B. thuringiensis toxins have been determined and published. In recent years, functional studies on the mode of action and resistance mechanism have been proposed, which notably promoted the developments of biological insecticides and insect-resistant transgenic crops. With the exploration of known pore-forming toxins (PFTs) structures, similarities between PFTs and B. thuringiensis toxins have provided great insights into receptor binding interactions and conformational changes from water-soluble to membrane pore-forming state of B. thuringiensis toxins. This review mainly focuses on the latest discoveries of the toxin working mechanism, with the emphasis on structural related progress. Based on the structural features, B. thuringiensis Cry, Cyt and parasporin toxins could be divided into three categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structures from each group are elucidated and discussed in relation to the latest data, respectively.Since the first X-ray structure of Cry3Aa was revealed in 1991, numerous structures of  Bacillus thuringiensis, as a Gram-positive soil bacterium, is a member of the B. cereus group that includes five other species: B. cereus, B. anthracis, B. mycoides, B. pseudomycoides, and B. weihenstephanensi. It produces parasporal crystal toxins of insecticidal properties during sporulation, which could be distinguished from other species [B. thuringiensis toxins have been demonstrated against many typical insect orders, such as Lepidoptera (butterflies and moths), Diptera (flies and mosquitoes) and Coleoptera (beetles and weevils). Newly emerging toxins have presented their toxicities towards other insect orders of Hymenoptera, Orthoptera, Hemiptera, Isoptera, Mallophaga, Thisanoptera, etc., and some pests, such as nematodes and mites [B. thuringiensis strains have been isolated and classified into at least 71 H serotypes by their flagellar immunological reactions [B. thuringiensis toxins have been distinguished and categorized into 73 cry and 3 cyt families [ species . With sind mites . These snd mites . Today, eactions ,5. Meanwfamilies ,7.B. thuringiensis produces two types of Crystal (Cry) and Cytolytic (Cyt) parasporal toxins: [B. thuringiensis that exhibits toxic effects to a target organism, or any protein that has obvious sequence similarity to a known Cry toxin [in vivo, but exhibits a broad range of cytotoxicity against dipteran and mammalian cells in vitro [B. thuringiensis that exhibits hemolytic activity, or any protein that has obvious sequence similarity to a known Cyt protein [During sporulation phase,  toxins: . Cry tox toxins: ,9. Cry try toxin . Cyt toxin vitro . Cyt tox protein .B. thuringiensis, has been demonstrated with strong cytocidal activity against human cancer cells [B. thuringiensis and related bacterial parasporal proteins that are non-hemolytic but capable of preferentially killing cancer cells” [Parasporin, which was isolated from non-insecticidal inclusion proteins of er cells . Althougr cells” .L. sphaericus [In addition, similar to parasporin, binary-like (Bin-like) and mosquitocidal-like (Mtx-like) toxins are related to Bin and Mtx toxins from haericus . Also, thaericus .B. thuringiensis protoxin would be solubilized in alkaline gut environment and proteolytically cleaved by gut proteases [B. thuringiensis toxins. To overcome the resistance, several strategies have been provided, including modifying receptor binding sites at the corresponding binding region or alternating receptor binding affinities by swapping domains within certain Cry toxins [B. thuringiensis toxin structures has contributed to and will continue to focus on the understanding of working mechanism and the development of long-term application in pest management.When digested by susceptible insect larvae, roteases . After broteases . Over throteases ,18. Howey toxins . ResearcB. thuringiensis toxins and pore-forming toxins (PFTs) share many common structural and functional features. PFTs is a major category of membrane-damaging toxins, which also could penetrate target cell membrane in a water-soluble form and generate pores, eventually leading to cell death [etc., are predicted to form β-barrel transmembrane pores [B. thuringiensis toxins and PFTs, we describe Cry, Cyt and parasporin toxins as three categories based on structural features: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. Structural features of these three categories, similarities to the PFTs and structure-related functional progress are elucidated in detail.ll death ,21. By tne pores . In thisB. thuringiensis Cry toxins have been reported  has revolutionized the understanding of protein structure at atomic level and the elucidation of their functions. By X-ray crystallography, several reported  [23,24,2These elucidated structures of Cry toxins exhibit various specific insecticidal toxicities against insect orders of Dipterta, Lepidoptera, Coleoptera, and nematode, which obviously are sharing similar three-domain architectures A. DomainComplete amino acid sequence alignment of the three-domain Cry toxins reveals that most of the toxins contain five conserved blocks . ConservBy structural alignment, domain I of Cry toxin shares a significant structural similarity with the pore-formation domain of α-PFTs colicin A A. TherefM. sexta and Bt-resistant P. gossypiella [M. sexta larvae [B. thuringiensis resistance in transgenic crops.After proteolytic digestion, interactions between activated Cry toxins and receptors may facilitate the cleavage of helix α1, and then promote oligomerization. However, recent data have identified that without cadherin receptor, two helix α1 deleted Cry1AbMod and Cry1AcMod mutants could form oligomers by themselves and still remained toxic towards cadherin-silenced sypiella . Moreovea larvae . EvidencA. aegypti larvae [As the conserved block 1 covers the central helix α5 and block 2 is corresponding to the C-terminal helix α7, these regions together with their surrounding areas may play a role in its action . Mutatioi larvae . Similari larvae . Based oi larvae . The umbIn the umbrella-like model, helix α7 shows a “semi-inserted” orientation and interacts with other helices, which may act as the binding sensor ,41. SiteDomain II is a β-prism of three antiparallel β-sheets and two short helices, with a hydrophobic core buried inside A. In CryL. dispar and enhanced binding affinity to the membrane, while by deleting the residue Asn372, toxicity and binding affinity were substantially reduced [M. sexta and H. virescens, which provided an example of interaction with the glycosyl-phosphatidylinositol(GPI)-anchored aminopeptidase-N(APN) receptor [1 from M. sexta [B. mori [H. virescens, while loop α8 and loop β6-β7 bound to M. sexta cadherin CR7 and CR11 fragments [1 and the monomer with APN in M. sexta [1 [M. sexta: domain II loop β10-β11 may be first involved with the highly abundant but low affinity APN receptor. In the following, loop β6-β7, loop β10-β11 and loop α8 interact with cadherin receptor Bt-R1, and by this interaction, removal of helix α1 promotes the oligomerization. Later, the oligomeric toxin binds to APN and ALP through other regions of the Cry toxin, such as strand β16-β22 in domain III, but the oligomer remains the binding to receptor Bt-R1. Finally, residues in loop β10-β11 participate in post-APN binding events, such as membrane insertion [Loop residues have been targeted by site-directed mutagenesis and membrane binding assays to investigate the molecular basis of insecticidal ability . Binding reduced . Mutantsreceptor ,49. Latereceptor . It has receptor . BesidesM. sexta . Recent [B. mori . Also, lragments ,53. It iM. sexta . Considesexta [1 ,55. Thernsertion .Domain III consists of two twisted anti-parallel β-sheets forming a face-to-face sandwich, with a “jelly-roll” topology ,26 FiguA. The shH. virescens, mutants in Cry1Ac were able to affect receptors’ binding affinities [M. sexta membrane [S. exigua larvae [H. armigera [The role of domain III has been determined by various mutagenesis or recombination experiments. In finities . Hybrid finities ,58. It wfinities . Moreovefinities . Mutagenmembrane . A simila larvae . Meanwhiarmigera . Domain P. gossypiella and P. xylostella, suggesting that domain III of Cry1C would be required for anti-resistance methods, while domain II of Cry1Ab alone might have limited contribution [In order to counter resistance or cross-resistance in transgenic crops, hybrid exchanges of domain III between Cry1C and Cry1Ab has been proven to reduce the cross-resistance in ribution . As domaB. thuringiensis stain A1190 was determined at 1.76 Å. It was described with limit sequence identity but significant structural similarity to the structure known in three-domain Cry toxins [2+ level rising after a few minutes of treatment [In 2005, structure of parasporin-1 from y toxins . Of prefreatment . It was reatment   from er cells . Sequencer cells . Recent er cells . Meanwhier cells   [E.coli by forming voltage-dependent channels [12 receptor; an N-terminal translocation domain that delivers pore-forming domain inserting into the periplasm; and a C-terminal pore-forming domain that forms a voltage-gated channel in the cytoplasmic membrane, that because of this, means it destroys the membrane potential [Based on the comparison between domain I of Cry toxins and pore-forming domain of α-PFTs, the three-domain Cry toxin was suggested to belong to the group of α-PFTs . The α-Polysins) . Colicinchannels . One of otential ,72,73. Totential . Accordiotential .Structural comparison by Dali server shows a Z-score  as high as 11.8 between domain II of Cry toxin and the structure of ZG16p protein (PDB ID 3APA), using domain II of Cry4Aa (PDB ID 2C9K) as template ,75. ZG16S. degradans (PDB ID 2CDP) [When using domain III of Cry4Aa as template, Dali comparison shows a Z-score of 14.7 with Aga16B-CBM6-2, a member of carbohydrate-binding module family 6 (CBM6) from ID 2CDP) ,80. CarbID 2CDP) . As a brID 2CDP) . StructuOver the past few years, the working mechanism of Cry toxins has continuously been the key topic of research publication and academic discussion. Valuable efforts have focused on gaining a comprehensive understanding of receptor binding and membrane insertion. Based on these findings, two main models of action have been proposed.M. sexta [M. sexta, at least two types of specific receptors are involved in binding activity. APN and ALP are supposed as the primary receptors. The activated Cry1Ab toxin first binds to APN and ALP with highly abundant but low affinity binding sites as to promote localization and concentration of the activated toxins in the midgut membrane before interacting with the second receptor of cadherin [The pore formation model of the three-domain Cry toxins was primarily put forward based on the interaction of Cry1Ab and brush border membrane vesicles (BBMV) of M. sexta . This mocadherin ,83. The cadherin . Later, cadherin ,83,85.In order to investigate the features of pores in the midgut cell membrane, a sequence of biochemical and biophysical experiments have been taken. However, limited by the challenges of obtaining and testing oligomers from water-soluble state to membrane-bound state, recent work is still lacking an unequivocal understanding of when oligomerization happens, the construction of oligomer, and the size of transmembrane pores. In the pore formation model, monomers oligomerize by sequentially binding to the specific receptors before penetrating the membrane. Whereas, the controversial evidence has questioned that Cry toxins insert into the membrane as monomers, then aggregate and assemble into oligomers within the membrane B. As des2+-dependent cellular signal cascade pathway that leads to the cell death [M. sexta [H. virescens larvae [B. thuringiensis toxins.On the other hand, a cadherin involved alternative model has been put forward. The signaling pathway model proposes that cytotoxicity is mediated by the specific binding with cadherin receptors, which activates a Mgll death . Moreovell death ,88. The M. sexta ,89,90. AM. sexta . Besidess larvae . Signal B. thuringiensis has two main toxin families, insect-specific Cry toxins and cytolytic Cyt toxins. Unlike Cry toxins, Cyt toxins show specific insecticidal activity towards dipteran insects (mosquito and black flies) in vivo but a broad range of cytotoxicity against dipteran and mammalian cells in vitro [B. thuringiensis toxins produced by B. thuringiensis subsp. israelensis (Bti) is the main choice for bioinsecticide used worldwide to control mosquitoes and black flies vectors. The toxin-encoding plasmid pBtoxis from Bti has been mapped: it encodes four Cry toxins  and three Cyt toxins  [in vitro ,93,94. A Cyt1Ca) . Due to Up until now, the known Cyt subfamily members include Cyt1 , Cyt2  and Cyt3Aa1. They share a high level of sequence identity . The monV. volvacea. It can cause cardiac arrest via activation of the Ca2+-dependent ATPase in the ventricular microsomal fraction, resulting in the hemolysis of human red blood cells and cytotoxicity against tumor cells and the mitochondria of liver cells [Crystal structures of Cyt toxins demonstrate a significant similarity with volvatoxin 2 (VVA2) (PDB ID 1VCY) , although primary sequence alignment shows very low identity (less than 17%)  75,96,9,100,101.er cells ,103. VVAer cells . Similarer cells ,100. In er cells . The proCyt toxin does not bind to specific receptors located on the midgut epithelial cells as Cry toxin, but directly interacts with saturated membrane lipid such as phophatidylcholine, phosphatidylethanolamine and sphingomyelin . Proteol196EIKVSAVKE204 (on helix α6 and strand β7) and 220NIQSLKFAQ228 (on strand β8) are involved in the binding to Cry11Aa [M. sexta [Besides the membrane binding activity, Cyt toxins could also bind to Cry toxins and enhance their specific binding activities to the membrane. The synergism examples were demonstrated between Cyt and Cry11Aa or Cry4Aa from Bti ,111,112. Cry11Aa . Additio Cry11Aa . This suM. sexta ,114. RecM. sexta ,116. UndB. thuringiensis was discovered, and the non-insecticidal parasporal inclusions were related to an unknown biological activity [B. thuringiensis strain A1190 (former strain 84-HS-1-11) and A1547 (former strain 90-F-45-14) were demonstrated with novel cytocidal toxicities against human cancer cells [Recently, a novel but unique category of activity ,117. In er cells . Up to der cells . This kier cells ,119. CurB. thuringiensis A1190 strain (formerly 84-HS-1-11) [50 of 0.12, 2.2 and 0.32 μg/mL, respectively. Cell membrane measurement of LDH leakage assay has demonstrated that parasporin-1 did not affect membrane permeability. Also, in HeLa cells, limited changes of fluorescence concentration after being treated with paraspron-1 has shown that parasporin-1 may not form pores in the membrane. On the other site, parasporin-1 could promote Ca2+ influx from the extracellular buffer, and the G-protein inhibitor, suramin, can suppress Ca2+ influx and the cytotoxicity of parasporin-1 [The first discovered parasporin-1 was isolated from HS-1-11) ,120. In HS-1-11) . These tsporin-1 ,120. Thesporin-1 ,118. Intsporin-1 . In mammsporin-1 . It playsporin-1 . The intB. thuringiensis A1547 (formerly 94-F-45-14), which belongs to B. thuringiensis serovar dakota (H15) [L. sphaericus [50 among 10–40 ng/mL. Early study proposed that parasporin-2 specifically targeted the receptor-like proteins in the membrane and induced cell damage [Parasporin-2 was first discovered in ta (H15) ,124. Thehaericus ,126. Prohaericus ,119,125.l damage . Experiml damage . Among tl damage ,129,130.B. thuringiensis stain A1462 (formerly 89-T-26-17) at a molecular weight around 88 kDa [orf2a and orf2b genes respectively and shared a high sequence homology of 88%, thus the ORF2a and ORF2b proteins were correspondingly designed as PS3Aa1 and PS3Ab1. It is worth mentioning that, although parasporin-3 has a very low sequence similarity with Cry or Cyt toxins, the five typical conserved block regions of the three-domain Cry toxin exist within this group [PS3Aa1 and PS3Ab1 of parasporin-3 are from d 88 kDa ,131. Whed 88 kDa . Primaryis group . Based ois group ,118.B. thuringiensis A1470 strain (formerly 89-T-34-22), belonging to the serovar shandongiensis [in vitro, such as MOLT-4, CACO-2 (human colon cancer), Sawano, TCS (human uterus cervix cancer), and HL60 cells [Parasporin-4 was isolated from the soil isolate ngiensis ,133. Whe60 cells ,136,137.60 cells ,138. On B. thuringiensis A1100 strain was discovered by Ekino and Shin [B. thuringiensis strain M019, which exhibits 56.4% sequence similarity to the three-domain Cry toxin. It may have extra peptide regions in the third domain. Parasporin-6 shows preferential cytotoxicity towards HepG2 and HeLa cells, with LC50 at 2.3 μg/mL and 7.2 μg/mL, respectively, which are lower than the toxicities of parasporin-1 and parasporin-2 against their corresponding human cancer cells [Parasporin-5 from and Shin . Paraspoer cells ,120,125.A. hydrophila and related Aeromonas species; (2) C. septicum epsilon-toxin and enterotoxin from C. perfringens; (3) Alpha-toxin (α-toxin) from C. septicum; (4) Enterolobin from E. contorisiliquum; (5) mosquitocidal toxins (MTx) from L. sphaericus; (6) anti-cancer parasporin-2 toxin from B. thuringiensis; (7) L. sulphureus lectin (LSL); and (8) Hydralysins from aquatic animals in the phylum Cnidaria [Since the first structure of proaerolysin toxin was solved in 1994, aerolysin-type toxins have been described as newly discovered category and have become the second largest family of PFTs after Cholesterol Dependent Cytolysins (CDCs) ,140. ThiCnidaria ,142. AerCnidaria .B. thuringiensis A1470 and shared 38% sequence identity with parasporin-4 [The crystal structures of parasporin-2 (PDB ID 2ZTB) and the nontoxic 26 kDa protein (PDB ID 2D42) were determined recently, the latter of which was a nontoxic protein from sporin-4 ,143,144.sporin-4 . A signiDomain I was suggested to play an important role in the binding interaction with GPI anchors . The α-hDomain II of parasporin-2 and the nontoxic 26 kDa protein shows highly similarity, which has one β-hairpin and one anti-parallel five-stranded β-sheet ,143. In Domain III of parasporin-2 and the nontoxic 26 kDa protein consists of an anti-parallel three-stranded β-sheet and an anti-parallel two-stranded β-sheet, packed as a β-sandwich, ,143. As Using parasporin-2 as a template, several structures with high similarity are found by DALI server: Z-score of ETX, nontoxic 26 kDa protein, LSL, proaerolysin, and CPE are 10.0, 9.7, 6.5, 6.3 and 5.5, respectively ,146,151.Domain I and domain II of aerolysin, and domain I of other members from aerolysin family present the most structural diversity. Differences within these regions probably result in the variation of receptor recognition and toxicity specificity . In the Some features of the pore-forming domain in aerolysin-type PFTs are shared in common. First of all, the amphipathic β-hairpin (or loop) resides in the center of the pore-forming domain. It is plausible that this long β-hairpin spans the lipid bilayer and forms the transmembrane β-barrel in the membrane . When thIn 1994, when the X-ray structure of proaerolysin was published, a pore-forming model of aerolysin was built . It is tB. thuringiensis Cry, Cyt and parasporin toxins into three different categories: three-domain type α-PFTs, Cyt toxin type β-PFTs and aerolysin type β-PFTs. By inter- and inner-comparison among B. thuringiensis toxins and PFTs or other structural related protein, an apparent outline of structural and functional features within these three groups has been drawn. Although numerous researches have been dedicated to investigating the role of specific structural components, and many clear interactions between membrane and toxins have been described, the mechanism of action still remains controversial, and there is still a long way to go before it can be elucidated unequivocally. Nevertheless, because of these contributions, applications of B. thuringiensis toxin and strategies for overcoming resistance or cross-resistance have developed rapidly.Based on known or predicted structures and structure–function relationships, we describe B. thuringiensis toxins would be described under these three categories or even according to other unidentified groups in the future. Comprehensive understanding of current structural related features may deepen the knowledge of novel toxins and accelerate the development of novel products in pest management and human health care.It is also worth mentioning that some toxins have been suggested with unique toxicities and possible distinct structures. As Cry6A, a nematicidal toxin shows low similarity to the three-domain Cry toxins and is lacking any of the five conserved blocks ,164. CryB. thuringiensis toxins [B. thuringiensis toxins.Moreover, primary sequences and structural similarity have predicted an evolutionary approach within s toxins . On the


Clostridium acetobutylicum is a protein of unknown function. Sequence analysis predicted that part of the protein contained a metallopeptidase-related domain. There are over 200 homologs of similar size in large sequence databases such as UniProt, with pairwise sequence identities in the range of ~40-60%. CA_C2195 was chosen for crystal structure determination for structure-based function annotation of novel protein sequence space.CA_C2195 from The structure confirmed that CA_C2195 contained an N-terminal metallopeptidase-like domain. The structure revealed two extra domains: an α+β domain inserted in the metallopeptidase-like domain and a C-terminal circularly permuted winged-helix-turn-helix domain.Based on our sequence and structural analyses using the crystal structure of CA_C2195 we provide a view into the possible functions of the protein. From contextual information from gene-neighborhood analysis, we propose that rather than being a peptidase, CA_C2195 and its homologs might play a role in biosynthesis of a modified cell-surface carbohydrate in conjunction with several sugar-modification enzymes. These results provide the groundwork for the experimental verification of the function. Clostridium acetobutylicum [UniProtKB:Q97H19_CLOAB] is a novel 434-residue protein of unknown function. Initial sequence analysis suggested that this protein could be a metallopeptidase. A PSI-BLAST   ~2.3 Å between CAll known Peptidase_M28 members bind two Zn ions, which are described as “co-catalytic” as both Zn ions participate in the catalytic activity. In contrast, CA_C2195 has one bound Zn ion. In an earlier study, it was found that HmrA [PDB:3ram] , a PeptiCA_C2195 does not possess conventional Peptidase_M28 active site residues, as both of the essential, invariant, active site residues have been replaced: Ser191 replaces the conserved Asp and Pro225 replaces the conserved Glu. Ser191 is conserved as Ser in 73 of the 82 homologs that were aligned and present as either Ala or Gly in the remaining 9 homologs. Pro225 is conserved as Pro in 81 of the homologs and present as Val in 1 homolog. All enzymes in Peptidase_M28, the closest known peptidase family by structure and sequence, have these residues conserved. There are over 550 non-peptidase M28 homologs in MEROPS, but only a few have been characterized. Those that have been characterized have evolved different functions, for example, the transferrin receptor proteins 1 and 2, and glutaminyl-peptide cyclotransferase. The glutaminyl-peptide cyclotransferase also has all five Zn-binding and both active site Asp and Glu residues conserved , therefoAneurinibacillus sp. strain AM-1 [PDB: 2ek8], suggests that its DUF2172 domain is very likely derived from the PA protein domain family  in CA_C2195 form a separate globular domain inserted into the DUF4910 domain. This insert domain adopts an α+β fold that does not closely match any other known structures. However, careful visual inspection shows  with other wHTH domains, although the sequence identities of these hits are in the 15-19% range . A Jackhmmer [One of the most interesting aspects of CA_C2195 and its homologs is the presence of a unique C-terminal circularly permuted wHTH domain in conjunction with the metallopeptidase domain. A search for other proteins using Fold that are similar to this domain (residues 356–434) results in very significant hits . The wHTH domain in Met-AP2 is inserted within a distinct peptidase domain belonging to the Peptidase_M24 family [Pfam:PF00557], which includes the creatinases and prolidases. In Met-AP2, the inserted wHTH domain has been shown to be important for the recognition and specificity of the substrate, namely, the amino-termini of proteins processed by the enzyme  . However, the presences of a C-terminal wHTH domain in CA_C2195, shows similarity to domain architectures found in Peptidase_M24 [PDB:1boa]. Analysis of sequence conservation reveals a cluster of non-sequential, highly conserved residues on the surface of the structure of CA_C2195, which are likely to be functionally important, some of which in the wHTH are involved in forming the protein interaction interface in the oligomeric form. It is possible that these proteins do not have any metallopeptidase activity because of the absence of all the catalytic residues that are expected from other characterized members of this peptidase clan. Based on gene neighborhood analysis, we propose that CA_C2195 and its homologs could be involved in the biosynthesis of modified carbohydrates. Given the importance of cell surface polysaccharides in inter-organismal interactions, further characterization of the biochemical activity of this protein is likely to be of interest in the case of pathogens that encode a CA_C2195 like gene, such as http://www.cgl.ucsf.edu/chimera) and all others were prepared using PyMOL [CA_C2195 gene or its homolog as anchors. This script uses either the PTT file (downloadable from the NCBI ftp site) or the Genbank file in the case of whole genome shot gun sequences to extract 20 gene neighbors on the 3’ and 5’ sides of a given query gene. The protein sequences of all neighbors were clustered using the BLASTCLUST program (ftp://ftp.ncbi.nih.gov/blast/documents/blastclust.html) to identify related sequences in gene neighborhoods. Each cluster of homologous proteins were then assigned an annotation based on the domain architecture or conserved shared domain which were detected using Pfam models and in-house profiles run using RPS-BLAST [-9. For all bioinformatics analyses that were performed using homologs within a family for comparison, the chosen sequences were well over the inclusion threshold for the family as built.Protein production and crystallization of CA_C2195 was carried out by standard JCSG protocols -38. Datang PyMOL . The topng PyMOL . Gene nePS-BLAST . This alhttp://www.wwpdb.org) with PDB accession code 3k9t (DOI:10.2210/pdb3k9t/pdb).Atomic coordinates and experimental structure factors for CA_C2195 have been deposited in the Protein Data Bank (The authors declare that they have no competing interests.DD performed x-ray structure determination; DD, LA, AGM, NDR, RDF and PC analyzed the sequence-structure-function relationships and prepared the manuscript; and AB and AG provided assistance on the analysis of DUF proteins and critically reviewed the manuscript. All authors read and approved the final manuscript.Conserved gene neighborhoods with representatives of other bacterial lineages. Description of data: Table describing genomic context in support of Table Click here for fileGene neighborhoods from all organisms containing an ortholog of CA_C2195. Description of data: Table describing genomic context in support of Table Click here for file


Chromosome conformation capture experiments have led to the discovery of dense, contiguous, megabase-sized topological domains that are similar across cell types and conserved across species. These domains are strongly correlated with a number of chromatin markers and have since been included in a number of analyses. However, functionally-relevant domains may exist at multiple length scales. We introduce a new and efficient algorithm that is able to capture persistent domains across various resolutions by adjusting a single scale parameter. The ensemble of domains we identify allows us to quantify the degree to which the domain structure is hierarchical as opposed to overlapping, and our analysis reveals a pronounced hierarchical structure in which larger stable domains tend to completely contain smaller domains. The identified novel domains are substantially different from domains reported previously and are highly enriched for insulating factor CTCF binding and histone marks at the boundaries. Additionally, each domain should have a larger interaction frequency within the domain than to its surrounding regions.Given a Dγ that optimizes the following objective:Specifically, we seek to identify a set of non-overlapping domains Dγ chosen from the set of all possible domains, and q is a function that quantifies the quality of a domain  at resolution γ. Here, the parameter γ is inversely related to the average domain size in Dγ: lower γ results in sets of larger domains and higher γ corresponds to sets of smaller domains. Since domains are required to contain consecutive fragments of the chromosome, this problem differs from the problem of clustering the graph of 3C interactions induced by A, since such a clustering may place non-contiguous fragments of the chromosome into a single cluster. In fact, this additional requirement allows for an efficient optimal algorithm.where A and a set of resolutions Γ={γ1,γ2,…}, identify a set of non-overlapping domains Dc that are most persistent across resolutions in Γ:Given Dc is the set of non-overlapping persistent domains across resolutions, and p is the persistence of domain  corresponding to how often it appears across resolutions.where Since each row and corresponding column in a 3C interaction matrix encodes a genomic position on the chromosome, we can write the solution to objective (1) as a dynamic program:1(l) is the optimal solution for objective (1) for the sub-matrix defined by the first l positions on the chromosome (OPT1(0)=0). The choice of k encodes the size of the domain immediately preceding location l. We define negative-scoring domains as non-domains and, as such, only domains with q>0 in the max term in (3) are retained.where OPTq is:Our quality function wherescaled density of the subgraph induced by the interactions Agh between genomic loci k and l. When γ=1, the scaled density is the weighted subgraph density  divided by the scaled length (l−k)γ of the domain. When γ=2, the scaled density is half the internal density of a graph cluster  in C as pi=(bi−ai)/L where L is the length of the chromosome. When computing the mutual information C and C′, we define the joint probability pij to be |∩|/L.A pairwise comparison of domain configurations displays regions of stability across multiple resolutions =H(C)+H(C′)−2I. Chromosome 1, for example, has three visually pronounced groups of resolutions within which domain sets tend to be more similar than across . If no domain is contained fully in any other domain the score h(·)=0. If, for every pair of domains, one of the domains is fully contained in the other, the score attains its maximum value h(·)=1. We empirically observe that randomly generated domains result in h(·)≈0.5.and K=10 optimal and near-optimal solutions for all chromosomes in human fibroblast cell line (IMR90) as well as mouse embryonic cells (mESC). The choice of K=10 is computationally beneficial given that even for such low K, the score for the next optimal solution drops off fast at lower γ, but for γ=0.5 the optimal score only changes by 0.02% (from 16774.7 to 16771.2) after 50000 solutions are considered. Alternatively, a weaker null hypothesis could be constructed that uses randomly shuffled Hi-C matrix. However, this approach does not control for the distribution of domain lengths — a previously established property of topological domains [h(·) for the identified set of domains is significantly larger than h(·) for the randomized domains . The mean value of the identified set of domains is ≈0.95 as opposed to ≈0.70 for 1,000 randomized sets of domains sampled from each resolution. Computing h(·) on the combined set of domains is conservative since it is likely that domains from multiple optimal and near-optimal solutions can overlap but may not be completely contained in one another within a length scale. This suggests that the multiple optimal and near-optimal domains across scales exhibit a hierarchical structure and that the ensemble of domains can be used as the basis of a more detailed analysis of the hierarchical organization of these genomes.To determine whether the set of all identified domains we observe is significantly more hierarchical than expected by chance, we randomly shuffle domains while maintaining the same domain and non-domain length distributions as the sets of domains we find . At each domains . This weIn this paper, we introduce an algorithm to identify topological domains in chromatin using interaction matrices from recent high-throughput chromosome conformation capture experiments. Our algorithm produces domains that display much higher interaction frequencies within the domains than in-between domains Figure  and for We define a consensus set to be a set of domains that persist across multiple resolutions and give an efficient algorithm that finds such a set optimally.Our method uses a score function that encodes the quality of putative domains in an intuitive manner based on their local density of interactions. Variations of the scoring function in (4), for example, by median centering rather than mean centering or by optimizing the homogeneity of interaction frequencies instead of total frequencies, can be explored to test the robustness of the enrichments described here.γmax. For our experiments, the parameter γmax was set based on the maximum domain sizes observed in Dixon et al.’s experiments so that we could easily compare our domains to theirs. This parameter can also be set intrinsically from properties of the Hi-C interaction matrices. For example, we observe similar enrichments in both human and mouse when we set γmax to be the smallest γ∈Γ such that the median domain size is >80kbp (two consecutive Hi-C fragments at a resolution of 40kbp). This is a reasonable assumption since domains consisting of just one or two fragments do not capture higher-order spatial relationships (e.g. triad closure) and interaction frequencies between adjacent fragments are likely large by chance [Our method is particularly appealing in that it requires only a single user-specified parameter y chance .We compared the fraction of the genome covered by domains identified by Dixon et al. vs. the domains obtained from our method at various resolutions. Dixon et al.’s domains cover 85% of the genome while our sets tend to cover less of the genome . The fact that our domain boundaries are more enriched for CTCF sites indicates that our smaller, more dense domains may be more desirable from the perspective of genome function. The dense, functionally-enriched domains discovered by our algorithm provide strong evidence that alternative chromatin domains exist and that a single length scale is insufficient to capture the hierarchical and overlapping domain structure visible in heat maps of 3C interaction matrices.γ since the domain sizes tend to be smaller. For this special case, it would be desirable to develop a method that more concisely characterizes these larger solution spaces, and this is an interesting direction for future work. The quantitative evidence of the hierarchical structure of topological domains also motivates the development of novel methods for domain discovery that directly account for such hierarchy in the models they assume and the functions they optimize.We provided the first quantitative analysis testing the hypothesis that the domain structure across scales is significantly hierarchically organized, suggesting that the domains we identify can be used as the basis for studying the hierarchical organization of genomes and how this structure impacts gene regulation. By incorporating multiple optimal and near optimal solutions into this analysis, we provide evidence that the observed hierarchical structure persists not only across scales but across a variety of plausible high-scoring domain sets. However, multiple optimal solutions are not necessary to quantify the hierarchical structure of the domains since single optimal solutions across scales can already reveal a hierarchical structure. There are many more near-optimal solutions at higher values of γ setting (with γ 0.0-0.9 with a step of 0.05) the computation finishes in 25 minutes 34 seconds.The method for discovering topological domains that we have introduced is practical for existing datasets. Our implementation is able to compute the consensus set of domains for the human fibroblast cell line and extract the consensus set in 24 minutes when run on a personal computer with 2.3GHz Intel Core i5 processor and 8Gb of RAM. Computing optimal and near-optimal solutions adds only a small overhead to overall running time: when computing 20 top optimal and near-optimal solutions per each A preliminary version of this manuscript appeared in the 2013 Workshop on Algorithms for Bioinformatics .http://www.cs.cmu.edu/~ckingsf/software/armatus/.A C++11 implementation of the algorithms and instructions for compilation and use are available at The authors declare that they have no competing interests.DF, RP, and GD contributed equally to the development of methods and manuscript. CK contributed to the manuscript. All authors reviewed the manuscript and approved it.


The zona pellucida (ZP) surrounding the oocyte is an extracellular fibrillar matrix that plays critical roles during fertilization including species-specific gamete recognition and protection from polyspermy. The mouse ZP is composed of three proteins, ZP1, ZP2, and ZP3, all of which have a ZP polymerization domain that directs protein fibril formation and assembly into the three-dimensional ZP matrix. Egg coats surrounding oocytes in nonmammalian vertebrates and in invertebrates are also fibrillar matrices and are composed of ZP domain-containing proteins suggesting the basic structure and function of the ZP/egg coat is highly conserved. However, sequence similarity between ZP domains is low across species and thus the mechanism for the conservation of ZP/egg coat structure and its function is not known. Using approaches classically used to identify amyloid including conformation-dependent antibodies and dyes, X-ray diffraction, and negative stain electron microscopy, our studies suggest the mouse ZP is a functional amyloid. Amyloids are cross-β sheet fibrillar structures that, while typically associated with neurodegenerative and prion diseases in mammals, can also carry out functional roles in normal cells without resulting pathology. An analysis of the ZP domain from mouse ZP3 and ZP3 homologs from five additional taxa using the algorithm AmylPred 2 to identify amyloidogenic sites, revealed in all taxa a remarkable conservation of regions that were predicted to form amyloid. This included a conserved amyloidogenic region that localized to a stretch of hydrophobic amino acids previously shown in mouse ZP3 to be essential for fibril assembly. Similarly, a domain in the yeast protein α-agglutinin/Sag 1p, that possesses ZP domain-like features and which is essential for mating, also had sites that were predicted to be amyloidogenic including a hydrophobic stretch that appeared analogous to the critical site in mouse ZP3. Together, these studies suggest that amyloidogenesis may be a conserved mechanism for ZP structure and function across billions of years of evolution. The zona pellucida (ZP) is an extracellular matrix surrounding the mammalian oocyte that carries out multiple functions during fertilization including protection from polyspermy and cross-species fertilization. The formation and structure of the mouse ZP in particular has been extensively studied. The mouse ZP is formed exclusively by three glycoproteins, ZP1, ZP2, and ZP3 , 5. All X-ray crystallographic studies of the ZP-N subdomain of mouse ZP3 showed it adopted a unique IgG-like fold with eight β-strands forming an antiparallel β-sandwich . SubsequProteins that are homologous to mammalian ZP proteins have been found in the egg coat surrounding oocytes in non-mammalian vertebrates as well as in invertebrates suggesting that the basic structure and function of the egg coat/ZP is conserved . Indeed,Studies in fish and in the silk moth have suggested that the egg coats (chorion) surrounding the oocytes are functional amyloid structures –14. MoreFunctional amyloids are also present within the reproductive tract. We have previously shown that functional amyloids are present in the mouse epididymal lumen and in the sperm acrosomal matrix suggesting roles in sperm maturation and in sperm-ZP interactions, respectively , 22. Her3-week old female C57BL/6 mice were purchased from Charles River Laboratories . Mice were housed under a 12 h light: 12 h dark cycle with free access to food and water. Mice (3 to 5-weeks old) were induced to superovulate by i.p. injection with 8 IU pregnant mare serum gonadotropin  at 5:00pm, followed 48 hours later with i.p. injection of 8 IU human chorionic gonadotropin . Oviducts were harvested 15–16 hours after hCG injection. All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health Guidelines for Care and Use of Experimental Animals. This work was approved by the Texas Tech University Health Sciences Center IACUC under protocol 94041.All steps were performed in embryological watch glasses using a dissecting microscope. One oviduct at a time was placed in sterile PBS  supplemented with 100 IU/ml penicillin and 100 μg/ml streptomycin. Oviducts were punctured with a 30 gauge needle to release cumulus-oocyte complexes. Complexes were then transferred using a pulled glass Pasteur pipette to PBS containing 0.3 mg/ml hyaluronidase  and incubated for 2 min at 37°C to allow cumulus cells to disperse. Cumulus-free oocytes were then transferred with pulled glass pipettes through two rinses of fresh PBS. ZP were isolated from oocytes by repeated pipetting of multiple oocytes through a glass pulled Pasteur pipette with inner diameter ~25 μm . Th. Th33]. We next analyzed the primary sequences of mouse ZP1, ZP2 and ZP3 using the algorithm Amylpred2 to identify potential amyloidogenic sites throughout each protein. Amylpred2 uses the consensus of 11 different methods that have been developed to predict features relating to amyloid fibrils including amyloidogenic sequence pattern, beta- strand contiguity, average packing density, and includes the amyloid prediction programs AGGRESCAN, TANGO, and WALTZ . AmyloidSeveral regions within the ZP polymerization domains of ZP1, ZP2, and ZP3 were predicted to form amyloid . Using tIn ZP1 and ZP2, regions proximal to the ZP polymerization domain also contained numerous sites that were predicted to form amyloid . The N-tTo examine more closely the potential role of the amyloid structure in ZP-N domain function, we used AmylPred 2 to identify amyloidogenic sites in the ZP-N domains from mouse ZP1, ZP2 and ZP3 as well as in abalone VERL ZP-N repeat 10 and the ZP-N like domain in yeast α-agglutinin/Sag 1p. As shown in A species comparison of egg coat/ZP proteins from abalone, fish, frog, quail, rat, and human demonstrated that they all shared a ZP polymerization domain . AlthougOur studies presented herein suggest the mouse ZP is a functional amyloid. Using approaches commonly used to identify amyloids, including conformation-dependent reagents, electron microscopy, and X-ray diffraction, our studies suggest that all three ZP proteins are amyloidogenic and contribute to the formation of the ZP amyloid. These results are consistent with previous reports indicating that all ZP protein can form fibrils and that ZP1-ZP3 and ZP2-ZP3 heterodimers contribute to the formation of the ZP matrix , 43. Ind2-microglobulin and the SH3 domain of c-src, leading to the suggestion that domain swapping may be an integral first step in amyloid assembly [The ZP polymerization domain is a C-terminal region of approximately 260 amino acids that is present in ZP/egg coat proteins as well as many other proteins of diverse function outside of the reproductive tract . Similarassembly –49. ThusDetailed studies of mutant and truncated mouse ZP3 identified a conserved duplicated motif of seven amino acids called the internal hydrophobic patch (IHP), which comprises the A β-strand of the ZP-C domain, and which is vital for ZP3 assembly , 50. TheIn addition to the ZP polymerization domain, our studies revealed that the ZP-N repeat domains also possess multiple stretches of amino acids that are predicted to form amyloid and that several of these sites are conserved across species. These sequences localize to defined β-strand structures some of which have proposed roles in protein-protein interactions. Indeed, studies both in mammals and other species suggest that the ZP-N repeat domains play important roles during gamete recognition –39. Our Saccharomyces cerevisiae mating protein, α-agglutinin, possessed several amyloidogenic regions that were similar to those present in egg coat/ZP proteins also suggesting a role for the amyloid structure in yeast mating. In particular, the ZP domains from all species, including yeast possessed a short amyloidogenic region that was analogous to the IHP described for mouse and human ZP3 [A comparison of the ZP polymerization domains from ZP3 homologs from several species including marine invertebrates and humans showed that many of the amyloidogenic regions we identified in mouse ZP3 were conserved suggesting that the egg coat/ZP from these species are also amyloid which is consistent with their also being described as fibrillar matrices. These results are also consistent with previous reports describing the fish and silk moth egg coat as amyloids –14. Althuman ZP3 . Conservuman ZP3 , 35. ThaKnowledge that many proteins unrelated to fertilization also contain ZP domains and are known to form fibrils and extracellular matrices would suggest that they too form amyloids for functional purposes. Many proteins that possess ZP domains are involved in cell-cell interactions and cell shape suggesting that amyloidogenesis may be a conserved mechanism for these basic cell biological processes , 52.Other evidence supports our conclusion that the mouse ZP is an amyloid. In particular, the mechanisms that regulate ZP assembly are very similar to those that mediate the formation of other functional amyloids including those produced by bacteria. Although poorly understood, proteins that form functional or pathological amyloids are thought to follow similar aggregation pathways progressing from monomeric to soluble oligomeric to mature amyloid fibrils. Because the soluble oligomeric forms of many amyloidogenic proteins are cytotoxic, functional amyloids are thought to form under controlled conditions that prevent or minimize these intermediate forms. Studies of functional amyloids have revealed several distinct mechanisms to control amyloid formation including sequestration of the amyloid within an organelle, the requirement for interactions with other family members which act as nucleators initiating amyloid formation, and activation of amyloidogenesis via protein processing by proprotein convertases , 53. AllThe functional significance of the ZP as an amyloid may be several-fold. First, the inherent stability of amyloid and its resistance to denaturants and proteases would allow the ZP to survive despite being surrounded by proteases and hydrolases associated with sperm that are undergoing, or have undergone, the acrosome reaction. Indeed, following sperm binding, the ZP does not dissolve but rather sperm penetrate the ZP via a small slit that occurs in the matrix . Because


Large scale molecular dynamics simulations starting from the crystal structures of human PDI (hPDI) in the oxidized and reduced states were performed. The results indicate that hPDI adopts more compact conformations in solution than in the crystal structures, which are stabilized primarily by inter-domain interactions, including the salt bridges between domains a and b' observed for the first time. A prominent feature of the compact conformations is that the two catalytic domains a and a' can locate close enough for intra-molecular electron transfer, which was confirmed by the characterization of an intermediate with a disulfide between the two domains. Mutations, which disrupt the inter-domain interactions, lead to decreased reductase activity of hPDI. Our molecular dynamics simulations and biochemical experiments reveal the intrinsic conformational dynamics of hPDI and its biological impact.Protein disulfide isomerase (PDI) composed of four thioredoxin-like domains  These pa'c and a) as a generous gift from Dr. Han Cheng in this lab et alThe expression plasmids of all hPDI mutants , except Reductase activity of hPDI and its mutants was determined by monitoring insulin reduction. The reaction was initiated by addition of hPDI proteins to 0.1 M potassium phosphate buffer (pH 7.5) containing 130 µM insulin, 2.5 mM EDTA and 0.1 mM DTT. The absorbance at 650 nm, that represents the light scattering resulted from the aggregation of reduced insulin chains, was immediately recorded on a SHIMADZU UV-2501PC spectrophotometer (Shimadzu) at 25°C. The activity was calculated by the maximal slope of the curve relative to the lag time All reaction components, except Ero1α, were freshly mixed in 100 mM Tris-HAc buffer containing 50 mM NaCl and 1 mM EDTA (pH 8.0) to a total volume of 0.5 ml in the reaction vessel of the Oxygraph Clark-type oxygen electrode (Hansatech Instruments), and the reaction was initiated by injection of 1 µM Ero1α.4HCO3 with and without 10 mM DTT respectively, for 45 min at 56°C. Then both samples were alkylated with 40 mM iodoacetamide in 25 mM NH4HCO3 for 45 min at room temperature in the dark and then digested overnight with trypsin (40 ng for each band) at 37°C. The reactions were terminated by adding trifluoroacetic acid to a final concentration of 1%, and desalted using C18 Zip-Tip microcolumns (Millipore) according to the protocol provided by the manufacturer. The samples were then loaded into the instrument in a crystalline matrix of α-cyano-4-hydroxycinnamic acid (5 mg/ml). Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric detection was achieved using an AXIMA-CFR Plus mass spectrometer from KRATOS Analytical (Shimadzu).The protein bands were manually excised from SDS-10% PAGE and divided into two aliquots. After dehydration, the gel plugs were incubated in 25 mM NHa and a'c in buffer A were incubated with 10 mM GSSG and 50 nM potassium ferricyanide respectively (oxidation) or with 100 mM DTT (reduction) for 2h at room temperature. The reactions were then exchanged to buffer B  by using a Hitrap Desalting column.The a domain of 40 µM was respectively incubated with 40 µM reduced and oxidized a'c in buffer B at 25°C. Aliquots of 30 µl from each reaction were removed at different times, quenched by 7.5 µl 5× SDS-loading buffer containing 10 mM 4-acetamido-4-male-imidylstilbene-2,2-disulfonic acid, and analyzed via non-reducing SDS-15% PAGE.The oxidized and reduced a and a' was occupied by the adjacent molecule in crystal packing. Since the packing effects are eliminated in the simulations, the observed compact conformations most likely represent the intrinsic stable states of a single hPDI molecule in solution without substrate or partner protein binding. In its resting state, various stable conformations of hPDI may be present in a dynamic equilibrium, including these compact ones observed in our simulations. And these compact forms may have important functional influence.We carried out four different large scale MD simulations, starting with the solved structures as shown in The compact conformations derived from different simulations are not identical, although the simulations carried out on the oxidized hPDI  or reduca, Glu22 to Thr133; b, Thr138 to Gln233; b', Ile238 to Gly349; a', Lys370 to Glu471. The RMSD values of these rigid portions kept at relatively low level (less than 2.5 Å) in all the simulations (a' were observed in the simulations starting from the reduced state (Sim 3 and Sim 4), and detailed analysis on the simulation trajectories indicated that these changes correspond to the conformational changes of the loop Thr428 to Phe440. The conformations of this loop in the crystal structures of oxidized and reduced hPDI are similar, yet the significance of the conformational change observed in MD simulation is not clear.In the following in-depth analysis of the simulations we used the geometrical center of the rigid portion of each domain as the center of the domain, because the N- or C-terminal boundary residues of each individual domain are relatively more flexible. The rigid portion of each domain was defined as: ulations , indicata and b' reduces to 32 Å from ∼45 Å in the oxidized and reduced crystal structures . Domain a is believed not to react with Ero1α directly b', which suggests a novel role of the a domain in regulating the accessibility of substrates or partner proteins to hPDI.Surprisingly, in the reaction with hPDI's oxidase Ero1α determined by oxygen consumption, both R97E and D180R/D181R showed higher reactivity than that of WT hPDI . Moreovea'a was reported to be partially oxidized in the reaction of hPDI with Ero1α a domain, which was dependent on the existence of the intact active site in domain a'a and a' in the Ero1/PDI pathway was hypothesized. According to the crystal structures a and a' was determined to be 40.3 Å in the oxidized state and 27.6 Å in the reduced state. The minimum distance between these two active sites so far reported was 16 Å using crosslinking technique a and a' by using three trapping mutants, C56A, C400A and C56A/C400A. These mutants showed the same mobility on reducing SDS-PAGE, but under non-reducing conditions a thick extra band appeared solely in C56A/C400A  of 3562.6 (43-Lys57 and Asn387-Lys401 peptide (exactly the same mass with the sum of the two peptides minus 2 Da). Under the reducing conditions this signal disappeared but with two new peptide signals instead. The m/z signals at 1853.9 and 1824.9 were 57 Da larger than the mass of Tyr43-Lys57 and Asn387-Lys401 respectively, due to cysteine alkylation. The above mass spectrometry data clearly demonstrates the existence of a disulfide between Cys53 and Cys397 in the C56A/C400A mutant, which provides prima facie evidence that domains a and a' in an hPDI molecule can move close enough to facilitate electron transfer.Although Ero1α preferentially oxidizes the active site in domain 6A/C400A , which wf 3562.6  was detea and a'c. As shown in a'c, domain a alone remained in reduced state for 15 min; in the presence of oxidized a'c, two extra bands corresponding to the oxidized domain a and reduced a'c appeared after 2 min, indicating the oxidation of the reduced a domain by oxidized a'c, which became reduced at the same time. At 15 min, only ∼15% of domain a was oxidized by a'c. In this respect, a small fraction of domain a in full-length PDI was reported to be also oxidized in the presence of Ero1α a'c by oxidized a is much more efficient, as the fraction of oxidized a'c appeared after only 30 seconds, and achieved ∼70% in 5 min. Although domains a and a' share high sequence identity and very similar reduction potentials a' domain, which was reported to be partly unfolded upon the formation of disulfide bond in the active site a by domain a' may keep the active site in domain a partially in the reduced state, which is required for performing isomerase and reductase activity of hPDI. At present, whether domain a' can be intra-molecularly oxidized by domain a and the physiological impacts, if any, still need require further study.Next, we characterized the disulfide exchange reaction between the active sites in the isolated domains b'xa', has been found to be the minimum redox-regulated cassette 326 of b' and Glu431 of a' and the cation-π interaction between Arg300 of b' and Trp396 of a' were observed in the reduced hPDI structure, while both were disrupted in the oxidized structure 326 and Glu431 was consistently observed in the compact conformations obtained from all our simulations .(TIF)Click here for additional data file.Figure S2RMSD of the hPDI molecule during MD simulations compared with the corresponding initial conformation (blue) and with the last snapshot of the simulations (red).(TIF)Click here for additional data file.Figure S3The time course of the closest distance between the residue pairs involved in the salt bridges between domains a and b or b'. The closest distance are the minimal distance between all acceptor and doner atoms of the two residues. The threshold to define the salt bridge is 3 Å (black dashed lines).(TIF)Click here for additional data file.Figure S4The time course of the closest distance between the residue pairs involved in the salt bridges between domains b' and a'. The black dashed lines are the 3 Å threshold to define the salt bridge.(TIF)Click here for additional data file.Figure S5Domains b and b' of hPDI undergo relative movement. The time course of RMSD of domains b (blue) and b' (red) when domain b (A) or b' (B) is structurally aligned with the corresponding crystal structure.(TIF)Click here for additional data file.Movie S1Representative movements of the domains of hPDI in Sim 4. With the b domain structurally aligned to the crystal structure, it is clearly shown that domains a and a' become much closer and the overall conformation becomes more compact as time evolves. Cysteine residues at the active sites are indicated as sticks with the sulfur atoms in yellow.(MPG)Click here for additional data file.


This paper evaluates the effect of a change in the quantity of money on relative prices in the U.S. economy based on quarterly time-series for the period of 1959 to 2013. We also estimate the implication of a change in relative prices on the rate of inflation and macroeconomic variables. The empirical results indicate that the change of money supply not only affects relative prices but also affects the inflation rate and real variables, such as investment, natural rate of unemployment and potential GDP, through the change in relative prices. The relevant finding of our study is that money is not neutral in a non-traditional sense because a change in the money supply disturbs relative prices and, consequently, the allocation of resources in the economy. This finding has serious implications that must be considered in the transmission mechanisms of monetary policy. Economists in general agree with the idea that the observed variations in the nominal price are related to changes in the quantity of money. The history of hyperinflation corroborates this view ,2. In thHowever, there are not enough studies regarding the specific role played by the change in the quantity of money on the structure of relative prices. It can be the case that even when money is neutral in the traditional approach of the quantity theory of money (QTM), changes in the money supply can modify relative prices in the economy.In other words, despite the macroeconomic effect considering the general price level, one cannot ignore the possibility that there exist microeconomic implications derived from a change in the money supply even in long run. In this sense, the traditional approach of QTM failed to establish the basis for microeconomic studies of monetary economy. Although some economists ,5,6,7 haThe equilibrium business cycle theories of Robert E. Lucas and others have opened new theoretical approaches, and among these, Hayek’s theory has reappeared. In his model,  shows thThere are some elucidative discussions on how the credit policy pursued by a central monetary authority can be a source of economy-wide distortions in the intertemporal allocation of resources and hence an important cause of business cycles based on the canonical Austrian Business Cycle Theory (ABCT) ,12,13,14Furthermore, there are several stylized facts concerning the US time structure of production business cycle during the 2002 through 2009, which are consistent with ABCT . The timIn the same vein,  show empIn the context of procyclical leverage and banking catastrophes,  have stuIt is noteworthy that  and 22,,22,23 usLater ,25 use aThe general price level is a statistical average of the prices in an economy. Therefore, it is difficult o assess changes in the structure of relative prices, if it really exists, and its consequence for resource allocation based on this concept.It is reasonable to accept the hypothesis that if the management of monetary policy is smooth it does not produce any relevant changes in relative prices. There are other situations where conducting monetary policy can generate relevant effects on the allocation of resources. In our view, this happened during the U. S. economic crisis of 2008, for example, where the Federal Reserve Bank kept the federal funds rate low for a long time and the government promoted regulatory policies to finance residential mortgages for low and middle-income people. For some economists the conjugation of these two facts resulted in a speculative bubble in the credit market, which can be perceived as responsible for the crisis ,27.Using quarterly data from 1959:2 to 2013:02 we perform an econometric analysis to evaluate the follow issues: i) the direct effect of the change in the money supply on the change in relative prices; ii) the indirect impact of the change of money supply on the inflation rate; iii) the indirect impact of the change of money supply on the variation of real investment; iv) the indirect impact of the change of money supply on the natural rate of unemployment; and v) the indirect impact of the change of money supply on the change in the real potential GDP.Our database allows plenty of distinct relative prices to be evaluated. We opt for the ratio between the producer price index and the consumer price index to represent the relative price variable. As we show in section 3, one can estimate how much a change in the money supply can distort relative prices.One of the postulates of the quantity theory of money is the dichotomy between relative prices and absolute prices, which is guaranteed if changes in relative prices are explained by changes in real variables such as GDP and employment, while absolute price movements are explained by changes in the money supply. This dichotomy means that given the supply of money, the velocity of money and the level of trade in goods, changes induced by a real shock in the relative prices produce compensatory changes in other relative prices, so that the absolute level of prices remain unchanged . HoweverThe econometric results display evidence that a change in the money supply affects the relative prices. This result does not corroborate the assumption that changes in relative prices only occur due to changes in real variables such as unemployment rate, technological shocks, etc. Moreover, a change in relative prices, resulting from changes in the quantity of money, must be considered when analyzing monetary policy transmission mechanisms because they indirectly affect real economic variables such as investment, natural rate of unemployment and potential output. In sum, we find empirical evidence that money is not neutral in a non-traditional sense because a change in the money supply disturbs relative prices and, consequently, the allocation of resources in the economy.MV = PY. This identity states that the price level (P) varies directly with the quantity of money (M) considering that the velocity (V) of circulation of money and the volume of transactions (Y) of final goods and services do not change. In this context,  although this effect disappears around twelve quarterlies ahead. Considering the results from Pi/Pj) on the other variables of interest .We now compare the results derived from the IRFs to GMM’s results. In The quantity theory of money assumes that relative price changes are caused only by real variables, such as changes in aggregate output, in employment or any changes that alter production costs, pressure from unions, monopoly power of firms or scarcity of goods, among others. The empirical results of this study show that changes in relative prices stem not only from changes in real variables but also from changes in the money supply. If monetary policy generates changes in relative prices then we can infer that it also alters the allocation of production factors and alters the goods and services produced, resulting in income redistribution.Regardless of whether money affects relative prices, most economists believe that money is not neutral in the short term. However, if money affects relative prices, policymakers have a major complicating factor to manage monetary policy. They will have to monitor the spread of money and credit throughout all sectors of the economy. They will have to map how different sectors of the economy will be affected differently. The simple fact that there are changes in relative prices will generate an income redistribution within the economy, which may result in an increase or decrease in income concentration. This effect is evident when we notice empirical evidence showing that changes in relative prices resulting from changes in the money supply affect the inflation rate, considering the monetary regime of greater tolerance for inflation in the 1960s and 1970s.Before reviewing the indirect effects of monetary policy via changes in relative prices on inflation rate, change of real investment and long-term variables, we will comment on one of the basic tenets of QTM, namely the dichotomy relative price/absolute prices.This dichotomy is linked to the fact that changes in real variables cause changes in relative prices, while changes in absolute prices are attributed to monetary causes. In other words, given the supply of money, the velocity of money and the level of transactions of goods, changes induced by a real shock in the relative prices produce compensatory measures in other prices, leaving unchanged the level of absolute prices.This strong assumption should startle readers because nothing guarantees that this compensatory effect will occur. The empirical results of this study do not support the postulate of TQM on the dichotomy between relative and absolute prices in a monetary regime of greater tolerance for inflation.The above-mentioned result initially shows a short-term effect of monetary expansion and changes in relative prices on the variation of investment. According to Austrian economists, when the central bank or commercial banks expand money and credit, the new money thus created is initially spent on specific goods and services. The demands for these products go up in relation to the demands of others, which increase their prices relative to other prices. As the new money spreads throughout the economy, demand for other goods increase and hence other prices will increase as well. Wealth and income redistribution is in favor of those who received the new currency at the beginning of the process at the expense of those who only receive it in the later stages. Therefore, there are two types of relative price changes. The first is the redistribution of income from late to first receivers of the new money, which occurs during the inflationary process. The second is the permanent changes in wealth and income that continue to occur even after the money has already spread throughout the economy. This permanent change in the redistribution of income and wealth shows the non-neutrality of money in the long run.Another relevant point concerning the work of the Austrian school is the development of the theory of the business cycle ,32. MiseEmpirical results  still show some indirect effects on the variation of investment through changes in relative prices. Based on model 2C we observe that a greater variation from the real product directly results in greater variation in relative prices and indirectly a higher variation of investment. Still, based on the 2C model we observe that in the 1960s and 1970s further monetary expansion directly resulted in greater variation in relative prices and indirectly in higher change of investment.They argue that excessive expansion of the money supply and consequent credit expansion distorts the system of incentives or signals received by investors due to the change in relative prices. Therefore, investors would not invest in a situation where the market credit does not suffer government interventions to monetize the economy. These resource allocations in investment projects with high return rates artificially explain an initial economic boom followed by a subsequent reduction in the level of economic activity, which can be seen in Tables Below we examine the effects of a change in the money supply through changes in relative prices on real long run variables . The empRegarding the potential output, we see that it responds positively to changes in relative prices. This result shows that increases in relative prices positively affect the growth rate of potential output. We observe that a greater variation of the supply of money in the 1960s and 1970s directly resulted in greater changes in relative prices and indirectly in a higher rate of potential GDP. See Tables This article evaluates the direct impact of the growth of the money supply on the change in relative prices, as well as the indirect impact of changes in relative prices on other variables of interest, considering the period (1960s and 1970s) in which the Federal Reserve Bank was more tolerant of inflation. For theoretical reasons, we use the ratio of the producer price index and consumer price index to represent relative prices. In other words, we investigate the direct impact of monetary policy on relative prices through a quarterly database of the U.S. economy for the period 1959:2 to 2013:2. Moreover, we also investigate the indirect impact of monetary policy via changes in relative prices on the rate of inflation, on the variation of real investment and on long-term variables such as the natural unemployment rate and real potential GDP changes.Based on estimates of equations obtained through GMM results we show that variations in the money supply in the 1960s and 1970s directly affected relative prices, which in turn indirectly affected the inflation rate, variation of real investment, the natural unemployment rate and the variation of real potential output.The empirical results of this study show that changes in relative prices stem not only from changes in real variables but also from changes in the money supply. If monetary policy generates changes in relative prices then we can infer that it also alters the allocation of production factors and alters the goods and services produced, resulting in income redistribution. In this context, the relevant achievement of our study is that money is not neutral in a non-traditional sense because a change in the money supply distorts the relative prices and consequently the allocation of resources in an economy.Furthermore, there exist a serious implication that must be considered in the issue of monetary policy transmission mechanisms, which occurs through changes in relative prices. It would be difficult for policymakers to predict the outcome that interactions of real and monetary shocks have on relative prices and, hence, on the variables of interest, such as inflation rate, variation of real investment, natural unemployment rate and real output growth.


Background. Recognizing the needs of cancer survivors is one of the important aspects in healthcare delivery. This study aimed to determine the prevalence of unmet supportive care needs and its associated factors among the breast cancer survivors of community-based support group in Kuching, Sarawak. Materials and Methods. This was a cross-sectional study using Supportive Care Needs Survey (SCNS-SF34). All the members of community-based breast cancer support groups in Kuching were invited. A total of 101 respondents were face-to-face interviewed after the consent was obtained. Data was entered and analyzed using SPSS version 20. Results. The respondents endorsed health system and information domain with the highest mean score . Top 10 items with “moderate to high” level unmet needs had a prevalence of 14.9% to 34.7% of respondents indicating need. Significantly higher level of unmet needs was associated with survivors who were younger (less than 60 years old), had higher education attainment, were unemployed, had survival duration of up to 5 years, and were undergoing active treatment. Conclusion. Systematic delivery of health information which is targeted, culturally sensitive, and linguistically appropriate for addressing younger age, education level, employment status, length of survivorship, and treatment stage should be considered not only at hospital-based setting but also at the community-based support groups. Breast cancer remains the most common cancer among women worldwide . SimilarA diagnosis of cancer can alter a person's perspective on health and life itself . Female Cancer survivors face a wide range of problems during and after their primary treatment which often persists in a chronic, long-term manner . During With the growing number of breast cancer survivors in the milieu of limited healthcare resources, there is a pressing demand to uncover the unmet needs of these survivors so that health systems may prioritize its service delivery in order to be more effective and efficient. Therefore, this study aims to describe the prevalence of unmet needs among breast cancer survivors in Kuching, Sarawak, and to assess relationship between their unmet needs and various associated factors.This descriptive cross-sectional study was conducted among breast cancer survivors recruited from the community-based nongovernmental organization (NGO) Sarawak Breast Cancer Support Group (SBCSG) in Kuching, Sarawak. Prior permission was obtained from the center in writing and all survivors in the support group were invited to participate in the study. Written informed consent was obtained prior to the interview assisted survey, which was conducted at the center from January 2014 to June 2014. This community-based approach, in which respondents were recruited, ensured that breast cancer survivors, whether they received treatment from public, private, or mixture of health facilities, were captured in this study.n = z2pq/d2, where z = 1.96, p = 0.2, q = 1 − p, and d = 0.1 .A total of 101 female breast cancer survivors participated in this study. The average age of respondents was 57.9 (SD 9.53) years. Majority of them were married (79.2%) and Chinese (74.3%). More than half of them were Christians (53.5%). Most of the respondents stay with 2 to 4 other persons at home (56.4%) and have an average household income of between RM 3001 to RM 5000 (43.6%). Approximately 80% of the respondents had primary and secondary school level education. Majority of the respondents were unemployed (70.3%) which include survivors who were housewives and retired from employment.The mean age at first diagnosis of breast cancer among the respondents in this study was 49.7 (SD 8.45) years with 54.5% of the respondents diagnosed at the age 50 years and older. Mean duration of survivorship was 8.2 (SD 5.72) years. Nearly two-thirds (63.4%) of the respondents had a survival duration of more than 5 years. Approximately 80% of the respondents had early stage (Stages I and II) breast cancer at time of diagnosis. Among the respondents, 72.3% were no longer undergoing any active treatment at time of study and were only on annual outpatient department follow-ups. The other information on the sociodemographic and medical characteristics of the respondents is depicted in  health systems and information domain was found to have the highest mean score , followed by psychological domain  and patient care and support domain . Meanwhile, the sexuality domain has the lowest mean score .Domain mean scores are identified by summing up the responses to each of the items within the domain and dividing the sum by the number of items in the domain. A higher score  would indicate higher level of needs in the domain.  having one member of hospital staff with whom you can talk about all aspects of your condition, treatment, and follow-up (34.7%), being given explanations on those tests about which you would like to get explanations (29.7%), and having access to professional counseling  if you, family, or friends need it (27.7%).The top 10 items that respondents have indicated a “moderate to high” level of need for help are as depicted in  feeling down or depressed (1%), feelings of sadness (1%), changes in sexual feelings (1%), changes in your sexual relationships (1%), and more choice about which hospital you attend (1%).The ten items with lowest “moderate to high” level unmet needs among the respondents are as shown in n = 57, 56.4%) reported significantly higher mean score across physical and daily living domain (p = 0.009), psychological domain (p = 0.002), sexuality domain (p < 0.001), and patient care and support domain (p = 0.038) compared to survivors aged 60 years and older . Ethnic Malays and Sarawak indigenous group recorded higher mean score in the physical and daily living domain (p = 0.017) and sexuality domain (p = 0.028) compared to the Chinese respondents. Survivors who are married reported higher mean score in the sexuality domain  compared to those who are never married/widowed/divorced/permanently separated  (p < 0.001). Respondents with primary education level reported a significantly lower domain mean score compared to those with postsecondary or tertiary education level across the domains of physical and daily living , sexuality , patient care and support , and health system and information . Respondents who were unemployed indicated a higher mean score in both the psychological domain  and the patient care and support domain  compared to those who were still employed.The supportive care needs (SCNs) domain mean scores are compared to the sociodemographic and medical characteristics of the respondents in  physical and daily living domain (p < 0.001), psychological domain (p < 0.001), sexuality domain (p = 0.019), and patient care and support domain (p = 0.008). Treatment status among respondents showed significant difference in mean scores across all 5 domains with survivors undergoing active treatment indicating a higher mean score compared to those who are not undergoing any current active treatment . There were no significant differences in supportive care needs domain mean scores among respondents with various cohabitation statuses, household income level, and age at diagnosis and between respondents who had early stage cancer compared to those with late stage cancer.Meanwhile, when comparing the SCNs domain mean scores to the medical characteristics and disease stages of the respondents in The present study is a cross-sectional analysis of the perceived needs among breast cancer survivors in Kuching, Sarawak, Malaysia. The survivors recruited were generally less than 60 years old, were mainly Chinese, were married, received at least secondary level education, and were unemployed .The age at diagnosis (mean age 49.7) and staging  were closely similar to various studies done in Malaysia . The mea health system and information domain , followed by psychological domain , and patient care and support domain . Meanwhile, sexuality domain was ranked lowest . This finding is consistent with recent research among breast cancer survivors in Singapore [ health system and information domain with the highest mean score. This tendency towards informational needs has been studied and established [The current study  revealedingapore , Hong Koingapore , and Koringapore  which raablished , with reablished . Furtherablished . health system and information domain compared to the other domains such as psychological domain. These findings however were in contrast to multiple Australian-based research outcomes, in which cancer survivors ranked the psychological domain as area with highest level unmet needs [Based on individual item ranking , there iet needs , 19. Sucet needs  and coul psychological domain in this study sample could be influenced by the fact that the study population was from a community-based breast cancer support group, in which their psychological and emotional needs would probably have been addressed. This finding highlights the importance of proper support group among cancer survivors [Furthermore, needs among survivors are dynamic and may change over time. Researchers discovered a shift in perceived needs from informational needs to psychological needs among cancer survivors, citing improvements in information delivery over the years, which have led to the shift . Assuminurvivors , as wellurvivors . sexuality, psychological, physical and daily living, and patient care and support. This observation is congruent with studies by researchers in Singapore, which demonstrated that the level of need is higher among survivors aged below 60 years [ sexuality, psychological, patient care and support, and health system and information domains compared to those who are 70 years or older [p = 0.643) in mean scores within the health system and information domain which may suggest that, across all age group, the informational need is ever present once a person is diagnosed with cancer.Younger respondents aged below 60 years old reported higher level of unmet needs across all 5 domains compared to their older counterparts , with si60 years . Similaror older . Comparaor older . Such dior older . Additio physical and daily living  and sexuality  domains. This variation could be influenced by the fact that almost 75% of the respondents in this study were of ethnic Chinese group, and the number of Malays and Sarawak indigenous groups were inadequate to be representative. In the absence of adequate research information, this finding is inconclusive. There may be an element of cultural background and variation in health beliefs which influence the level of needs in these domains. Further exploration into this area is needed to understand these variations. Comparison with other studies involving ethnic Chinese survivors revealed similar lower scores in sexuality domain [ physical and daily living domain [ sexuality domain scores could be due to underreporting of sexuality needs, as it is regarded as an intimate matter and not for discussion within the Asian Chinese community. Specific cultural norms among the Chinese could also account for lower physical and daily living needs, as it is believed that personal problems or outcry for help reflects badly on the family and may bring shame to the family name [Significant difference in mean domain scores was found between Malays and Sarawak indigenous groups compared to ethnic Chinese survivors in both they domain , 14 and g domain . Lower sily name ; therefo sexuality domain was endorsed by survivors who are married compared to others (p < 0.001). This was consistent with a Korean study [p = 0.03) were significantly more likely to indicate the need for help in this domain. Furthermore, this finding makes intuitive sense, given that married survivors have spouse in whom they need to confront their sexuality needs more frequently, hence giving rise to needs.Higher level of unmet needs in thean study  whereby  psychological, sexuality, patient care and support, and health system and information domains. This finding is in agreement with other studies which concluded that education level was found to be predictor of higher level of unmet needs among breast cancer survivors [Higher education attainment was associated with greater level of unmet needs among the respondents  with meaurvivors , 26. Sur psychological and patient care and support domains. There may be underlying factors which influence this outcome. A survivor may be unemployed due to various reasons. Among them could be complications from treatment of breast cancer with residual deficits, which would hamper their ability to work and thus be reported as “unemployed.” Such disadvantage may increase their needs in the psychological and patient care and support domain.Current study revealed that survivors who were unemployed reported a higher domain mean score across all 5 domains compared to those who were employed  with sig health system and information domain  . This difference can be attributed to the fact that survivors receiving active treatment were still coping with physical side effects of treatment modalities such as surgery and chemotherapy and hence had much greater need in this domain compared to those who were not on any active treatment any more.Being under active treatment is significantly associated with higher mean scores across all 5 domains in this study o 0.019) . This fi . This fp = 0.097–0.866) (p = 0.044) [There was no significant difference in mean scores across all 5 domains in relation to cohabitation status of the survivors (7–0.866) . This fi= 0.044) . This co physical and daily living domain (p = 0.019) [Household income level did not show any significant difference in any of the 5 domains mean scores. This finding differs from a Korean study which concluded that less income predicted higher needs in= 0.019) . It was = 0.019) . Majoritp value range 0.627 to 0.964). This however was different from other studies which reported that tumor size was significant in predicting needs in the psychological and health system and information domains [ health system and information domain, Early stage  and later stage . The present finding may highlight a lack of understanding among survivors about the differences of cancer staging which also is indirectly reflected in the high level of unmet needs in the health system and information domain. On the other hand, there may be some form of underlying passive coping mechanism in play, whereby survivors choose to keep themselves occupied and not to think too much about their condition [Current study revealed that, in relation to cancer stage at time of diagnosis, there was no significant difference in mean scores across all domains , that survivors may be less than expected. This could be attributed to the fact that late stage survivors have shorter life span and might not be in the best health to participate regularly in support group activities. Second, the Chinese-majority ethnic representation in this study, despite not being the majority population in the country, concurs with other local studies and characterizes the higher breast cancer incidence rate among the Chinese in Malaysia. Third, the inherent limitation of being a cross-sectional study means that cause-effect relationships could not be assessed, and changes in unmet needs could not be evaluated over time. Despites these limitations, this study has its strength in the fact that samples were drawn from community-dwelling breast cancer survivors which is more likely to represent their needs in the actual lived-in environment compared to samples obtained purely from hospitals.In conclusion, this study provided valuable insights into the characteristics of breast cancer survivors in Kuching, Sarawak, and their associated unmet supportive care needs with gaps in the informational domain emphasized. Efforts should include the systematic delivery of health information which is targeted, culturally sensitive, and linguistically appropriate, especially to breast cancer survivors who were younger, had higher education attainment, were unemployed, had survivorship of 5 years or less, and were undergoing active treatment. Future research should recruit samples from both community-based support group and survivors receiving treatment. A longitudinal exploration of the supportive care needs relative to survival duration is also recommended.


CDK5 or p35 (CDK5R1) genes induces abnormal neuronal layering in various regions of the mouse brain via impaired neuronal migration, which may be relevant for mental retardation in humans. Accordingly, mutations in the p35 gene were reported in patients with nonsyndromic mental retardation; however, their effect on the biochemical function of p35 has not been examined. Here, we studied the biochemical effect of mutant p35 on its known properties, i.e., stability, CDK5 activation, and cellular localization, using heterologous expression in cultured cells. We also examined the effect of the mutations on axon elongation in cultured primary neurons and migration of newborn neurons in embryonic brains. However, we did not detect any significant differences in the effects of the mutant forms of p35 compared with wild-type p35. Therefore, we conclude that these p35 mutations are unlikely to cause mental retardation.p35 is an activation subunit of the cyclin-dependent kinase 5 (CDK5), which is a Ser/Thr kinase that is expressed predominantly in neurons. Disruption of the  Cyclin-dependent kinases (CDKs) are a family of Ser/Thr kinases that are activated by binding to a regulatory subunit called cyclin and function in cell-cycle progression in proliferating cells . CDK5 isp35 gene have been reported in the coding and 3′-untranslated region (UTR) in patients with nonsyndromic mental retardation  ATP at 35°C for 1 h [COS-7 cells expressing CDK5 and p35 or its mutants were lysed in 20 mM MOPS, pH 6.8, 1 mM EGTA, 0.1 mM EDTA, 0.3 M NaCl, 1 mM MgCl for 1 h . The reaCOS-7 cells expressing CDK5 and p35 or its mutants were cultured on cover slips and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature, which was followed by incubation with an anti-myc (1:1000) antibody in PBS containing 5% normal goat serum and 0.1% Triton X-100 for 1 h and a second incubation with a secondary antibody conjugated to Alexa Fluor 488 (1:500) . Nuclei In utero electroporation was performed as described previously [in utero. Mice were sacrificed at E18 and brains were fixed for 24 h at 4°C in 4% paraformaldehyde. Brains were sliced and observed using a macro zoom microscope MVX-10 . GFP-positive neurons were counted using Image J software.eviously . Plasmid2, as described previously [SDS–PAGE was carried out on a 12.5% polyacrylamide gel and Phos-tag SDS–PAGE was performed on a 7.5% acrylamide gel containing 50 μM Phos-tag and 100 μM MnCleviously ,23. Membp35 is a protein with a short half-life, and its levels are regulated by proteasomal degradation ,24. Firsp35 is cleaved by calpain between Phe98 and Ala99, to produce the stable C-terminal fragment p25 . The A10A theoretically possible effect of the p35 mutations is on the interaction of Cdk5 with the activator p39. However, we consider this as unlikely because the p35 mutations did not affect the expression level of p35. Further, Cdk5 is expressed at much higher levels than its activators in neurons [in vitro. We assessed the CDK5 activity in cells by phosphorylation of p35 at Ser8 [Next, we examined whether mutations alter the binding or activation ability of p35 to CDK5. CDK5 was immunoprecipitated from the lysates of cells expressing CDK5 and p35 wt or its mutants, and the amount of p35 bound to CDK5 was estimated by immunoblotting, whereas kinase activity was assessed by histone H1 phosphorylation . There w at Ser8 . Phospho at Ser8 . We comp at Ser8 . Nonphosp35-CDK5 is a membrane-bound kinase. We studied the effect of these mutations on the subcellular distribution of p35 in Neuro2A and COS-7 cells. We transfected p35  together with knCDK5 in these cells, and observed the subcellular distribution of p35 by immunofluorescent staining. Since the similar results were obtained, the results of Neuro2A cells are shown in It is known that CDK5-p35 regulates axon outgrowth. We addressed functional effects of p35 mutations on axon outgrowth of primary cortical neurons. Overexpression of p35 increased the length of axons , consistin utero electroporation. Neurons were labeled with EGFP, which was co-introduced with p35 or its mutants at E14. Most of EGFP-positive neurons migrated to the outer cortical layer when observed at E18 and no difference was detected between the wt and mutants  in the C-terminal p25 region , box. Ac297DKKRLLL303 sequence matches the classical di-leucine motif of (D/E)xxxL(L/I), which determines the endosomal or lysosomal trafficking of transmembrane proteins [2+ ATPase 4b (PMCA4b) [Leu302 is located in the C-terminal end of p35, which is dispensable for CDK5 binding and activation , 33. Theproteins . Althoug(PMCA4b) , 25, 36.(PMCA4b) . Even ifImpairment of neuronal migration during brain development may be a cause of mental retardation . CDK5 ishttp://www.ncbi.nlm.nih.gov/snp/?term=Cdk5r1+human)  , SNPs. TCDK5R1) gene. However, Venturin et al. (2006) also reported several mutations in the 3’-UTR region. The 3’-UTR includes DNA sequences involved in post-transcriptional regulation of gene expression such as the stability and translational efficiency of mRNA. Several 3’-UTR sequence-binding proteins are recently reported for p35 mRNA [In this study, we focused on the missense mutations found in the coding region of p35 (p35 mRNA . However