNA . A primordial molecule principle was put forth as early as 1968 by  Crick , [ 30 ]  and lead to what is now known as  The RNA World Hypothesis . [ 31 ]   DNA  is found, predominantly, as fully  base paired  double helices, while biological  RNA  is single stranded and often exhibits complex base-pairing interactions. These are due to its increased ability to form  hydrogen bonds , a fact which stems from the existence of the extra  hydroxyl  group in the  ribose  sugar.


In the 1970s, Stein and  M. Waterman  laid the groundwork for the combinatorics of  RNA  secondary structures. [ 32 ]   Waterman  gave the first graph theoretic description of   RNA  secondary structures and their associated properties, and used them to produce an efficient  minimum free energy  (MFE) folding algorithm. [ 33 ]  An  RNA  secondary structure can be viewed as a diagram over N labeled vertices with its Watson-Crick  base pairs  represented as non-crossing arcs in the upper half plane. Therefore, a  secondary structure  is a scaffold having many sequences compatible with its implied  base pairing constraints.  Later,  Smith and Waterman  developed an algorithm that performed local sequence alignment. [ 34 ]  Another prediction algorithm for  RNA  secondary structure was given by  Nussinov [ 35 ]  Nussinov's algorithm described the folding problem over a two letter alphabet as a planar graph optimization problem, where the quantity to be maximized is the number of matchings in the sequence string.


Come the year 1980, Howell et al. computed a generating function of all foldings of a sequence [ 36 ]  while  D. Sankoff  (1985) described algorithms for alignment of finite sequences, the prediction of RNA secondary structures (folding), and the reconstruction of proto-sequences on a phylo-genetic tree. [ 37 ]  Later,  Waterman  and  Temple  (1986) produced a  polynomial time   dynamic programming  (DP) algorithm for predicting general  RNA  secondary structure. [ 38 ]  while in the year 1990, John McCaskill presented a polynomial time DP algorithm for computing the full equilibrium partition function of an RNA secondary structure. [ 39 ]  This changed the dominant calculation of RNA folding from a mapping of sequence to a particular 3D structure, to a mapping of sequence to a whole weighted ensemble of structures, which smooths RNA fitness, which depends on sequence via folding, facilitating more nearly neutral nets.


M. Zuker, implemented algorithms for computation of MFE  RNA  secondary structures [ 40 ]  based on the work of   Nussinov  et al., [ 35 ]   Smith and Waterman [ 34 ]  and Studnicka, et al. [ 41 ]  Later L. Hofacker (et al., 1994), [ 42 ]  presented The  Vienna RNA package , a software package that integrated  MFE folding and the computation of the partition function as well as base pairing probabilities.


Peter Schuster  and W. Fontana (1994) shifted the focus towards sequence to structure maps  ( genotype–phenotype ) . They used an inverse folding algorithm, to produce computational evidence that  RNA  sequences sharing the same structure are distributed randomly in  sequence space . They observed that common structures can be reached from a random sequence by just a few mutations. These two facts lead them to conclude that the sequence space seemed to be  percolated  by neutral networks of nearest neighbor mutants that fold to the same structure. [ 43 ]


In 1997, C. Reidys  Stadler and  Schuster  laid the mathematical foundations for the study and modelling of neutral networks of  RNA  secondary structures. Using a  random graph model  they proved the existence of a threshold value for connectivity of random sub-graphs in a configuration space, parametrized by λ, the fraction of neutral neighbors. They showed that the networks are connected and  percolate  sequence space if the fraction of neutral nearest neighbors exceeds  λ*,  a threshold value. Below this threshold the networks are partitioned into a largest  giant component  and several smaller ones. Key results of this analysis where concerned with threshold functions for density and connectivity for neutral networks as well as  Schuster 's shape space conjecture. [ 43 ] [ 44 ] [ 45 ]


The  nik operon  is an  operon  required for uptake of nickel ions into the cell. It is present in many bacteria, but has been extensively studied in  Helicobacter pylori .  Nickel  is an essential nutrient for many microorganisms, where it participates in a variety of cellular processes. However, excessive levels of nickel ions in cell can be fatal to the cell. Nickel ion concentration in the cell is regulated through the  nik  operon.


The  nik  operon consists of six genes. The first five genes  nikABCDE  encode components of a typical  ABC transport system  and the last gene  nikR  encodes a DNA-binding protein that represses transcription of  nikABCDE  when sufficient Ni 2+  is present. The  nikR  gene is located 5 bp downstream of the end of  nikE , transcribed in the same direction as  nikABCDE . The following table summarizes the structure of the  nik  operon:


Regulation of expression of the  nikR  gene is achieved by two  promoters . The first is through the  FNR regulon . The FNR controlled regulation of  nikABCDE–nikR  occurs at a FNR box located upstream of  nikA  at a putative NikR binding site. [ 1 ]  The second promoter element regulating  nikR  expression occurs 51 bp upstream of the  nikR  transcription start site and results in low-level constitutive expression. There is also evidence that  nikR  expression is partially autoregulated. [ 2 ]


Ni 2+  is taken up into prokaryotic cells by one of two types of high-affinity transport systems. [ 3 ]  The first method involves ABC-type transporters (discussed in this article) and the second mechanism makes use of transition-metal permeases (such as HoxN of  Ralstonia eutropha ). The ABC-type transporter system consists of five proteins, NikA–E, that carry out the ATP-dependent transport of Ni 2+ . [ 1 ]  NikA is a soluble, periplasmic, Ni-binding protein; NikB and NikC form a transmembrane pore for passage of Ni; and NikD and NikE hydrolyze ATP and couple this energy to Ni 2+ -transport. When Ni 2+  is available in excess, NikR protein represses transcription of  nikABCDE . [ 2 ]


Using profile-based sequence database searches, NikR was shown to be a member of the ribbon-helix-helix (RHH) family of transcription factors. [ 4 ]  It has been demonstrated that the N-terminal domain of NikR is responsible for binding to DNA and that it only binds in presence of Ni 2+ . NikR has two sites for binding to Ni 2+  ions. Binding of Ni 2+  at concentrations that allow full occupancy of only the high-affinity sites is sufficient for operator binding, but the affinity for the operator is increased 1000-fold and the operator footprints are larger when both nickel-binding sites are occupied. [ 5 ]  These results, combined with estimates of intracellular Ni 2+  and NikR concentrations, lead to the conclusion that NikR is able to sense Ni 2+  and regulate the  nik  operon expression over a range of intracellular Ni 2+  concentrations from as low as one to as high as 10,000 molecules per cell.


Nondisjunction  is the failure of  homologous chromosomes  or  sister chromatids  to separate properly during  cell division  ( mitosis / meiosis ). There are three forms of nondisjunction: failure of a pair of  homologous chromosomes  to separate in  meiosis I , failure of sister chromatids to separate during  meiosis II , and failure of sister chromatids to separate during  mitosis . [ 1 ] [ 2 ] [ 3 ]  Nondisjunction results in daughter cells with abnormal chromosome numbers ( aneuploidy ).


Calvin Bridges  and  Thomas Hunt Morgan  are credited with discovering nondisjunction in  Drosophila melanogaster  sex chromosomes in the spring of 1910, while working in the Zoological Laboratory of Columbia University. [ 4 ]


In general, nondisjunction can occur in any form of cell division that involves ordered distribution of chromosomal material. Higher animals have three distinct forms of such cell divisions:  Meiosis I  and   meiosis II  are specialized forms of cell division occurring during generation of  gametes  (eggs and sperm) for sexual reproduction,   mitosis  is the form of cell division used by all other cells of the body. [ citation needed ]


Ovulated eggs become arrested in metaphase II until  fertilization  triggers the second meiotic division. [ 5 ]  Similar to the segregation events of  mitosis , the pairs of sister  chromatids  resulting from the separation of bivalents in  meiosis I  are further separated in  anaphase  of  meiosis II . In oocytes, one sister chromatid is segregated into the second polar body, while the other stays inside the egg. During  spermatogenesis , each meiotic division is symmetric such that each primary  spermatocyte  gives rise to 2 secondary spermatocytes after meiosis I, and eventually 4  spermatids  after meiosis II. Meiosis II-nondisjunction may also result in  aneuploidy  syndromes, but only to a much smaller extent than do  segregation  failures in meiosis I. [ 6 ]


Division of  somatic  cells through mitosis is preceded by replication of the genetic material in  S phase . As a result, each chromosome consists of two sister  chromatids  held together at the  centromere . In the  anaphase  of  mitosis , sister  chromatids  separate and migrate to opposite cell poles before the cell divides. Nondisjunction during  mitosis  leads to one daughter receiving both sister  chromatids  of the affected chromosome while the other gets none. [ 2 ] [ 3 ]  This is known as a  chromatin bridge  or an anaphase bridge. Mitotic nondisjunction results in somatic  mosaicism , since only daughter cells originating from the cell where the nondisjunction event has occurred will have an abnormal number of  chromosomes . [ 3 ]  Nondisjunction during mitosis can contribute to the development of some forms of  cancer , e.g.,  retinoblastoma  (see below). [ 7 ]  Chromosome nondisjunction in mitosis can be attributed to the inactivation of  topoisomerase II ,  condensin , or  separase . [ 8 ]  Meiotic nondisjunction has been well studied in  Saccharomyces cerevisiae . This yeast undergoes mitosis similarly to other  eukaryotes . Chromosome bridges occur when sister chromatids are held together post replication by DNA-DNA topological entanglement and the  cohesion  complex. [ 9 ]  During anaphase,  cohesin  is cleaved by separase. [ 10 ]  Topoisomerase II and condensin are responsible for removing  catenations . [ 11 ]


The  spindle assembly checkpoint  (SAC) is a molecular safe-guarding mechanism that governs proper  chromosome segregation  in eukaryotic cells. [ 12 ]  SAC inhibits progression into anaphase until all homologous chromosomes (bivalents, or tetrads) are properly aligned to the  spindle apparatus . Only then, SAC releases its inhibition of the  anaphase promoting complex  (APC), which in turn irreversibly triggers progression through anaphase. [ citation needed ]


Surveys of cases of human aneuploidy syndromes have shown that most of them are maternally derived. [ 5 ]  This raises the question: Why is female meiosis more error prone? The most obvious difference between female oogenesis and male spermatogenesis is the prolonged arrest of oocytes in late stages of  prophase I  for many years up to several decades. Male gametes on the other hand quickly go through all stages of meiosis I and II. Another important difference between male and female meiosis concerns the frequency of recombination between homologous chromosomes: In the male, almost all chromosome pairs are joined by at least one  crossover , while more than 10% of human oocytes contain at least one bivalent without any crossover event. Failures of recombination or inappropriately located crossovers have been well documented as contributors to the occurrence of nondisjunction in humans. [ 5 ]


Due to the prolonged arrest of human oocytes, weakening of cohesive ties holding together chromosomes and reduced activity of the SAC may contribute to maternal age-related errors in  segregation  control. [ 6 ] [ 13 ]  The  cohesin  complex is responsible for keeping together sister chromatids and provides binding sites for spindle attachment. Cohesin is loaded onto newly replicated chromosomes in  oogonia  during fetal development. Mature  oocytes  have only limited capacity for reloading cohesin after completion of  S phase . The prolonged arrest of human oocytes prior to completion of meiosis I may therefore result in considerable loss of cohesin over time. Loss of cohesin is assumed to contribute to incorrect  microtubule - kinetochore  attachment and chromosome segregation errors during meiotic divisions. [ 6 ]


The result of this error is a cell with an imbalance of chromosomes. Such a cell is said to be  aneuploid . Loss of a single chromosome (2n-1), in which the daughter cell(s) with the defect will have one chromosome missing from one of its pairs, is referred to as a  monosomy . Gaining a single chromosome, in which the daughter cell(s) with the defect will have one chromosome in addition to its pairs is referred to as a  trisomy . [ 3 ]  In the event that an aneuploidic gamete is fertilized, a number of syndromes might result. [ citation needed ]


The only known survivable monosomy in humans is  Turner syndrome , where the affected individual is monosomic for the  X chromosome  (see below). Other monosomies are usually lethal during early fetal development, and survival is only possible if not all the cells of the body are affected in case of a  mosaicism  (see below), or if the normal number  of chromosomes is restored via duplication of the single monosomic chromosome ("chromosome rescue"). [ 2 ]


Complete loss of an entire X chromosome accounts for about half the cases of  Turner syndrome . The importance of both X chromosomes during embryonic development is underscored by the observation that the overwhelming majority (>99%) of fetuses with only one X chromosome ( karyotype  45, X0) are spontaneously aborted. [ 14 ]


The term autosomal trisomy means that a chromosome other than the sex chromosomes X and Y is present in 3 copies instead of the normal number of 2 in diploid cells. [ citation needed ]


Down syndrome , a trisomy of chromosome 21, is the most common anomaly of chromosome number in humans. [ 2 ]  The majority of cases result from nondisjunction during maternal meiosis I. [ 14 ]  Trisomy occurs in at least 0.3% of newborns and in nearly 25% of  spontaneous abortions . It is the leading cause of pregnancy wastage and is the most common known cause of  intellectual disability . [ 15 ]  It is well documented that  advanced maternal age  is associated with greater risk of meiotic nondisjunction leading to Down syndrome. This may be associated with the prolonged meiotic arrest of human oocytes potentially lasting for more than four decades. [ 13 ]


Human autosomal trisomies compatible with live birth, other than  Down syndrome  (trisomy 21), are  Edwards syndrome  (trisomy 18) and  Patau syndrome  (trisomy 13). [ 1 ] [ 2 ]  Complete trisomies of other chromosomes are usually not viable and represent a relatively frequent cause of miscarriage. Only in rare cases of a  mosaicism , the presence of a normal cell line, in addition to the trisomic cell line, may support the development of a viable trisomy of the other chromosomes. [ 2 ]


The term  sex chromosome aneuploidy  summarizes conditions with an abnormal number of sex chromosomes, i.e., other than XX (female) or XY (male). Formally, X chromosome monosomy ( Turner syndrome , see above) can also be classified as a form of sex chromosome aneuploidy. [ citation needed ]


Klinefelter syndrome  is the most common sex chromosome aneuploidy in humans. It represents the most frequent cause of  hypogonadism  and  infertility  in men. Most cases are caused by nondisjunction errors in paternal meiosis I. [ 2 ]  About eighty percent of individuals with this syndrome have one extra X chromosome resulting in the  karyotype  XXY. The remaining cases have either multiple additional sex chromosomes (48,XXXY; 48,XXYY; 49,XXXXY), mosaicism (46,XY/47,XXY), or structural chromosome abnormalities. [ 2 ]


The incidence of  XYY syndrome  is approximately 1 in 800–1000 male births. Many cases remain undiagnosed because of their normal appearance and fertility, and the absence of severe symptoms. The extra Y chromosome is usually a result of nondisjunction during paternal meiosis II. [ 2 ]


Trisomy X  is a form of sex chromosome aneuploidy where females have three instead of two X chromosomes. Most patients are only mildly affected by neuropsychological and physical symptoms. Studies examining the origin of the extra X chromosome observed that about 58–63% of cases were caused by nondisjunction in maternal meiosis I, 16–18% by nondisjunction in maternal meiosis II, and the remaining cases by post-zygotic, i.e., mitotic, nondisjunction. [ 16 ]


Uniparental disomy  denotes the situation where both chromosomes of a chromosome pair are inherited from the same parent and are therefore identical. This phenomenon most likely is the result of a pregnancy that started as a trisomy due to nondisjunction. Since most trisomies are lethal, the fetus only survives because it loses one of the three chromosomes and becomes disomic. Uniparental disomy of chromosome 15 is, for example, seen in some cases of  Prader-Willi  syndrome and  Angelman syndrome . [ 14 ]


Mosaicism  syndromes can be caused by mitotic nondisjunction in early fetal development. As a consequence, the organism evolves as a mixture of cell lines with differing  ploidy  (number of chromosomes). Mosaicism may be present in some tissues, but not in others. Affected individuals may have a patchy or asymmetric appearance. Examples of mosaicism syndromes include  Pallister-Killian syndrome  and  Hypomelanosis of Ito . [ 14 ]


Development of cancer often involves multiple alterations of the cellular genome ( Knudson hypothesis ). Human  retinoblastoma  is a well studied example of a cancer type where mitotic nondisjunction can contribute to malignant transformation: Mutations of the RB1 gene, which is located on chromosome 13 and encodes the tumor suppressor  retinoblastoma protein , can be detected by cytogenetic analysis in many cases of retinoblastoma. Mutations of the RB1 locus in one copy of chromosome 13 are sometimes accompanied by loss of the other wild-type chromosome 13 through mitotic nondisjunction. By this combination of lesions, affected cells completely lose expression of functioning tumor suppressor protein. [ 7 ]


Pre-implantation genetic diagnosis (PGD or PIGD)  is a technique used to identify genetically normal  embryos  and is useful for couples who have a family history of genetic disorders. This is an option for people choosing to procreate through  IVF . PGD is considered difficult due to it being both time consuming and having success rates only comparable to routine IVF. [ 17 ]


Karyotyping  involves performing an  amniocentesis  in order to study the cells of an unborn fetus during metaphase 1.  Light microscopy  can be used to visually determine if aneuploidy is an issue. [ 18 ]


Polar body diagnosis  (PBD) can be used to detect maternally derived chromosomal aneuploidies as well as translocations in oocytes. The advantage of PBD over PGD is that it can be accomplished in a short amount of time. This is accomplished through zona drilling or laser drilling. [ 19 ]


Blastomere  biopsy is a technique in which blastomeres are removed from the  zona pellucida .  It is commonly used to detect aneuploidy. [ 20 ]  Genetic analysis is conducted once the procedure is complete. Additional studies are needed to assess the risk associated with the procedure. [ 21 ]


Exposure of spermatozoa to lifestyle, environmental and/or occupational hazards may increase the risk of aneuploidy. Cigarette smoke is a known  aneugen  ( aneuploidy  inducing agent). It is associated with increases in aneuploidy ranging from 1.5 to 3.0-fold. [ 22 ] [ 23 ]  Other studies indicate factors such as alcohol consumption, [ 24 ]  occupational exposure to  benzene , [ 25 ]  and exposure to the insecticides  fenvalerate [ 26 ]  and  carbaryl [ 27 ]  also increase aneuploidy.


Noninvasive genotyping  is a modern technique for obtaining DNA for genotyping that is characterized by the indirect sampling of specimen, not requiring harm to, handling of, or even the presence of the organism of interest. Beginning in the early 1990s, with the advent of  PCR , researchers have been able to obtain high-quality  DNA  samples from small quantities of hair, feathers, scales, or excrement. These  noninvasive  samples are an improvement over older  allozyme  and DNA sampling techniques that often required larger samples of tissue or the destruction of the studied organism. Noninvasive genotyping is widely utilized in conservation efforts, where capture and sampling may be difficult or disruptive to behavior. [ 1 ]  Additionally, in medicine, this technique is being applied in humans for the diagnosis of genetic disease and early detection of tumors. In this context, invasivity takes on a separate definition where noninvasive sampling also includes simple blood samples.


In conservation, noninvasive genotyping is an important part of implementing the  3Rs principles . [ 2 ] [ 3 ]  Modern DNA amplification methods allow researchers to use a variety of animal material collected in the field, including feces, [ 4 ] [ 5 ]  hair, [ 6 ]  and feathers, [ 7 ]  to gain insights into effective population size, gene flow, and hybridization. [ 8 ]  Despite the potential that noninvasive genotyping has in  conservation genetics  efforts, it is still not broadly used, [ 9 ]  potentially due to problems with degradation, contamination or a lower DNA quality in comparison with blood or tissue samples. However, optimized laboratory protocols and specialized extraction kits can help overcome these issues. [ 10 ] [ 11 ]


The most common use of noninvasive genotyping in medicine is  non-invasive prenatal diagnosis  (NIPD), which provides an alternative to riskier techniques such as  amniocentesis . With the discovery of  cell-free fetal DNA  in maternal plasma, NIPD became a popular method for determining sex, paternity,  aneuploidy , and the occurrence of  monogenic diseases  as it requires only a simple blood sample. [ 12 ] [ 13 ]  One NIPD provider maintains that a 10 mL blood sample will provide 99% accurate detection of basic genomic abnormalities as early as 10 weeks into pregnancy. [ 14 ]  The  karyotype  below is that of an individual with trisomy 21, or  Down Syndrome , which is what is most routinely checked for by NIPD screens.


This same technique is also utilized to identify the incidence of tumor DNA in the blood, which can both provide early detection of tumor growth and indicate relapse in cancer.  Circulating tumor DNA  can be found in the blood before  metastasis  occurs and, therefore, detection of certain mutant alleles may enhance survival rates in cancer patients. [ 15 ] [ 16 ]  In a recent study, ctDNA was shown to be "a broadly applicable, sensitive, and specific biomarker that can be used for a variety of clinical and research purposes in patients with multiple different types of cancer". [ 17 ]  This technique is often referred to as a  liquid biopsy , and has not been widely implemented in clinical settings although its impact could be quite large. [ 18 ]  Although blood-borne ctDNA remains the most clinically significant noninvasive cancer detection, other studies have emerged that investigate other potential methods, including detection of colorectal cancer via fecal samples. [ 19 ]


The method by which samples are collected in noninvasive genotyping is what separates the technique from traditional genotyping, and there are a number of ways that this is accomplished. In the field, procured samples of tissue are captured, the tissue is dissolved, and the DNA is  purified , although the exact procedure differs between different samples. [ 20 ]  Following the collection of DNA samples, PCR technology is utilized to amplify particular genetic sequences, with PCR  primer  specificity avoiding contamination from other DNA sources. Then, the DNA can be analyzed using a number of genomic techniques, similarly to traditionally obtained samples.


Nuclease protection assay  is a laboratory technique used in  biochemistry  and  genetics  to identify individual  RNA  molecules in a heterogeneous RNA sample extracted from  cells . The technique can identify one or more RNA molecules of known sequence even at low total  concentration . The extracted RNA is first mixed with  antisense  RNA or  DNA  probes that are complementary to the sequence or sequences of interest and the complementary strands are  hybridized  to form double-stranded RNA (or a DNA-RNA hybrid). The mixture is then exposed to  ribonucleases  that specifically cleave only  single -stranded RNA but have no activity against double-stranded RNA. When the reaction runs to completion, susceptible RNA regions are degraded to very short  oligomers  or to individual  nucleotides ; the surviving RNA fragments are those that were complementary to the added antisense strand and thus contained the sequence of interest.


The probes are prepared by cloning part of the gene of interest in a vector under the control of any of the following promoters, SP6, T7 or T3. These promoters are recognized by DNA dependent RNA polymerases originally characterized from bacteriophages. The probes produced are radioactive as they are prepared by in vitro transcription using radioactive UTPs. Uncomplemented DNA or RNA is cleaved off by nucleases. When the probe is a DNA molecule,  S1 nuclease  is used; when the probe is RNA, any single-strand-specific ribonuclease can be used. Thus the surviving probe-mRNA complement is simply detected by autoradiography.


Nuclease protection assays are used to map  introns  and  5'  and  3'  ends of transcribed  gene  regions.  Quantitative  results can be obtained regarding the amount of the target RNA present in the original cellular extract - if the target is a  messenger RNA , this can indicate the level of  transcription  of the gene in the cell.


They are also used to detect the presence of double stranded RNA, presence of which could mean  RNA interference .


Northern blotting  is a laboratory technique that produces similar information. It is slower and less quantitative, but also produces accurate information about the  size  of the target RNA. Nuclease protection assay products are limited to the size of the initial probes due to the destruction of the non-hybridized RNA during the nuclease digestion step.


Nucleomodulins  are a family of bacterial proteins that enter the nucleus of  eukaryotic cells . [ 1 ]


This term comes from the contraction between "nucleus" and "modulins", which are microbial molecules that modulate the behaviour of eukaryotic cells. Nucleomodulins are produced by pathogenic or symbiotic bacteria. They act on various processes in the  nucleus : remodelling of the  chromatin  structure, [ 2 ] [ 3 ] [ 4 ] [ 5 ] [ 6 ] [ 7 ] [ 8 ] [ 9 ] [ 10 ] [ 11 ] [ 12 ] [ excessive citations ]   transcription , [ 13 ] [ 14 ]   splicing of pre-messenger RNA , [ 15 ] [ 16 ]   cell division . [ 17 ]


The identification of nucleomodulins in several species of bacterial pathogens of humans, animals and plants has led to the emergence of the concept that direct control of the nucleus is one of the most sophisticated strategies used by microbes to bypass host defences. Nucleomodulins can be directly secreted into the intracellular medium after entry of the bacteria into the cell, like  Listeria monocytogenes , or they can be injected from the extracellular medium or intracellular  organelles  using a type  III  or  IV  bacterial secretion system, also known as a "molecular syringe". [ citation needed ]


More recently, it has been shown that some of them, such as YopM from  Yersinia pestis  and IpaH9.8 from  Shigella flexneri , can autonomously penetrate eukaryotic cells thanks to a membrane transduction domain. [ 18 ]


The diversity of molecular mechanisms triggered by nucleomodulins  [ 1 ] [ 19 ]  is a source of inspiration for new  biotechnologies . They are true nano-machines capable of hijacking a multitude of nuclear processes. In research, nucleomodulins are the subject of in-depth studies that have led to the discovery of new human nuclear regulators, such as the  epigenetic  regulator  BAHD1 . [ 8 ]


Agrobacterium tumefaciens , responsible for crown gall disease, produces an arsenal of Vir proteins, including VirD2 and VirE2, enabling the precise integration of a piece of its DNA, called  T-DNA , into that of the host plant  [ 20 ]


Listeria monocytogenes , responsible for listeriosis, can modulate the expression of immunity genes. One of the mechanisms at play involves the bacterial protein LntA, which inhibits the function of the epigenetic regulator BAHD1. The action of this nucleomodulin is associated with chromatin decompaction and activation of an interferon response genes. [ 8 ] [ 21 ]


Shigella flexneri , responsible for shigellosis, secretes the IpaH9.8 protein targeting a  mRNA  splicing protein that disrupts the production of protein isoforms and the inflammatory response in humans. [ 16 ]


Legionella pneumophila , responsible for  legionellosis , secretes an  enzyme  with histone methyltransferase activity capable of methylating  histones  at different  chromosome  loci  [ 22 ]  or at the level of  ribosomal DNA  (rDNA) in the nucleolus. [ 23 ]


Ohno's law  was proposed by a Japanese-American biologist  Susumu Ohno , saying that the  gene  content of the  mammalian   species  has been conserved over species not only in the  DNA  content but also in the genes themselves. That is, nearly all mammalian species have conserved the  X chromosome  from their primordial X chromosome of a common ancestor. [ 1 ]


Mammalian X chromosomes in various species, including  human  and  mouse , have nearly the same size, with the content of about 5% of the genome. Additionally, for individual  gene loci , a number of X-linked genes are common through mammalian species.  Examples include  glucose-6-phosphate dehydrogenase  (G6PD), and the genes for   Factor VIII  and  Factor IX . Moreover, no instances were found where an  X-linked  gene in one species was located on an  autosome  in the other species. [ 1 ]


The content of a chromosome would be changed mainly by mutation after duplication of the chromosome and translocation with other chromosomes. However, in mammals, since the chromosomal  sex-determination  mechanism would have been established in their earlier stages of  evolution ,  polyploidy  would have not occurred due to its incompatibility with the sex-determining mechanism. Moreover, X-autosome  translocation  would have been prohibited because it might have resulted in detrimental effects for survival to the organism. Thus in mammals, the content of X chromosomes has been conserved after typical 2 round duplication events at early ancestral stages of evolution, at the fish or amphibia ( 2R hypothesis ). [ 1 ] [ 2 ]


Genes on the long arm of the human X are contained in the monotreme X and genes on the short arm of the human X are distributed on the autosomes of marsupials. [ 3 ]  Ohno commented to the result that  monotremes  and  marsupials  were not considered to be ancestors of true mammals, but they have diverged very early from the main line of mammals. [ 4 ]   Chloride channel  gene ( CLCN4 ) was mapped to the human X but on chromosome 7 of C57BL/6 mice, species of  Mus musculus , though the gene is located on X of  Mus spretus  and  rat . [ 5 ]


Oogenesis  ( / ˌ oʊ . ə ˈ dʒ ɛ n ɪ s ɪ s / ) or  ovogenesis  is the differentiation of the  ovum  (egg cell) into a cell competent to further develop when fertilized. [ 1 ]  It is developed from the primary oocyte by maturation. Oogenesis is initiated in the embryonic stage.


In  mammals , the first part of oogenesis starts in the  germinal epithelium , which gives rise to the development of  ovarian follicles , the functional unit of the  ovary .


Oogenesis consists of several sub-processes:  oocytogenesis ,  ootidogenesis , and finally maturation to form an ovum (oogenesis proper).  Folliculogenesis  is a separate sub-process that accompanies and supports all three oogenetic sub-processes.


Oogonium —(Oocytogenesis)—> Primary Oocyte —(Meiosis I)—> First  Polar body  (Discarded afterward) + Secondary oocyte —(Meiosis II)—> Second Polar Body (Discarded afterward) + Ovum


Oocyte meiosis, important to all animal life cycles yet unlike all other instances of animal cell division, occurs completely without the aid of  spindle -coordinating  centrosomes . [ 2 ] [ 3 ]


The creation of  oogonia  traditionally does not belong to oogenesis proper, but, instead, to the  common process  of  gametogenesis , which, in the female human, begins with the processes of  folliculogenesis , oocytogenesis, and  ootidogenesis . Oogonia enter meiosis during embryonic development, becoming oocytes. Meiosis begins with DNA replication and meiotic crossing over. It then stops in early prophase.


Mammalian oocytes are maintained in meiotic prophase arrest for a very long time—months in mice, years in humans. Initially, the arrest is due to lack of sufficient cell cycle proteins to allow meiotic progression. However, as the oocyte grows, these proteins are synthesized, and meiotic arrest becomes dependent on  cyclic AMP . [ 4 ]  The cyclic AMP is generated by the oocyte by adenylyl cyclase in the oocyte membrane. The adenylyl cyclase is kept active by a constitutively active G-protein-coupled receptor known as  GPR3  and a G-protein, Gs, also present in the oocyte membrane. [ 5 ]


Maintenance of meiotic arrest also depends on the presence of a multilayered complex of cells, known as a follicle, that surrounds the oocyte. Removal of the oocyte from the follicle causes meiosis to progress in the oocyte. [ 6 ]  The cells that comprise the follicle, known as granulosa cells, are connected to each other by proteins known as gap junctions, that allow small molecules to pass between the cells. The granulosa cells produce a small molecule,  cyclic GMP , that diffuses into the oocyte through the gap junctions. In the oocyte, cyclic GMP prevents the breakdown of cyclic AMP by the phosphodiesterase PDE3, and thus maintains meiotic arrest. [ 7 ]  The cyclic GMP is produced by the guanylyl cyclase NPR2. [ 8 ]


As follicles grow, they acquire receptors for luteinizing hormone, a pituitary hormone that reinitiates meiosis in the oocyte and causes ovulation of a fertilizable egg. Luteinizing hormone acts on receptors in the outer layers of granulosa cells of the follicle, causing a decrease in cyclic GMP in the granulosa cells. [ 4 ]  Because the granulosa cells and oocyte are connected by gap junctions, cyclic GMP also decreases in the oocyte, causing meiosis to resume. [ 9 ]  Meiosis then proceeds to second metaphase, where it pauses again until fertilization. Luteinizing hormone also stimulates gene expression leading to ovulation. [ 10 ]


Oogenesis starts with the process of developing primary oocytes, which occurs via the transformation of  oogonia  into primary [oocyte]s, a process called  oocytogenesis . [ 11 ]  From one single oogonium, only one mature oocyte will rise, with 3 other cells called polar bodies. Oocytogenesis is complete either before or shortly after birth.


It is commonly believed that, when oocytogenesis is complete, no additional primary oocytes are created, in contrast to the male process of spermatogenesis, where gametocytes are continuously created. In other words, primary oocytes reach their maximum development at ~20  weeks of gestational age, when approximately seven million primary oocytes have been created; however, at birth, this number has already been reduced to approximately 1-2 million per ovary. At puberty, the number of oocytes decreases even more to reach about 60,000 to 80,000 per ovary, and only about 500 mature oocytes will be produced during a woman's life, the others will undergo atresia (degeneration). [ 12 ]   Two publications have challenged the belief that a finite number of oocytes are set around the time of birth generation in adult mammalian ovaries by putative germ cells in bone marrow and peripheral blood. [ 13 ] [ 14 ]  The renewal of ovarian follicles from germline stem cells (originating from bone marrow and peripheral blood) has been reported in the postnatal mouse ovary. In contrast, DNA clock measurements do not indicate ongoing oogenesis during human females' lifetimes. [ 15 ]
Thus, further experiments are required to determine the true dynamics of small follicle formation.


The succeeding phase of  ootidogenesis  occurs when the  primary oocyte  develops into an  ootid . This is achieved by the process of meiosis. In fact, a primary oocyte is, by its biological definition, a cell whose primary function is to divide by the process of meiosis. [ 16 ]


However, although this process begins at prenatal age, it stops at  prophase I . In late fetal life, all oocytes, still primary oocytes, have halted at this stage of development, called the  dictyate . After  menarche , these cells then continue to develop, although only a few do so every  menstrual cycle .


Meiosis I  of ootidogenesis begins during embryonic development, but halts in the  diplotene  stage of prophase I until puberty. The mouse oocyte in the dictyate (prolonged diplotene) stage actively repairs DNA damage, whereas DNA repair is not detectable in the pre-dictyate ( leptotene ,  zygotene  and  pachytene ) stages of meiosis. [ 17 ]  For those primary oocytes that continue to develop in each menstrual cycle, however,  synapsis  occurs and  tetrads  form, enabling  chromosomal crossover  to occur. As a result of meiosis I, the primary oocyte has now developed into the  secondary oocyte .


Immediately after meiosis I, the  haploid  secondary oocyte initiates  meiosis II . However, this process is also halted at the  metaphase II  stage until  fertilization , if such should ever occur. If the egg is not fertilized, it is disintegrated and released ( menstruation ) and the secondary oocyte does not complete meiosis II (and does not become an  ovum ). When meiosis II has completed, an ootid and another polar body have now been created. The polar body is small in size.


The ovarian cycle is divided into several phases:


The uterine cycle [ 20 ]  occurs parallel to the ovarian cycle and is induced by estrogen and progesterone. The  endometrium , formed by a monostratified cylindrical epithelium, with  uterine glands  (simple tubular), connective with a functional superficial layer (divided into a spongy layer, a compact layer, and a deeper basal layer, which is always maintained, presents four phases:


If, instead, there is fertilization, the uterine mucosa is modified to accommodate the fertilized egg, and the secretive phase is maintained.


Both polar bodies disintegrate at the end of Meiosis II, leaving only the ootid, which then eventually undergoes maturation into a mature ovum.


The function of forming polar bodies is to discard the extra haploid sets of chromosomes that have resulted as a consequence of meiosis.


In vitro maturation  ( IVM ) is the technique of letting  ovarian follicles  mature  in vitro . It can potentially be performed before an  IVF . In such cases,  ovarian hyperstimulation  is not essential. Rather, oocytes can mature outside the body prior to IVF. Hence, no (or at least a lower dose of) gonadotropins have to be injected in the body. [ 21 ]  Immature eggs have been grown until maturation  in vitro  at a 10% survival rate, but the technique is not yet clinically available. [ 22 ]  With this technique, cryopreserved ovarian tissue could possibly be used to make oocytes that can directly undergo  in vitro fertilization . [ 22 ]


By definition it means, to recapitulate mammalian oogenesis and producing fertilizable oocytes in vitro.it is a complex process involving several different cell types, precise follicular cell-oocyte reciprocal interactions, a variety of nutrients and combinations of cytokines, and precise growth factors and hormones depending on the developmental stage. [ 23 ]  In 2016, two papers published by Morohaku et al. and Hikabe et al. reported in vitro procedures that appear to reproduce efficiently these conditions allowing for the production, completely in a dish, of a relatively large number of oocytes that are fertilizable and capable of giving rise to viable offspring in the mouse. This technique can be mainly benefited in cancer patients where in today's condition their ovarian tissue
is cryopreserved for preservation of fertility. Alternatively to the autologous transplantation, the development of culture systems that support oocyte development from the primordial follicle stage represent a valid strategy to restore fertility. Over time, many studies have been conducted with the aim to optimize the characteristics of ovarian tissue culture systems and to better support the three main phases: 1) activation of primordial follicles; 2) isolation and culture of growing preantral follicles; 3) removal from the follicle environment and maturation of oocyte cumulus complexes. While complete oocyte in vitro development has been achieved in mouse, with the production of live offspring, the goal of obtaining oocytes of sufficient quality to support embryo development has not been completely reached into higher mammals despite decades of effort. [ 24 ]


BRCA1  and  ATM  proteins are employed in  repair of DNA  double-strand break during  meiosis . These proteins appear to have a critical role in resisting  ovarian aging . [ 25 ]   However,  homologous recombinational  repair of DNA double-strand breaks mediated by BRCA1 and ATM weakens with age in  oocytes  of humans and other species. [ 25 ]   Women with BRCA1  mutations  have lower  ovarian reserves  and experience earlier  menopause  than women without these mutations.  Even in woman without specific BRCA1 mutations, ovarian aging is associated with depletion of ovarian reserves leading to menopause, but at a slower rate than in those with such mutations.  Since older premenopausal women ordinarily have normal progeny, their capability for meiotic recombinational repair appears to be sufficient to prevent deterioration of their  germline  despite the reduction in ovarian reserve.  DNA damages may arise in the germline during the decades long period in humans between early oocytogenesis and the stage of meiosis in which  homologous chromosomes  are effectively paired ( dictyate  stage).  It has been suggested that such DNA damages may be removed, in large part, by mechanisms dependent on chromosome pairing, such as homologous recombination. [ 26 ]


Some  algae  and the  oomycetes  produce eggs in  oogonia . In the brown alga  Fucus , all four egg cells survive oogenesis, which is an exception to the rule that generally only one product of female meiosis survives to maturity.


In  plants , oogenesis occurs inside the female  gametophyte  via  mitosis . In many plants such as  bryophytes ,  ferns , and  gymnosperms , egg cells are formed in  archegonia . In  flowering plants , the female gametophyte has been reduced to an eight-celled  embryo sac  within the  ovule  inside the  ovary  of the flower. Oogenesis occurs within the embryo sac and leads to the formation of a single egg cell per ovule.


In  ascaris , the oocyte does not even begin meiosis until the  sperm  touches it, in contrast to mammals, where meiosis is completed in the  estrus  cycle.


In female  Drosophila  flies,  genetic recombination  occurs during  meiosis .  This recombination is associated with formation of  DNA double-strand breaks  and the  repair  of these breaks.
 [ 27 ]   The repair process leads to  crossover recombinants  as well as at least three times as many noncrossover recombinants (e.g. arising by  gene conversion  without crossover). [ 27 ]


Cho WK, Stern S, Biggers JD. 1974. Inhibitory effect of dibutyryl cAMP on mouse oocyte maturation in vitro. J Exp Zool.187:383-386


Pathogenicity islands  ( PAIs ), as termed in 1990, are a distinct class of  genomic islands  acquired by  microorganisms  through  horizontal gene transfer . [ 1 ] [ 2 ]  Pathogenicity islands are found in both animal and plant pathogens. [ 2 ]  Additionally, PAIs are found in both  gram-positive  and  gram-negative bacteria . [ 2 ]  They are transferred through horizontal gene transfer events such as transfer by a  plasmid ,  phage , or conjugative  transposon . [ 3 ]  Although the general makeup of pathogenicity islands (PAIs) might vary throughout bacterial pathogen strains, all PAIs are known to have characteristics with all genomic islands, which includes virulence genes, functional mobility elements, and areas of homology to tRNA genes and direct repeats. [ 2 ] [ 4 ]  Therefore, PAIs enables microorganisms to induce disease and also contribute to microorganisms' ability to evolve. The spread of antibiotic resistance and, more generally, the conversion of non-pathogenic strains in natural environments to strains that infect animal and plant hosts with disease are two examples of the evolutionary and ecological changes brought about by the transmission and acquisition of PAIs among bacterial species. [ 5 ]  However, It is impossible to overlook their impact on bacterial evolution, though, since if a PAI is acquired and is stably absorbed, it can irreversibly change the  bacterial genome . [ 2 ] [ 3 ]


One species of bacteria may have more than one PAI. For example,  Salmonella  has at least five. [ 6 ]  An analogous genomic structure in  rhizobia  is termed a  symbiosis island .


Pathogenicity islands (PAIs) are gene clusters incorporated in the  genome , chromosomally or extrachromosomally, of pathogenic organisms, but are usually absent from those  nonpathogenic organisms  of the same or closely related species. [ 2 ] [ 7 ] [ 8 ]  They may be located on a bacterial chromosome or may be transferred within a  plasmid  or can be found in bacteriophage genomes. [ 2 ]  Every genomic island has the following characteristics; a  GC- content  that differs from the surrounding DNA sequence, a connection with tRNA genes, the presence of repeats on both ends (flanking), and the capacity to recombine, which is usually shown by the presence of an  integrase . [ 5 ]  The GC-content and codon usage of pathogenicity islands often differs from that of the rest of the genome, potentially aiding in their detection within a given DNA sequence, unless the donor and recipient of the PAI have similar GC-content. [ 2 ]


The most basic kind of mobile genetic element is an  insertion sequence (IS) , which usually just has one or two  open reading frames  that encode genes to make transposition easier. [ 5 ]  Sections inside the PAI may be rearranged or deleted with the use of IS components. [ 2 ]  These changes encourages adaption and aid in the generation of alternative strains. [ 5 ]  PAIs also contain transposons, which are more sophisticated forms of IS elements. The majority are surrounded by brief terminal inverted repeats that serve as  homologous recombination  sites, enhancing a PAI's stability. [ 5 ]  Bacteriophage integrases also found on pathogenicity islands (PAIs) are enzymes produced by bacteriophages to enable site-specific recombination between two  recognition sequences , serving as another form of mobility element to enable PAIs insertion into host DNA. [ 5 ]  PAIs are often associated with  tRNA  genes, which target sites for this integration event. [ 2 ]  Given that integration may result in tRNA truncation, it is probable that only non-essential tRNA loci found in multiple locations, or those possessing wobble capacity (the ability of a 5' base of a tRNA anticodon to mispair with the thrid base of an mRNA codon) can become common integration sites. [ 2 ]  They can be transferred as a single unit to new bacterial cells, thus conferring virulence to formerly benign strains. [ 7 ]


Pathogenicity islands carry genes encoding one or more virulence factors, including, but not limited to,  adhesins ,  secretion systems  (type III and IV secretion system),  toxins ,  invasins ,  modulins ,  effectors ,  superantigens , iron uptake systems, o-antigen synthesis, serum resistance,  immunoglobulin A  proteases,  apoptosis ,  capsule  synthesis, and plant tumorigenesis via  Agrobacterium tumefaciens . [ 2 ]  Type III and type IV secretion systems, which are both expressed in Gram-negative bacteria, are the secretion systems most frequently linked to PAIs. [ 5 ]  The bacterial membranes contain the  type III secretion system  (T3SS), which functions essentially as a molecular syringe. The needle-like apparatus secretes effectors, which go from the bacterial cell to the host cell via the tip of the apparatus, creating a hole in the membrane of the host cell. [ 5 ]


There are various combinations of regulation involving pathogenicity islands. The first combination is that the pathogenicity island contains the genes to regulate the virulence genes encoded on the PAI. [ 2 ]  The second combination is that the pathogenicity island contains the genes to regulate genes located outside of the pathogenicity island. [ 2 ]  Additionally, regulatory genes outside of the PAI may regulate virulence genes in the pathogenicity island. [ 2 ]  Regulation genes typically encoded on PAIs include AraC-like proteins and two-component response regulators. [ 2 ]


PAIs can be considered unstable DNA regions as they are susceptible to deletions or mobilization. [ 2 ]  This may be due to the structure of PAIs, with direct repeats, insertion sequences and association with tRNA that enables deletion and mobilization at higher frequencies. [ 3 ]  Additionally, deletions of pathogenicity islands inserted in the genome can result in disrupting tRNA and subsequently affect the metabolism of the cell. [ 7 ]


Patome  is a database of  biological sequence  data of issued  patents  and/or published applications. [ 1 ]


Penetrance  in  genetics  is the proportion of individuals carrying a particular variant (or  allele ) of a  gene  ( genotype ) that also expresses an associated trait ( phenotype ). In  medical genetics , the penetrance of a  disease -causing  mutation  is the proportion of individuals with the mutation that exhibit clinical  symptoms  among all individuals with such mutation. [ 1 ]  For example: If a mutation in the gene responsible for a particular  autosomal dominant  disorder has 95% penetrance, then 95% of those with the mutation will go on to develop the disease, showing its phenotype, whereas 5% will not.


Penetrance only refers to whether an individual with a specific genotype exhibits any phenotypic signs or symptoms, and is not to be confused with  variable expressivity  which is to what extent or degree the symptoms for said disease are shown (the expression of the phenotypic trait). Meaning that, even if the same disease-causing mutation affects separate individuals, the expressivity will vary. [ 1 ] [ 2 ]   [ 3 ]


If 100% of individuals carrying a particular genotype express the associated trait, the genotype is said to show complete penetrance. [ 1 ]   Neurofibromatosis type 1 (NF1) , is an autosomal dominant condition which shows complete penetrance, consequently everyone who inherits the disease-causing variant of this gene will develop some degree of symptoms for NF1. [ 4 ]


The penetrance is said to be reduced if less than 100% of individuals carrying a particular genotype express associated traits, and is likely to be caused by a combination of genetic, environmental and lifestyle factors. [ 1 ] [ 3 ]   BRCA1  is an example of a genotype with reduced penetrance. By age 70, the mutation is estimated to have a breast cancer penetrance of around 65% in women. Meaning that about 65% of women carrying the gene will develop breast cancer by the time they turn 70. [ 5 ]


Many factors such as age, sex, environment, epigenetic modifiers, and modifier genes are linked to penetrance. These factors can help explain why certain individuals with a specific genotype exhibit symptoms or signs of disease, whilst others do not.  [ 1 ] [ 3 ]


If clinical signs associated with a specific genotype appear more frequently with increasing age, the penetrance is said to be age dependent. Some diseases are non-penetrant up until a certain age and then the penetrance starts to increase drastically, whilst others exhibit low penetrance at an early age and continue to increase with time. For this reason, many diseases have a different estimated penetrance dependent on the age. [ 1 ]


A specific hexanucleotide repeat expansion within the  C9orf72  gene said to be a major cause for developing  amyotrophic lateral sclerosis  (ALS) and  frontotemporal dementia  (FTD) is an example of a genotype with age dependent penetrance. The genotype is said to be non-penetrant until the age of 35, 50% penetrant by the age of 60, and almost completely penetrant by age 80.  [ 1 ] [ 7 ]


For some mutations, the phenotype is more frequently present in one  sex  and in rare cases mutations appear completely non-penetrant in a particular gender. This is called gender-related penetrance or sex-dependent penetrance and may be the result of allelic variation, disorders in which the expression of the disease is limited to organs only found in one sex such as testis or ovaries, or sex steroid-responsive genes. [ 1 ] [ 3 ] [ 9 ]  Breast cancer caused by the BRCA2 mutation is an example of a disease with gender-related penetrance. The penetrance is determined to be much higher in women than men. By age 70, around 86% of females in contrast to 6% of males with the same mutation is estimated to develop breast cancer. [ 9 ]


In cases where clinical symptoms or the phenotype related to a genetic mutation are present only in one sex, the disorder is said to be sex-limited.  Familial male-limited precocious puberty  (FMPP) caused by a mutation in the LHCGR gene, is an example of a genotype only penetrant in males. Meaning that males with this particular genotype exhibit symptoms of the disease whilst the same genotype is nonpenetrant in females. [ 3 ] [ 9 ] [ 10 ]


Genetic modifiers are genetic variants or mutations able to modify a primary disease-causing variant's phenotypic outcome without being disease causing themselves. [ 11 ]  For instance, in single gene disorders there is one gene primarily responsible for development of the disease, but modifier genes inherited separately can affect the phenotype. Meaning that the presence of a mutation located on a  loci  different from the one with the disease-causing mutation, may either hinder manifestation of the phenotype or alter the mutations effects, and thereby influencing the penetrance. [ 1 ] [ 3 ]


Exposure to environmental and lifestyle factors such as  chemicals ,  diet ,  alcohol intake ,  drugs  and  stress  are some of the factors that might influence disease penetrance. [ 1 ] [ 12 ]  For example, several studies of BRCA1 and BRCA2 mutations, associated with an elevated risk of  breast  and  ovarian cancer  in women, have examined associations with environmental and behavioral modifiers such as  pregnancies , history of  breast feeding ,  smoking , diet, and so forth. [ 13 ]


Sometimes, genetic alterations which can cause genetic disease and phenotypic traits, are not from changes related directly to the  DNA  sequence, but from  epigenetic  alterations such as DNA  methylation  or  histone modifications . Epigenetic differences may therefore be one of the factors contributing to reduced penetrance. [ 1 ] [ 6 ] [ 14 ]  A study done on a pair of genetically identical  monozygotic twins , where one twin got diagnosed with  leukemia  and later on  thyroid carcinoma  whilst the other had no registered illnesses, showed that the affected twin had increased methylation levels of the BRCA 1 gene. The research concluded that the family had no known  DNA-repair syndrome  or any other hereditary diseases in the last four generations, and no genetic differences between the studied pair of monozygotic twins were detected in the BRCA1 regulatory region. This indicates that epigenetic changes caused by environmental or behavioral factors had a key role in the cause of promotor hypermethylation of the BRCA1 gene in the affected twin, which caused the cancer. [ 15 ]


It can be challenging to estimate the penetrance of a specific genotype due to all the influencing factors. In addition to the factors mentioned above there are several other considerations that must be taken into account when penetrance is determined:


Penetrance  estimates  can be affected by ascertainment  bias  if the sampling is not systematic. [ 16 ]  Traditionally a phenotype-driven approach focusing on individuals with a given condition and their family members has been used to determine penetrance. However, it may be difficult to transfer these estimates over to the general population because family members may share other genetic and/or environmental factors that could influence manifestation of said disease, leading to ascertainment bias and an overestimation of the penetrance. Large-scale population-based studies, which use both  genetic sequencing  and phenotype data from large groups of people, is a different method for determining penetrance. This method offers less upward bias compared to family-based studies and is more accurate the larger the sample population is. These studies may contain a healthy-participant-bias which can lead to lower penetrance estimates. [ 16 ] [ 17 ] [ 18 ]


A genotype with complete penetrance will always display the clinical phenotypic traits related to its mutation (taking into consideration the expressivity), but the signs or symptoms displayed by a specific affected individual can often be similar to other unrelated phenotypical traits. Taking into consideration the effect that environmental or behavioral modifiers have, and how they can impact the cause of a mutation or epigenetic alteration, we now have the cause as to how different paths lead to the same phenotypic display. When similar phenotypes can be observed but by different causes, it is called  phenocopies .  Phenocopies is when environmental and/or behavioral modifiers causes an illness which mimics the phenotype of a genetic inherited disease. Because of phenocopies, determining the degree of penetrance for a genetic disease requires full knowledge of the individuals attending the studies, and the factors that may or may not have caused their illness. [ 6 ]


For example, new research on  Hypertrophic Cardiomyopathy  ( HCM )  based on a technique called  Cardiac Magnetic Resonance  (CMR), describes how various genetic illnesses that showcase the same phenotypic traits as HCM, are actually phenocopies. Previously these phenocopies were all diagnosed and treated, thought to arrive from the same cause, but because of new diagnostic methods, they can now be separated and treated more efficiently. [ 19 ]


1SK3 ,  1SK4 ,  1TWQ ,  2APH


Peptidoglycan recognition protein 3  ( PGLYRP3 , formerly PGRP-Iα) is an antibacterial and anti-inflammatory  innate immunity   protein  that in humans is encoded by the  PGLYRP3   gene . [ 5 ] [ 6 ] [ 7 ] [ 8 ]


PGLYRP3 (formerly PGRP-Iα), a member of a family of human  Peptidoglycan Recognition Proteins  (PGRPs), was discovered in 2001 by  Roman Dziarski  and coworkers who cloned and identified the genes for three human PGRPs, PGRP-L, PGRP-Iα, and PGRP-Iβ (named for long and intermediate size transcripts), [ 5 ]  and established that human  genome  codes for a family of 4 PGRPs: PGRP-S (short PGRP or PGRP-S) [ 9 ]  and PGRP-L, PGRP-Iα, and PGRP-Iβ. [ 5 ]  Subsequently, the Human Genome Organization Gene Nomenclature Committee changed the gene symbols of PGRP-S, PGRP-L, PGRP-Iα, and PGRP-Iβ to  PGLYRP1  ( peptidoglycan recognition protein 1 ),  PGLYRP2  ( peptidoglycan recognition protein 2 ),  PGLYRP3  (peptidoglycan recognition protein 3), and  PGLYRP4  ( peptidoglycan recognition protein 4 ), respectively, and this nomenclature is currently also used for other  mammalian  PGRPs.


PGLYRP3 has similar expression to PGLYRP4 ( peptidoglycan recognition protein 4 ) but not identical. [ 5 ] [ 6 ]  PGLYRP3 is constitutively expressed in the skin, in the eye, and in the  mucous membranes  in the  tongue ,  throat , and  esophagus , and at a much lower level in the remaining parts of the  intestinal tract . [ 5 ] [ 6 ] [ 10 ] [ 11 ]   Bacteria  and their products increase the expression of PGLYRP3 in  keratinocytes  and oral  epithelial cells . [ 6 ] [ 12 ]  Mouse PGLYRP3 is also differentially expressed in the developing brain and this expression is influenced by the intestinal  microbiome . [ 13 ]  PGLYRP3 is secreted and forms  disulfide -linked dimers. [ 6 ]


PGLYRP3, similar to PGLYRP4, has two  peptidoglycan-binding type 2 amidase domains  (also known as PGRP domains), which are not identical (have 38% amino acid identity in humans) [ 5 ] [ 14 ]  and do not have  amidase   enzymatic  activity. [ 15 ]  PGLYRP3 is secreted, it is  glycosylated , and its glycosylation is required for its bactericidal activity. [ 6 ]  PGLYRP3 forms disulfide-linked homodimers, but when expressed in the same cells with PGLYRP4, it forms PGLYRP3:PGLYRP4 disulfide-linked heterodimers. [ 6 ]


The C-terminal  peptidoglycan -binding domain of human PGLYRP3 has been crystallized and its structure solved [ 16 ]  and is similar to human PGLYRP1. [ 17 ]  PGLYRP3 C-terminal PGRP domain contains a central β-sheet composed of five β-strands and three α-helices and N-terminal segment unique to PGRPs and not found in  bacteriophage  and  prokaryotic  amidases. [ 16 ]


Human PGLYRP3 C-terminal PGRP domain, similar to PGLYRP1, [ 17 ]  has three pairs of  cysteines , which form three disulfide bonds at positions 178–300, 194–238, and 214–220. [ 16 ]  The Cys214–Cys220 disulfide is broadly conserved in  invertebrate  and  vertebrate  PRGPs, the Cys178–Cys300 disulfide is conserved in all mammalian PGRPs, and the Cys194–238 disulfide is unique to mammalian PGLYRP1, PGLYRP3, and PGLYRP4, but not found in the amidase-active PGLYRP2. [ 5 ] [ 15 ] [ 16 ] [ 17 ]  The structures of the entire PGLYRP3 molecule (with two PGRP domains) and of the disulfide-linked dimer are unknown.


PGLYRP3 C-terminal PGRP domain contains peptidoglycan-binding site, which is a long cleft whose walls are formed by α-helix and five β-loops and the floor by a β-sheet. This site binds muramyl-tripeptide (MurNAc-L-Ala-D-isoGln-L-Lys), but can also accommodate larger peptidoglycan fragments, such as disaccharide-pentapeptide. [ 18 ]  Located opposite the peptidoglycan-binding cleft is a large  hydrophobic  groove, formed by residues 177–198 (the PGRP-specific segment). [ 18 ]


The PGLYRP3 protein plays an important role in the innate immune responses.


PGLYRP3 binds peptidoglycan, a polymer of β(1-4)-linked  N -acetylglucosamine  (GlcNAc) and  N -acetylmuramic acid  (MurNAc) cross-linked by short  peptides , the main component of  bacterial   cell wall . [ 5 ] [ 6 ] [ 18 ] [ 19 ] [ 20 ]  The smallest peptidoglycan fragment that binds to human PGLYRP3 is MurNAc-tripeptide (MurNAc-L-Ala-D-isoGln-L-Lys), which binds with low affinity (Kd = 4.5 x 10 −4  M), whereas a larger fragment, MurNAc-pentapeptide (MurNAc-L-Ala-γ-D-Gln-L-Lys-D-Ala-D-Ala), binds with higher affinity (Kd = 6 x 10 -6   M). [ 19 ] [ 20 ] [ 21 ]  Human PGLYRP3, in contrast to PGLYRP1, does not bind  meso-diaminopimelic acid  ( m -DAP) containing fragment (MurNAc-L-Ala-γ-D-Gln-DAP-D-Ala-D-Ala). [ 19 ] [ 20 ] [ 21 ]   m -DAP is present in the third position of peptidoglycan peptide in  Gram-negative  bacteria and  Gram-positive  bacilli, whereas L- lysine  is in this position in peptidoglycan peptide in Gram-positive cocci. Thus, PGLYRP3 C-terminal PGRP domain has a preference for binding peptidoglycan fragments from Gram-positive cocci. Binding of MurNAc-pentapeptide induces structural rearrangements in the binding site that are essential for entry of the ligand and locks the ligand in the binding cleft. [ 22 ]  The fine specificity of the PGLYRP3 N-terminal PGRP domain is not known.


Human PGLYRP3 is directly bactericidal for both Gram-positive ( Bacillus subtilis ,  Bacillus licheniformis ,  Bacillus cereus ,  Lactobacillus acidophilus ,  Listeria monocytogenes ,  Staphylococcus aureus ,  Streptococcus pyogenes ) and Gram-negative ( Escherichia coli ,  Proteus vulgaris ,  Salmonella enterica ,  Shigella sonnei ,  Pseudomonas aeruginosa ) bacteria. [ 6 ] [ 23 ] [ 24 ] [ 25 ]


The mechanism of bacterial killing by PGLYRP3 is based on induction of lethal envelope stress, which eventually leads to the shutdown of  transcription  and  translation . [ 24 ]  PGLYRP3-induced killing involves simultaneous induction of three stress responses in both Gram-positive and Gram-negative bacteria: oxidative stress due to production of  reactive oxygen species  ( hydrogen peroxide  and  hydroxyl radicals ),  thiol  stress due to depletion ( oxidation ) of cellular thiols, and metal stress due to an increase in intracellular free (labile) metal ions. [ 24 ] [ 25 ]  PGLYRP3-induced bacterial killing does not involve cell membrane permeabilization, which is typical for  defensins  and other  antimicrobial peptides , cell wall hydrolysis, or  osmotic shock . [ 6 ] [ 23 ] [ 24 ]  Human PGLYRP3 has synergistic bactericidal activity with antibacterial peptides. [ 23 ]


PGLYRP3 plays a limited role in host defense against  infections .  Intranasal  administration of PGLYRP3 protects mice from lung infection with  S. aureus  and  E. coli , [ 6 ] [ 26 ]  but  PGLYRP3 -deficient mice do not have altered sensitivity to  Streptococcus pneumoniae -induced  pneumonia . [ 27 ]


Mouse PGLYRP3 plays a role in maintaining healthy  microbiome , as  PGLYRP3 -deficient mice have significant changes in the composition of their intestinal microbiome, which affect their sensitivity to  colitis . [ 11 ] [ 28 ] [ 29 ]


Mouse PGLYRP3 plays a role in maintaining anti- and pro-inflammatory homeostasis in the intestine and skin.  PGLYRP3 -deficient mice are more sensitive than wild type mice to dextran sodium sulfate (DSS)-induced colitis, which indicates that PGLYRP3 protects mice from DSS-induced colitis. [ 11 ] [ 29 ]  The anti-inflammatory effect of PGLYRP3 on DSS-induced colitis depends on the PGLYRP3-regulated intestinal microbiome, because this greater sensitivity of  PGLYRP3 -deficient mice to DSS-induced colitis could be transferred to wild type  germ-free  mice or to antibiotic-treated mice by microbiome transplant from  PGLYRP3 -deficient mice [ 11 ] [ 29 ]  or by  PGLYRP3 -regulated bacteria. [ 28 ]  PGLYRP3 is also directly anti-inflammatory in  intestinal   epithelial cells . [ 30 ] [ 31 ] [ 32 ]


PGLYRP3 -deficient mice are more sensitive than wild type mice to experimentally induced  atopic dermatitis . [ 33 ]  These results indicate that mouse PGLYRP3 is anti-inflammatory and protects skin from  inflammation . This anti-inflammatory effect is due to decreased numbers and activity of  T helper 17  (Th17) cells and increased numbers of  T regulatory  (Treg) cells. [ 33 ]


Genetic  PGLYRP3  variants are associated with some diseases. Patients with  inflammatory bowel disease  (IBD), which includes  Crohn's disease  and  ulcerative colitis , have significantly more frequent missense variants in  PGLYRP3  gene (and also in the other three  PGLYRP  genes) than healthy controls. [ 14 ]   PGLYRP3  variants are also associated with  Parkinson's disease [ 34 ]  and  psoriasis . [ 35 ] [ 36 ]  These results suggest that PGLYRP3 protects humans from these diseases, and that mutations in  PGLYRP3  gene are among the genetic factors predisposing to these diseases.  PGLYRP3  variants are also associated with the composition of airway microbiome. [ 37 ]


2EAV ,  2EAX


NM_001165968 NM_207263


NP_001159440 NP_997146


Peptidoglycan recognition protein 4  ( PGLYRP4 , formerly PGRP-Iβ) is an antibacterial and anti-inflammatory  innate immunity   protein  that in humans is encoded by the  PGLYRP4   gene . [ 5 ] [ 6 ] [ 7 ] [ 8 ]


PGLYRP4 (formerly PGRP-Iβ), a member of a family of human  Peptidoglycan Recognition Proteins  (PGRPs), was discovered in 2001 by  Roman Dziarski  and coworkers who cloned and identified the genes for three human PGRPs, PGRP-L, PGRP-Iα, and PGRP-Iβ (named for long and intermediate size  transcripts ), [ 5 ]  and established that human  genome  codes for a family of 4 PGRPs: PGRP-S (short PGRP or PGRP-S [ 9 ] ) and PGRP-L, PGRP-Iα, and PGRP-Iβ. [ 5 ]  Subsequently, the Human Genome Organization Gene Nomenclature Committee changed the gene symbols of PGRP-S, PGRP-L, PGRP-Iα, and PGRP-Iβ to  PGLYRP1  ( peptidoglycan recognition protein 1 ),  PGLYRP2  ( peptidoglycan recognition protein 2 ),  PGLYRP3  ( peptidoglycan recognition protein 3 ), and  PGLYRP4  (peptidoglycan recognition protein 4), respectively, and this nomenclature is currently also used for other  mammalian  PGRPs.


PGLYRP4 has similar expression to PGLYRP3 ( peptidoglycan recognition protein 3 ) but not identical. [ 5 ] [ 6 ]  PGLYRP4 is constitutively expressed in the  skin , in the  eye , in the  mucous membranes  in the  tongue ,  throat , and  esophagus , in the  salivary glands  and  mucus -secreting cells in the throat, and at a much lower level in the remaining parts of the  intestinal tract . [ 5 ] [ 6 ] [ 10 ] [ 11 ]  Bacteria and their products increase the expression of PGLYRP4 in  keratinocytes  and oral  epithelial  cells. [ 6 ] [ 12 ]  Mouse PGLYRP4 is also differentially expressed in the developing  brain  and this expression is influenced by the intestinal  microbiome . [ 13 ]  PGLYRP4 is secreted and forms  disulfide -linked dimers. [ 6 ]


PGLYRP4, similar to PGLYRP3, has two  peptidoglycan-binding type 2 amidase domains  (also known as PGRP domains), which are not identical (have 34% amino acid identity in humans) [ 5 ] [ 14 ]  and do not have  amidase  enzymatic activity. [ 15 ]  PGLYRP4 is secreted, it is  glycosylated , and its glycosylation is required for its bactericidal activity. [ 6 ]  PGLYRP4 forms disulfide-linked homodimers, but when expressed in the same cells with PGLYRP3, it forms PGLYRP3:PGLYRP4 disulfide-linked heterodimers. [ 6 ]


The C-terminal peptidoglycan-binding domain of human PGLYRP4 has been crystallized and its structure solved (in a free form and in a complex with  peptidoglycan  fragment,  disaccharide -pentapeptide) [ 16 ]  and is similar to human PGLYRP1 [ 17 ]  and PGLYRP3. [ 18 ] [ 19 ]  PGLYRP4 C-terminal PGRP domain contains central β-sheet composed of six β-strands surrounded by three α-helices and three short helices and N-terminal segment unique to PGRPs and not found in  bacteriophage  and  prokaryotic  amidases. [ 16 ]  PGLYRP4 C-terminal PGRP domain contains three disulfide bonds, one broadly conserved in  invertebrate  and  vertebrate  PRGPs, one conserved in all mammalian PGRPs, and one unique to mammalian PGLYRP1, PGLYRP3, and PGLYRP4, but not found in the amidase-active PGLYRP2. [ 5 ] [ 15 ] [ 16 ] [ 17 ] [ 18 ]  The structures of the entire PGLYRP4 molecule (with two PGRP domains) and of the disulfide-linked dimer are unknown.


The PGLYRP4 protein plays an important role in the innate immune responses.


PGLYRP4 binds peptidoglycan, a polymer of β(1-4)-linked  N -acetylglucosamine  (GlcNAc) and  N -acetylmuramic acid  (MurNAc) cross-linked by short  peptides , the main component of  bacterial   cell wall . [ 5 ] [ 6 ] [ 16 ]  PGLYRP4 (its C-terminal PGRP domain) binds peptidoglycan fragment, MurNAc-pentapeptide (MurNAc-L-Ala-γ-D-Gln-L-Lys-D-Ala-D-Ala), with Kd = 1.2 x 10 −5 , but similar to PGLYRP3 (and unlike PGLYRP1) does not bind  meso-diaminopimelic acid  ( m -DAP)-containing fragment (MurNAc-L-Ala-γ-D-Gln-DAP-D-Ala-D-Ala). [ 16 ]   m -DAP is present in the third position of peptidoglycan peptide in  Gram-negative bacteria  and  Gram-positive  bacilli, whereas L- lysine  is in this position in peptidoglycan peptide in Gram-positive cocci. Thus, PGLYRP4 C-terminal PGRP domain has a preference for binding peptidoglycan fragments from Gram-positive cocci. The fine specificity of the PGLYRP4 N-terminal PGRP domain is not known.


Human PGLYRP4 is directly  bactericidal  for both Gram-positive ( Bacillus subtilis ,  Bacillus licheniformis ,  Bacillus cereus ,  Lactobacillus acidophilus ,  Listeria monocytogenes ,  Staphylococcus aureus ) and Gram-negative ( Escherichia coli ,  Proteus vulgaris ,  Salmonella enterica ) bacteria [ 6 ] [ 20 ] [ 21 ] [ 22 ] [ 23 ] [ 24 ]  and is also active against  Chlamydia trachomatis . [ 25 ]


In Gram-positive bacteria, human PGLYRP4 binds to the separation sites of the newly formed daughter cells, created by bacterial peptidoglycan-lytic  endopeptidases , LytE and LytF in  B. subtilis , which separate the daughter cells after cell division. [ 21 ]  These cell-separating endopeptidases likely expose PGLYRP4-binding muramyl peptides, as shown by co-localization of PGLYRP4 and LytE and LytF at the cell-separation sites, and no binding of PGLYRP4 to other regions of the cell wall with highly cross-linked peptidoglycan. [ 21 ]  This localization is necessary for the bacterial killing, because  mutants  that lack LytE and LytF endopeptidases and do not separate after cell division, do not bind PGLYRP4, and are also not readily killed by PGLYRP4. [ 21 ]


The mechanism of bacterial killing by PGLYRP4 is based on induction of lethal envelope stress, which eventually leads to the shutdown of  transcription  and  translation . [ 21 ]  PGLYRP4-induced killing involves simultaneous induction of three stress responses in both Gram-positive and Gram-negative bacteria: oxidative stress due to production of  reactive oxygen species  ( hydrogen peroxide  and  hydroxyl radicals ),  thiol  stress due to depletion (oxidation) of cellular thiols, and metal stress due to an increase in intracellular free (labile) metal ions. [ 21 ] [ 22 ]  PGLYRP4-induced oxidative and thiol stress involve malfunction of the respiratory  electron transport chain  in bacteria. [ 23 ] [ 24 ] [ 26 ]  PGLYRP4-induced bacterial killing does not involve  cell membrane  permeabilization, which is typical for  defensins  and other  antimicrobial peptides , cell wall hydrolysis, or  osmotic shock . [ 6 ] [ 20 ] [ 21 ]  Human PGLYRP4 has synergistic bactericidal activity with antibacterial peptides. [ 20 ]


PGLYRP4 plays a limited role in host defense against  infections .  Intranasal administration  of PGLYRP4 protects mice from  lung  infection with  S. aureus  and  E. coli [ 6 ] [ 27 ]  and  PGLYRP4 -deficient mice are more sensitive to  Streptococcus pneumoniae -induced  pneumonia . [ 28 ]


Mouse PGLYRP4 plays a role in maintaining healthy microbiome, as  PGLYRP4 -deficient mice have significant changes in the composition of their intestinal microbiome, [ 11 ] [ 28 ] [ 29 ]  which affects their increased sensitivity to lung  inflammation  and severity of  S. pneumoniae -induced pneumonia. [ 28 ]


Mouse PGLYRP4 plays a role in maintaining anti- and pro-inflammatory homeostasis in the intestine, skin, and lungs.  PGLYRP4 -deficient mice are more sensitive than wild type mice to dextran sodium sulfate (DSS)-induced  colitis , which indicates that PGLYRP4 protects mice from DSS-induced colitis. [ 11 ]


PGLYRP4 -deficient mice are also more sensitive than wild type mice to experimentally induced  atopic dermatitis . [ 30 ]  These results indicate that mouse PGLYRP4 is anti-inflammatory and protects skin from inflammation. This anti-inflammatory effect in the skin is due to decreased numbers and activity of  T helper 17  (Th17) cells and increased numbers of  T regulatory  (Treg) cells. [ 30 ]   PGLYRP4 -deficient mice also have increased inflammatory responses in the lungs during  S. pneumoniae -induced pneumonia associated with impaired bacterial clearance [ 28 ]  and more severe pulmonary inflammation following  Bordetella pertussis  infection, [ 31 ]  indicating anti-inflammatory role of PGLYRP4 in the lungs.


Genetic  PGLYRP4  variants are associated with some diseases. Patients with  inflammatory bowel disease  (IBD), which includes  Crohn's disease  and  ulcerative colitis , have significantly more frequent missense variants in  PGLYRP4  gene (and also in the other three  PGLYRP  genes) than healthy controls. [ 14 ]   PGLYRP4  variants are also associated with  Parkinson's disease , [ 32 ] [ 33 ]   psoriasis , [ 34 ] [ 35 ]  and  ovarian cancer . [ 36 ]  These results suggest that PGLYRP4 protects humans from these diseases, and that  mutations  in  PGLYRP4  gene are among the genetic factors predisposing to these diseases.  PGLYRP4  variants are also associated with the composition of airway microbiome. [ 37 ]


A  phene  is an individual genetically determined characteristic or  trait  which can be possessed by an  organism , such as eye colour, height, behavior, tooth shape or any other observable characteristic.


The term 'phene' was evidently coined as an obvious parallel construct to 'gene'.  Phene is to  Phenotype  as  Gene  is to  Genotype , and Similarly Phene is to  Phenome  as Gene is to  Genome . More specifically, a Phene is an abstract concept describing a particular characteristic which can be possessed by an organism. Whereas Phenotype refers to a collection of Phenes possessed by a particular organism, and Phenome refers to the entire set of Phenes that exist within an organism or species.


Genome wide association studies  use "phenes" or "traits" (symptoms) to distinguish groups in the human population. These groups are then employed to identify associations with genetic alleles that are more common in the symptomatic group than in the asymptomatic control group. Allen et al. report that with respect to Schizophrenia "Research in molecular genetics has focused on detecting multiple genes of small effect" [ 1 ]  This indicates the importance of discovering individual traits or "phenes" that are governed by single genes. Schizophrenia or bipolar disorder may be described as a phenotype but how many individual traits or "phenes" contribute to these phenotypes? Very large genome wide association studies have not found many significant gene linkages. On the contrary the results of these studies implicate a large number of gene alleles that have a very small effect (phene). [ 2 ]