Chrnb2−/− mutants and from wildtype mice were obtained at postnatal day 4 (P4), during the normal period of segregation of eye-specific afferents to the LGN. Microarray analysis reveals reduced expression of genes located on the cell membrane or in extracellular space, and of genes active in cell adhesion and calcium signaling. In particular, mRNA for cadherin 1 (Cdh1), a known axon growth regulator, is reduced to nearly undetectable levels in the LGN of P4 mutant mice and Lypd2 mRNA is similarly suppressed. Similar analysis of retinal tissue shows increased expression of crumbs 1 (Crb1) and chemokine (C-C motif) ligand 21 (Ccl21) mRNAs in Chrnb2−/− mutant animals. Mutations in these genes are associated with retinal neuronal degeneration. The retinas of Chrnb2−/− mutants are normal in appearance, but the increased expression of these genes may also be involved in the abnormal projection patterns of RGC to the LGN. These data may provide the tools to distinguish the interplay between neural activity and molecular expression. Finally, comparison of the transcriptomes of the two different Chrnb2−/− mutant strains reveals the effects of genetic background upon gene expression.Mice lacking expression of the ß2 subunit of the neuronal nicotinic acetylcholine receptor (CHRNB2) display abnormal retinal waves and a dispersed projection of retinal ganglion cell (RGC) axons to their dorsal lateral geniculate nuclei (dLGNs). Transcriptomes of LGN tissue from two independently generated  Chrnb2−/− mutants) have served as a popular model for studying visual system development Chrnb2−/− mutants the projections of the two eyes remain intermingled in the LGN at P8 but form an altered eye specific segregated pattern by P14 Chrnb2−/− mutants compared to wild type (WT) animals Mutant mice provide an invaluable tool for studying the development and organization of the mammalian visual system Chrn2b−/− mutant mice were intitally reported to lack retinal waves Chrnb2−/− mutants do manifest retinal waves, though the waves are not normal in their spatial or temporal characteristics Coordinated firing of action potentials that sweep across the retina in a wavelike manner  occur in the WT retina from the late embryonic stage to eye opening in the mouse Chrnb2−/− mutant animals are thought to reflect the abnormal retinal activity that occurs during the developmental period when key features of the visual system are being established. Whether or not the structural and functional abnormalities that have been documented in the Chrnb2−/− mutants are driven by abnormal patterns of retinal activity during development, the aberrations displayed by the visual system of the Chrnb2−/− mutants presumably also reflect the abnormal expression of molecules that play a role in forming the patterns of connections in the developing visual system. As a first step in probing this matter, in the present study we have used microarray technology to compare the expression of molecules in the retina and the LGN of the Chrnb2−/−mutants with those of WT animals during the period of eye specific segregation in the LGN.The aberrations in the structural and functional organization of the Chrnb2−/− mutant mice were a kind gift from Dr. M Picciotto All experiments were performed in accordance with NIH and institutional guidelines regarding animal use and were approved by the campus animal use and care committee of the University of California, Davis. WT (C57BL/6J) mice were obtained from Jackson Laboratory , Picciotto (“Pic”) Chrnb2−/−, Xu Chrnb2−/−, or C57BL/6J WT animals. Retinas from each animal were separated from the sclera and were combined. RNA was isolated with the RNAeasy Mini kit (Qiagen). A total of 500 ng of RNA was amplified with Ambion MessageAmp II-Biotin Enhanced reagents (AM1791) and aRNA yields were 57–122 µg. Twenty micrograms of each aRNA target  were fragmented and hybridized to Affymetrix GeneChip Mouse 430 2.0 expression arrays using the Affymetrix GeneChip Fluidics Station and Affymetrix reagents (Affymetrix). Adult retinal tissue was prepared as above but only two male littermates of each type were sampled.Total retinas from three male P4 littermates from timed pregnancies were harvested and immediately placed on dry ice. Tissue was maintained at −80°C until RNA was prepared. Mice were homozygous Picciotto Total LGN  was isolated from a second set of male P4 littermates under the dissecting microscope. The cortex was removed and the LGNs were visualized and excised using an 18G needle. The LGN was isolated as a discrete entity without any surrounding thalamic tissue attached. Left and right LGN were combined for each animal and frozen on dry ice. RNA was prepared and amplified as described for retinas, but the aRNA yield from 500 ng of LGN RNA was much lower, 10–31 µg. Ten micrograms of each aRNA target  were fragmented and hybridized as described for retinal samples.Both the retinal and LGN tissue contain a mixture of cell types.Chip data were analyzed with dChip software Following euthanasia, eyes from WT and mutant animals were enucleated then fixed in 4% PFA in PBS for 30–45 minutes. After cryoprotection in a 25% sucrose solution each eye was embedded in OCT , sectioned at 10 µm on a Leica  cryostat, and mounted on glass slides. Brains from WT and mutant animals were harvested and the unfixed brain was embedded in OCT, frozen on dry ice, sectioned at 15 µm on a cryostat (Leica), mounted on glass slides and stored at −80°C until used. Before processing for immunohistochemistry, brain section slides were warmed to room temperature, fixed in 4% PFA for 2–4 minutes and washed in PBS.For immunostaining, sections were blocked in 10% normal donkey serum, 2% bovine serum albumin, and 0.3% Triton X in PBS for 2 hours, then incubated in blocking solution overnight at 4°C with primary antibodies used as follows: Anti-CHRNB2 M270 , anti-CHRNB2 , anti-CCL21 , anti-CDH1  and anti-SPP1 , followed by incubation with Alexa Fluor 568 or 594 fluorescent secondaries , or CY3  for 1 hour. Nuclei were visualized with DAPI  and sections were coverslipped using Vectashield . For control slides primary antibodies were omitted. Images were acquired on an Olympus FV500 confocal microscope . Brightness and contrast were adjusted using Adobe Photoshop.Retinal projections were traced by making intraocular injections of cholera toxin-ß (CTB) conjugated to fluorescent dyes (Molecular Probes). Twenty four hours prior to the required time points,  mice were anesthetized by immersion in ice water and intraocular injections of 1 µl of CTB conjugated to Alexa 488 (left eye) and Alexa 594 (right eye) in 0.5% saline were made into the far temporal region of the eye with a micro pipette. The eyes were treated with ophthalmic antibiotic and the animals placed on a warm surface until full movement was regained. After euthanasia, the brains were removed and immersion fixed with 4% paraformaldehyde for 48 hours. The brains were then sectioned at 50 µm on a vibratome (Leica), mounted on glass slides, cover slipped, then imaged on a Nikon Eclipse E600 upright microscope equipped with an ORCA-ER C4742-90 CCD camera (Hamamatsu Photonics) using a 10× objective. Images were pseudo colored using Wasabi software (Hamamatsu).Qualitative RT-PCR was performed to validate the microarray assay for selected genes of interest. Qiagen OneStep RT-PCR reagents were used with the protocol recommended by the manufacturer. Ten to 20 ng of each template RNA was used per 25 µl reaction volume. Negative controls were run without added template. At 20–35 cycles aliquots of each reaction were withdrawn and run on agarose gels. Gel images have been adjusted for brightness and contrast with Adobe Photoshop. Actin control RT-PCRs are shown in Chrnb2 expression vs. those resulting from inter-strain differences. The importance of the contribution of the mouse background strain to the transcriptional profile has been noted by others Erdr1 and Traf4) differ between the two mutants in the P4 retina  reveals the genes truly affected by lack of 4 retina , while h4 retina . HoweverXlr3a gene, which is overexpressed in P4 mutant LGN (see below), is located on the X chromosome. The two Chrnb2−/−mutant strains potentially express combinations of X alleles from the 129/SvEv, HM1, DBA/2 and/or C57BL/6J lines Pisd-ps3 pseudogene family is reduced in mutants for both tissues and ages examined. However, two probe sets (1453144_at and 1453145_at) are also detected significantly less in Picciotto than in Xu Chrnb2−/−mutant mice, indicating a potential background strain effect for at least some of these loci.Expression of at least two genes which show significantly different expression between mutant and WT animals may also be affected by background strain. The Chrnb2−/−mutants are created by two distinct procedures. In the Picciotto mutant a small portion of the gene around the start codon and the signal peptide has been replaced with a lacZ-neomycin resistance construct Chrnb2 mRNA was dramatically lower in microarray analysis of tissues from both mutants  and six have known cell adhesion functions . Cdh1  expression is remarkably suppressed:  with significantly altered expression in P4 retina show an increased expression in the Chrnb2−/−mutants  and outer and inner plexiform layers at P4 compared to WT  shows reduced expression in both mutants in P4 LGN and in P4 and adult retinas by the selection criteria chosen , or the reduced expression in P4 LGN is localized. Expression of the immune response indicator CCL21 (chemokine (C-C motif) ligand 21; 6CKINE) is increased in P4 Chrnb2−/−mutant retina, but not in adult mutant retina , we assayed RNA populations in both mutant and WT (C57BL/6J) mouse LGN and retinal tissue at P4 by microarray hybridizations. Adult retinas were also assayed and gene expression compared to age matched WT animals.Recently, this laboratory has reported that two strains of Chrnb2−/− mutants is heterozygous for at least one chromosomal locus Chrnb4−/− mutants with C57BL/6J in a microarray analysis. The Chrnb4−/− mutants used were generated by a process similar to the Xu Chrnb2−/− mutants presented in this work Chrnb4 gene on chromosome 9 While the use of single-gene mutants to elucidate gene function is a powerful tool, many authors have cautioned that the methods by which these knockout animals are prepared may not reveal the true contribution of the knocked out gene to the transcriptional profile compared with “control” animals Chrnb2 is located on Chr3 and examination of our data reveals that four genes we score as having significantly different expression are also located on chromosome 3: S100a10 and Cldn11 in P4 LGN, Cp in P4 retina, and Gstm1 in adult retina. Of these, S100a10 and Gstm1 lie very close to Chrnb2 at Chr3F and differences in their expression may be a result of the mutation process rather than the lack of CHRNB2.Chrnb4 gene, but our tissue, ages, and selection criteria all vary from that report Chrnb4 does lead to changes in expression of calcium ion binding proteins similar to our results for Chrnb2, indicating that this may be a pathway generally sensitive to alterations in nAChR subunit composition. While comparing both independently produced Chrnb2−/− animals to the selected WT strain does not completely correct for allelic effects, our analysis of two independently generated mutants allows a more certain determination of transcripts affected by the lack of CHRNB2. At least two genes with consistently different expression between both mutants and WT . CCL21 is a ligand expressed by injured neurons that activates microglia as part of the inflammatory response. Retinas from mice lacking CHRNB2 show 8-fold overexpression of Ccl21 by microarray analysis. CCL21 is overexpressed in the OS, OPL and IPL of P4 Chrnb2−/− retinas compared with WT. Photic injury to the adult retina has been reported to increase expression of one member of the CCL21a/b/c chemokine family, Ccl21a (aka Scya21a) by more than four-fold in the ONL In P4 retinas, mutation of Chrnb2−/− mice, which have not been exposed to abnormally high light levels, synthesize large amounts of CCL21 in their retinas is not known. Unlike the pyknotic nuclei and other cellular abnormalities reported in photic injured retinas Chrnb2−/− mutant mice. Adult brains of Picciotto Chrnb2−/− mutants have been reported to show increased neuronal atrophy and microgliosis Why the P4 Cp) mRNA has also been reported to be upregulated in response to photic damage Cp mRNA in P4 Chrnb2−/− mutant retina relative to WT  may be eliminated by lack of CHRNB2 in the retina, and that this transient defect in the adherens junction leads to subsequent compensatory overexpression of CRB1.Mutation of CRB1 (crumbs1) is associated with Leber's congenital retinopathy and retinitis pigmentosa in humans 2+ ion flow at the synapse. CRB1 is required to prevent light-induced retinal degradation in Drosophila Chrnb2−/− LGN.CRB1 shares calcium binding properties with CHRNB2 and activation of nicotinic AChRs increases calcium trafficking at the synapse Chrnb2 is associated with a decrease in transcript expression in the P4 LGN as 32 of 36 transcripts with altered expression show a reduction of expression in the mutants , LYPD2  and CDH1, proteins with known axon guidance properties, are significantly lower in P4 mutant LGN compared to the age matched WT. Various effects of SPP1 on axon growth have been reported. Ries et al in vitro assay using purified rat E20 RGCs while in an in vitro growth assay of effects on dorsal root ganglia showed SPP1 in the substrate inhibited outgrowth and caused fasciculation Spp1−/− mutant mice are grossly normal Spp1−/− mutant mice appears equivalent to WT. Reduced SPP1 does not cause the segregation defect.Given the displacement of RGC axonal projections in the Ccl21 and Crb1, in Chrnb2−/− mutant retinas at P4 and decreased expression of Spp1 mRNA in mutant LGNs at P4. Zoli et al Chrnb2−/− mutant mice. Spp1 expression appears to be tissue-dependent in Chrnb2−/− mutant mice, but whether it plays a role in neuronal injury response in these animals is not known.The association of SPP1 expression with neuronal degeneration in the rodent brain Chrnb2−/− LGNs. LYPD2 is an Ly6 superfamily member closely related to LYNX1, a known modulator of α4ß2 nAChRs Expression of Lypd2 is markedly downregulated in Chrnb2−/− mutant LGN. While more detailed analysis is required to address whether genes isolated in the microarray experiments are associated with the reported visual system defects, the finding that CDH1 is specifically down-regulated in the Chrnb2−/− mutant LGN is novel and intriguing. CDH1 and CDH2 (N-cadherin) are prototypes of a large family of adhesion molecules that play important roles in the development of the nervous system Cdh1 transcript was not found in embryonic or postnatal retinas tested until P14 CDH1 expression is strongly downregulated and is not detectable in P4 dh2 mRNA in retina and LGN is high and unaltered in P4 Chrnb2−/− mutant mice. It has been established that cadherins, including CDH1 and CDH2, regulate cell-cell interactions through homophilic binding Chrnb2−/− mutant visual system.Our microarray data show expression of CChrnb2−/− mutants. Matter et al 2+ activation of transcription is abnormal and expression of CDH1 or LYPD2 in LGN neurons is weakened. The accuracy of neuronal connections in the LGN therefore may be compromised by the altered expression of axon guidance and adhesion molecules at the thalamic level.We recognize the limitations of extrapolating from microarray and RT-PCR analysis to protein expression. Nontheless, based on these RNA analyses, it is tempting to hypothesize that deficits of the axon growth regulators CDH1 and LYPD2 are responsible for the diffuse localization of ipsilateral RGC projections in the LGNs of Figure S1Actin controls for RT-PCRs. Actin amplification controls for RT-PCRs shown in (TIF)Click here for additional data file.Table S1Excel spreadsheets of present calls for all the microarrays.(XLS)Click here for additional data file.Table S2Excel spreadsheets of all mutant vs WT comparisons.(XLS)Click here for additional data file.Table S3Excel spreadsheets of all Pic vs Xu comparisons.(XLS)Click here for additional data file.Table S4Gene Abbreviations and Names from ,  and .(XLS)Click here for additional data file.


SNF3 gene in the yeast Saccharomyces cerevisiae encodes a low glucose sensor that regulates expression of an important subset of the hexose transporter (HXT) superfamily. Null mutations of snf3 result in a defect in growth on low glucose concentrations due to the inability to relieve repression of a subset of the HXT genes. The snf3 null mutation phenotype is suppressed by the loss of either one of the downstream co-repressor proteins Rgt1p or Mth1p. The relief of repression allows expression of HXT transporter proteins, the resumption of glucose uptake and therefore of growth in the absence of a functional Snf3 sensor.The RGT1 and MTH1 genes (RGT1/rgt1Δ MTH1/mth1Δ snf3Δ/snf3Δ) but homozygous for the snf3∆ were found to grow on low glucose. Since null alleles in the heterozygous state lead to suppression, MTH1 and RGT1 display the phenomenon of combined haploinsufficiency. This observed haploinsufficiency is consistent with the finding of repressor titration as a mechanism of suppression of snf3. Mutants of the STD1 homolog of MTH1 did not display haploinsufficiency singly or in combination with mutations in RGT1. HXT gene reporter fusion assays indicated that the presence of heterozygosity at the MTH1 and RGT1 alleles leads to increased expression of the HXT2 gene. Deletion of the HXT2 gene in a heterozygous diploid, RGT1/rgt1Δ MTH1/mth1Δ snf3Δ/snf3Δ hxt2Δ/hxt2Δ, prevented the suppression of snf3Δ.Strains heterozygous for both the HXT2 is the gene responsible for restoration of growth under these conditions suggests that the numbers of repressor binding domains found in the regulatory regions of members of the HXT family may have biological relevance and enable differential regulation.These findings support the model of relief of repression as the mechanism of restoration of growth on low glucose concentrations in the absence of functional Snf3p. Further, the observation that  Saccharomyces cerevisiae. Glucose transport occurs via facilitated diffusion mediated by a group of homologous transmembrane hexose transporters, encoded by the HXT genes  prion allows growth on alternate substrates in the presence of glucose due to a specific interaction of the Pma1p and Std1p proteins fostering the creation of a stable Std1p-Rgt1p complex [HXT gene expression under specific physiological conditions.Thus unctions ,24. In can Std1p ,16,17 bu complex  stronglysnf3 function results in the inability to grow on low glucose solid media, it results in a dramatic increase in the time it takes to adapt to low glucose liquid media and a diminished capacity to achieve the same final OD of wild type strains [While the loss of  strains . The sporgt1 and mth1 in the snf3 null strains were examined to determine if these mutations can completely suppress the adaptation defect. The adaptation time of the snf3/snf3 mth1/mth1 (UCD2883), snf3/snf3 rgt1/rgt1 (UCD2884) strains and wild type (YPH501) are equivalent , , snf3/snHXT2 promoter was the most sensitive reporter to changes in regulatory machinery and promoter copy numbers, it was used to compare the strains that demonstrated some amount of single haploinsufficiency as well as the homozygous nulls of mth1 and rgt1 . Null of HXT2 were also constructed in a series of haploid mutations to assess the impact of loss of this transporter on suppression of the snf3 growth defect in a variety of backgrounds. The hxt2 null strains were transformed with the HXT2 gene and used as a control. Loss of the HXT2 gene did not impact growth of the wild type strain on low glucose, YPH500 . The growth of both haploid strains, snf3∆ hxt2∆ mth1∆  (Sector B) and snf3∆ hxt2∆ rgt1∆  (Sector C) was improved by transformation with the HXT2 gene (Panel B). Growth of the heterozygous diploid, snf3∆ / snf3∆ hxt2∆ / hxt2∆ mth1∆ /MTH1 rgt1∆ /RGT1 (Sector F) was greatly reduced by deletion of the HXT2 gene and became similar to the s∆ nf3 mutation (compare sector E to sector F). Thus the expression of combined haploinsufficiency requires the presence of a wild type copy of the HXT2 gene. The fact that growth of strains carrying the snf3 suppressors mth1∆  and rgt1∆  in a haploid state also requires the presence of the HXT2 gene suggests that expression of this transporter is important for growth under these low glucose conditions.The analysis of the impact of loss of repressor gene dosage on HXT gene promoter regions revealed a direct connection between the number of binding sites and the level of repression. Addition of binding sites to a reporter gene construct increased repression 10 to 50 fold [HXT2 promoter was shown to carry only three Rgt1 binding sites while the promoters of the HXT1 and HXT3 genes carried 13 and 15 putative binding sites for Rgt1p-mediated repression respectively [HXT2 repression. When considered in combination with the previous reports that 2 micron plasmids containing only the promoters of genes regulated by this complex can suppress the snf3 growth defect [Analysis of the distribution of Rgt1 binding sites in  50 fold . These aectively . Our finh defect , the cleHXT2 protein may be an important transporter for the transition between substrate concentrations and that the affinity of the transporter may be impacted by other factors within the cell. It is also possible that under very high substrate conditions the Hxt2 transporter plays an important regulatory role and that putative regulatory role rather than level of transporter protein expression is the key factor enabling growth of the heterozygous strain. A rapid induction of HXT2 may therefore confer a distinct growth advantage to the cells during adaptation to differing substrate concentrations. Differences in numbers of repressor binding sites could play a role in allowing expression of some transporters to occur under otherwise limiting conditions.These observations also suggest that repressor binding site number may represent yet another mechanism to differentially regulate the different hexose transporters. The Hxt2 transporter has been shown to exist in forms with differing affinity for substrate  althoughThis information adds unique insight to the model that has formed over the past decade since the roles of the corepressor proteins were first elucidated and this intriguing mechanism of transcriptional regulation began to be understood ,16,17. OSaccharomyces cerevisiae used in this study are listed in Table Saccharomyces Genome Deletion Project was purchased from Open Biosystems and used to make all complete null alleles that contain the KanMX resistance marker that provides resistance to G418. Genomic DNA was purified by using the MasterPure™ Yeast DNA purification kit (Epicentre). Genomic DNA of the respective null strain from the deletion set was used as the template for PCR mediated gene disruption by amplifying approximately 200 base pairs of homology flanking each side of the deleted ORF. For all deletions that contain the HphMX resistance marker specifying resistance to Hygromycin B, the pYC140 plasmid [The strains of  plasmid  was used plasmid . All del plasmid . DiploidYeast strains were grown on Yeast Extract Peptone Media (YP) consisting of 1% yeast extract, 2% Bacto peptone and either 2% glucose, 2% galactose, or 0.05% glucose. Synthetic complete (SC) or synthetic dropout (SD) media were made according to established protocols  with theSTD1 was cloned into the p416TEF vector which contains the URA3 selectable marker [STD1 preceded by the strong constitutive TEF promoter and flanked by the CYC1 terminator sequence.The complete open reading frame of e marker  by usingHXT2, HXT3, and HXT4 were made in the CEN/ARS vector pRS416 which contains the URA3 selectable marker [HXT2, HXT3 and HXT4 were PCR amplified with 5’ SacI and 3’NotI overhangs and cloned into pRS416 to create pKD-HXT2, pKD-HXT3 and pKD-HXT4. The resulting plasmids were then sequenced to ensure the fidelity of the cloning. The E. coli LacZ gene from the one hybrid reporter vector described in [E. coli LacZ gene. All plasmids were transformed into the respective yeast strains as described in [The LacZ reporter fusions to the promoters of e marker . The proribed in  was amplribed in .HXT2 from YPH500 was constructed in the CEN/ARS vector pRS316, which contains the URA3 yeast selectable marker [HXT2 gene, along with 562bp of the upstream region, was PCR amplified using HiFi Taq (Invitrogen #11304-011) with primers containing 5’ XhoI (5’-CTCGAGTTTCCGTGAAATAGATTC-3’) and 3’ XbaI (5’-TCTAGATATTAGTAGCCATTAGCC-3’) overhangs. The resulting PCR product was cloned into the MCS of pRS316. The plasmid, now carrying a functional copy of HXT2 under its native promoter, was transformed into previously hxt2 null strains [A plasmid carrying a copy of e marker . The HXT strains .Strains were streaked out from glycerol stocks to YPD media and a separate single colony was used to inoculate three replicate starter cultures in Synthetic Complete (SC) 2% glucose media. Starter cultures were grown for 48 hours and used to inoculate 200 mL of SC 0.05% glucose plus Antimycin A to an OD580 of 0.02. Cultures were grown in 500 mL non baffled Erlenmeyer flasks at 30°C with shaking on an orbital shaker at 200 RPM and the OD monitored at 580 nm. All strains were examined in triplicate and each figure represents one experiment where all strains shown were grown at the same time. Error bars indicate the standard deviation of the average of the three triplicates.Overnight cultures of strains were grown in YP 2% glucose, washed once with sterile water and then re-suspended in sterile water to an OD580 of 1.0. The strains were then subjected to a 10 fold serial dilution and 10 μL of each was spotted on YP or SC plus 0.05% glucose and Antimycin A. Growth was assessed after 3 days of incubation at 30°C.LacZ data were performed using SAS .Strains with reporter plasmids were grown to mid log phase in SD minus uracil with 2% galactose, harvested by centrifugation and then resuspended in either SD minus uracil with 0.05% and Antimycin A or SD minus uracil with 2% galactose and allowed to grow for 3 hours in the respective media. Approximately 15 OD units were harvested by centrifugation and resuspended in assay buffer and assayed as described in . β-galacThere are no financial or non-financial competing interests for any of the authors.KLD and LFB planned all experiments. KLD did the work with the exception of the data provided in Figure KLD current address: Amyris Biotechnologies, 5885 Hollis Street, Suite 100, Emeryville, CA


Within the gut the autonomous enteric nervous system (ENS) is able to sense mechanical stimuli and to trigger gut reflex behaviour. We previously proposed a novel sensory circuit in the ENS which consists of multifunctional rapidly adapting mechanosensitive enteric neurons (RAMEN) in the guinea pig. The aim of this study was to validate this concept by studying its applicability to other species or gut regions.++/high Mg++. Activity levels of RAMEN increased with the degree of ganglion deformation. Recruitment of more RAMEN with stronger stimuli may suggest low and high threshold RAMEN. The majority of RAMEN were cholinergic but most lacked expression of NF145kD or calretinin.We deformed myenteric ganglia in the mouse small and large intestine and recorded spike discharge using voltage sensitive dye imaging. We also analysed expression of markers hitherto proposed to label mouse sensory myenteric neurons in the ileum (NF145kD) or colon . RAMEN constituted 22% and 15% of myenteric neurons per ganglion in the ileum and colon, respectively. They encoded dynamic rather than sustained deformation. In the colon, 7% of mechanosensitive neurons fired throughout the sustained deformation, a behaviour typical for slowly adapting echanosensitive neurons (SAMEN). RAMEN and SAMEN responded directly to mechanical deformation as their response remained unchanged after synaptic blockade in low CaWe showed for the first time that fundamental properties of mechanosensitive enteric neurons, such as firing pattern, encoding of dynamic deformation, cholinergic phenotype and their proportion, are conserved across species and regions. We conclude that RAMEN are important for mechanotransduction in the ENS. They directly encode dynamic changes in force as their firing frequency is proportional to the degree of deformation of the ganglion they reside in. The additional existence of SAMEN in the colon is likely an adaptation to colonic motor patterns which consist of phasic and tonic contractions. The discovery that the distension induced motor behaviour of the isolated intestine is regulated by the enteric nervous system (ENS) Recent concepts suggested that mechanosensitivity in the ENS is a property of many different classes of neurons Previous work mainly focused on mechanosensitive enteric neurones in the guinea pig. There are only a few studies which addressed the question of whether AH/Dogiel type II neurons behave as mechanosensors in regions outside the ileum or in mice. AH/Dogiel type II neurons were described in the mouse small and large intestine We argue that the functional relevance of RAMEN and the validity of the concept of multifunctional enteric neurons require that their existence is confirmed in other species and gut regions. The aim of this study was therefore to characterise properties of mechanosensitive myenteric neurons in the mouse ileum and colon. Mice were chosen for the obvious advantage to perform in the future studies in knockout models.All animal procedures are approved by the ethical committee of the government of Oberbayern (Germany) (55.2-1-54-2531.8-210-09) and according to the German guidelines for animal protection and animal welfare.2, 5% CO2; pH = 7.40) Krebs solution containing (in mM): 117 NaCl, 4.7 KCl, 1.2 MgCl2 6 H2O, 1.2 NaH2 PO4, 25 NaHCO3, 2.5 CaCl2 2 H2O, 11 glucose. The mucosa and submucosa were gently removed in order to obtain myenteric plexus preparations still embedded between the two muscle layers. The preparations (5×10 mm) were pinned onto a silicone ring that was placed in a recording chamber continuously perfused with 37°C carbogen bubbled Krebs solution with a rate of perfusion of 11 ml/min. To block synaptic transmission 20 min perfusion of the entire tissue with a low Ca++/high Mg++ Krebs solution, containing (in mM) 98 NaCl, 4.7 KCl, 16 MgCl2 6 H2O, 1.2 NaH2 PO4, 25 NaHCO3, 0.25 CaCl2 H2O, 11 glucose, was used.Male mice C57BI/6  16 weeks old were killed by cervical dislocation followed by exsanguination. The ileum or the distal colon was quickly removed and further dissected in Carbogen aerated (95% On-octylamino)-6-naphthyl]vinyl] pyridinium betaine) by local application through a microejection pipette loaded with 20 µM Di-8-ANEPPS dissolved in DMSO and pluronic F-127 containing Krebs solution. The dye staining did not change the electrophysiological properties of the nerve cells 2 or ∼4 µm2 per pixel, respectively. The fluorescent images were acquired and processed by the Neuroplex 8.3 software (RedShirt Imaging). Individual cells can be identified since the dye incorporates into the membrane revealing the outline of individual cell bodies  which was connected to a motorized micromanipulator  to control advancement of the hair. The hair was placed 1 to 2 µm above the ganglion and then advanced to create ganglion deformation with a step size of 10 µm, then left in place for 1 min and finally retracted with a 10 µm step. In preliminary experiments mechanical stimulations were repeated at least three times at 5–10 min intervals to ensure that there was a genuine response rather than cell damage induced spike discharge. In addition, after the experiments, any cell damage could be excluded by immunohistochemical staining that allowed us to verify intact cell morphology.3 (0.1%)/horse serum  for 1 h at room temperature followed by 24 h and 3 h incubation with the primary and secondary antibody, respectively. As primary antibodies we used goat anti-choline acetyltransferase , rabbit anti-Neurofilament 145 kDa , rabbit anti-nitric oxide synthase  and goat anti-calretinin . As secondary antibodies we used donkey anti-rabbit conjugated to Carbocyanin , donkey anti-goat conjugated to Carbocyanin  or donkey anti-rabbit conjugated with biotin  and then streptavidin AMCA . Finally, specimens were washed in PBS, mounted on poly-l-lysine-coated slides and cover slipped with a solution of PBS (pH 7.0)/NaN3 (0.1) containing 65% glycerol. The preparations were examined with an epifluorescence microscope (Olympus), equipped with appropriate filter blocks. Pictures were acquired with a video camera connected to a computer and controlled by Scion image software . Frame integration and contrast enhancement were employed for image processing.In order to study the neurochemical code of the mechanosensitive neurons immunohistochemistry was performed. Tissue specimens were fixed 4 h at room temperature in a solution containing 4% paraformaldehyde and 0.2% picric acid in 0.1 mol/l phosphate buffer and then washed (3×10 min) in phosphate buffer. Whole mount preparations were first incubated in phosphate buffered saline (PBS)/NaNWe counted the number of dye-labeled neurons in the ganglia and analysed the number of mechanosensitive neurons per ganglion and the frequency of action potentials discharge. In addition, spike discharge frequency during the dynamic deformation was separately analysed. These values were then correlated with the degree of ganglion deformation. The duration of spike discharge and the occurrence of the last action potential were calculated. We additionally analysed the instantaneous frequency  and averaged the values for dynamic and sustained deformation as well as for the entire period.For signal and image analysis we used Neuroplex 8.3.2 (RedShirt Imaging), Igor Pro 6.03  and Image J 1.43u  software. For the movie editing the program Adobe Premiere Pro  was used. The statistical analyses and graphics were performed with Sigmaplot 12.0 . All data are presented as mean ± standard deviation or, when not normally distributed, as median values together with the 25% and 75% quartiles given in brackets. Differences in the spike frequency between several control stimulations were performed with a one way repeated measures analysis of variance (ANOVA) test. Action potential frequency fired from the same neuron after 2 subsequent stimuli were tested with a paired t-test. Correlation analyses were performed with Pearson Product Moment Correlation. Comparisons between data from the small and large intestine were made with Mann-Whitney Rank Sum Test. To test the differences in action potential frequency before and after blockade of synaptic transmission we used repeated measures ANOVA on ranks. Differences and correlations were considered significant when P was <0.05.In the ileum mechanical stimulation by intraganglionic volume injection was performed in 51 ganglia from 12 mice. The intraganglionic injection resulted in an initial dynamic deformation of the ganglia which lasted 306 [153/401] ms, followed by a sustained deformation which lasted for 150±40 s; during this time the volume was redistributed in the ganglionic network and the ganglion regained its original shape see .The mean number of neuronal cell bodies per ganglion was 19±6. Ganglion deformation evoked a spike discharge in 22±11% neurons per ganglion (range 5–55%). Of these mechanosensitive neurons 20% (47 neurons) fired one action potential only. All other 188 mechanosensitive neurons fired at least 2 and up to 59 spikes  which fired during the dynamic deformation with at comparable frequencies (5 [5/5] Hz versus 3 [1.7/8.7] Hz) .The degree of deformation expressed as percentage change in the ganglionic area after intraganglionic volume injection was 5.1 [3.8/8.9] % (23 ganglia from 8 mice). Of note, the percentage of mechanosensitive neurons as well as the spike frequency during the dynamic deformation positively correlated (p<0.05) with the degree of ganglion deformation .Once started, the spike discharge lasted on average 239 [100/402] ms. The time of occurrence of the last action potential during burst firing was 389 [243/686] ms after the beginning of the dynamic deformation. To check for possible late onset responses we used longer recording periods in 4 ganglia and found that none of the responding neurons fired action potentials beyond 2 s after the stimulus.The spike discharge pattern was rapidly adapting see  and veryIn order to study the reproducibility of the responses we performed experiments (5 ganglia from 4 mice) with 3 mechanical stimulations 10–15 min apart. These stimulations provoked each time a similar change in ganglionic area which varied between the stimuli less than 0.21%. In these experiments we found a high reproducibility of the responses: the number of mechanosensitive neurons per ganglion  and the overall spike discharge  remained stable.In the distal colon mechanical stimulation was performed in 91 myenteric ganglia from 14 mice. Like for the ganglia in the ileum the intraganglionic volume injection resulted in a two phase’s deformation. An initial dynamic deformation of the ganglia which lasted for 320 [248/433] ms was followed by a sustained deformation which lasted for 175±35 s.The mean number of neuronal cell bodies per ganglion was 22±10. Of these, 14±9% neurons per ganglion responded to the mechanical stimulation (range 2–26%) . About 2Like RAMEN in the ileum, two subsequent stimuli within an interval of 2 s activated the same mechanosensitive neurons in the colon  without significant changes in the firing rate during the dynamic deformation (5 [2.5/10] Hz versus 7.5 [5/12] Hz).The degree of deformation calculated as percentage change in the ganglionic area was 3.4 [1.6/5.0] % (29 ganglia from 6 mice) and significantly smaller than in the ileum. Like in the ileum the percentage of mechanosensitive neurons as well as the firing frequency during the dynamic deformation was positively correlated  with the degree of ganglion deformation . In two Once started, the spike discharge lasted on average 540  ms. The last spike occurred 1,067  ms after the beginning of the dynamic deformation. The majority of mechanosensitive neurons (127 out of 159) showed a rapidly adapting spike discharge typical for RAMEN . The remLike their counterparts in the guinea pig myenteric plexus, RAMEN in the mouse myenteric plexus respond to von Frey hair probing with spike discharge during dynamic deformation. As already described in our study in guinea pig myenteric plexus, intraganglionic volume injection was much superior to von Frey hair stimulation regarding reliability and reproducibility of the response. This was mainly due to the inability to repeatedly position the hair exactly on the same spot. The disadvantage of the deformation evoked by volume injection was the slow recovery. Therefore, onset and rapid offset of the stimulus can only be controlled with von Frey hair probing. We used von Frey hair probing to specifically address the question whether RAMEN respond to loading (onset of deformation by advancing the hair) as well as to unloading (offset of the stimulus by retracting the hair). Such experiments were performed in 20 mechanosensitive neurons (17 ganglia from 3 mice). The spike frequency of RAMEN was identical between loading and unloading (1.4 [0.7/2.8] versus 1.4 [0.5/1.8] Hz) .We performed experiments  where we used in the same ganglia the von Frey hair probing and the intraganglionic volume injection. With the von Frey hair one deforms a relatively small area of the ganglion while distant areas of the ganglion showed no noticeable deformation see also .With the++/high Mg++ did not change in the ileum the number of mechanosensitive neurons responding to ganglion deformation . There was also no change in the overall spike discharge rate (3.5 [1.5/6.2] Hz versus 2.9 [1.5/4.9] Hz) or in action potential frequency during the dynamic deformation (7.7 Hz versus 6.7 Hz) (The blockade of synaptic transmission with low Ca 6.7 Hz) .++/high Mg++ (9.4 and 5.9 Hz).Likewise, in the colon the percentage of mechanosensitive neurons per ganglion remained unchanged . Moreover, the action potential frequency during the dynamic deformation 10.0 Hz versus 8.1 Hz  and the ++/high Mg++.Of note, the percentage of mechanosensitive neurons as well as the firing frequency during the dynamic deformation was still positively correlated (p<0.05) with the degree of ganglion deformation during synaptic blockade by low Ca+ concentrations are in equilibrium during continuous Krebs perfusion. We nevertheless performed experiments with intraganglionic injection of low K+ (1 mM) Krebs solution in order to account for the possible buffering capacity of glia cells. Intraganglionic injection of low K+ solution (12 colonic ganglia in preparations from 4 mice) activated 16±9% neurons per ganglion which fired at 7.5 [3.2/9.8] Hz during the dynamic and at 2.8 [2.0/5.8] Hz during the sustained deformation. Both the percentages of mechanosensitive neurons as well as their firing frequencies were comparable to injection of Krebs solution containing 4.7 mM K+ (see data above).We assumed that the intra- and extraganglionic KBased on previous studies, the putative mechanosensitive myenteric neurons in the mouse (AH/Dogiel type II neurons) can be identified by NF145kDa-IR in the ileum Additional markers where used to identify the cholinergic or nitrergic phenotype of RAMEN. In the ileum (16 ganglia from 2 mice) double staining revealed that 79% of mechanosensitive neurons (62 out of 78 ) were ChAT-IR, 12% (9 out of 78) were NOS-IR and the remaining 9% (7 out of 78) were negative for both markers . ChAT-IRSmall neurons may be easier to activate because of their lower input resistance; but different cell sizes did not affect the identification of RAMEN. There was no difference in cells size between mechanosensitive neurons  and those that did not respond to ganglionic deformation .2 versus 61,232  µm2). This may explain why the degree of deformation was on average significantly higher in the ileal myenteric plexus (p = 0.005). Nevertheless, the duration of the dynamic deformation was similar in both regions. Although the overall spike frequency as well as the firing frequency during the dynamic deformation did not significantly differ between the two regions, colonic RAMEN fired for a longer period than RAMEN in the ileum (p = 0.003).The behaviour of most mechanosensitive neurons in the ileum and distal colon was comparable and characterised by rapidly adapting spike discharge. Therefore, the use of the term RAMEN seems appropriate. The number of neurons per ganglion was comparable in the ileum (19±6) and distal colon (22±10), despite the finding that myenteric ganglia in the ileum were significantly smaller than those in the colon  than in the in the colon (14±9%). This is likely an experimental bias as the degree of ganglion deformation was in general smaller in the colon. Analysis of only those ganglia that were deformed to a similar degree (variation of ≤0.3% in ganglion area) revealed that neither the percentage of responding neurons nor the firing frequency during dynamic deformation was significantly different between the two regions: 19±8% responding neurons in the ileum fired at 5.8 [4.2/9.9] Hz and 14±6% responding neurons fired at 8.8 [6.9/9.6] Hz in the colon.This study identified in the mouse small and large intestine mechanosensitive neurons with characteristics and properties comparable to previously identified RAMEN in the guinea pig ileum By analogy to the guinea pig, mouse RAMEN fired action potentials during the dynamic deformation. The response declined with the decay in deformation (rapid adaptation) and ceased when the ganglion entered the phase of sustained deformation. This behaviour is not due to desensitization because spike discharge occurred in response to two consecutive mechanical stimuli applied within 2 seconds. This pattern of response is likely to reflect the type of muscle activity present in the small intestine. Phasic contractions are in fact dominant in the ileum Several results extended our previous findings in the guinea pig ileum. Firstly, RAMEN fired during the onset of ganglionic deformation by advancing the von Frey hair but also when releasing the force by retracing the hair. This suggested that they are able to encode dynamic deformations independent of whether we apply or release the force. Secondly, the proportion of RAMEN responding to deformation and their firing frequency strongly correlated with the degree of ganglionic deformation. Based on the result that there is still a positive correlation between firing rate of RAMEN and ganglion deformation after synaptic blockade, we conclude that sensitivity to deformation is not influenced by neural tone but rather an intrinsic property of RAMEN. Thirdly, we demonstrated that firing rate of mechanosensitive enteric neurons not only relates to the stimulus strength but, even more important, to the degree of ganglion deformation as a result of varying stimulus strengths. Our results are in line with previous suggestion that guinea pig enteric neurons respond to ganglion deformation that occurs during contraction and relaxation of the gut Markers for putative sensory Dogiel Type II neurons in the mouse ileum NK145KDa  and coloIn summary, our findings support the existence and functional relevance of RAMEN across species and regions. Basic properties of RAMEN, such as firing pattern in response to ganglion deformation, their proportion and cholinergic phenotype are almost identical in mouse and guinea pig. This suggests that RAMEN are fundamental for mechanosensitivity in the ENS.Movie S1Activation of enteric mechanosensitive neurons by ganglionic deformation. Slow motion movie of the ganglionic deformation during intraganglionic volume injection . The outlines of the neurons are visible because the voltage sensitive dye Di-8-ANEPPS incorporates into the outer membrane. The movie shows the deformation during the volume injection and at the same time the colour coded action potential discharge (red flashes). Locations of the 2 neurons that fire action potentials in response to intraganglionic volume injection are indicated by red arrows. Note that with this magnification activity in one neuron is detected by several pixels. The two traces to the left show the responses of the two mechanosensitive neurons. Each action potential corresponds to the colour coded activity (red flashes). In the upper part of the movie onsets of the dynamic and sustained deformations are indicated.(AVI)Click here for additional data file.


Saccharomyces cerevisiae msh4/5 point mutants were identified recently that show two fold reduction in crossing over, compared to wild-type without affecting chromosome segregation. Three distinct classes of msh4/5 point mutations could be sorted based on their meiotic phenotypes. These include msh4/5 mutations that have a) crossover and viability defects similar to msh4/5 null mutants; b) intermediate defects in crossing over and viability and c) defects only in crossing over. The absence of a crystal structure for the Msh4–Msh5 complex has hindered an understanding of the structural aspects of Msh4–Msh5 function as well as molecular explanation for the meiotic defects observed in msh4/5 mutations. To address this problem, we generated a structural model of the S. cerevisiae Msh4–Msh5 complex using homology modeling. Further, structural analysis tailored with evolutionary information is used to predict sites with potentially critical roles in Msh4–Msh5 complex formation, DNA binding and to explain asymmetry within the Msh4–Msh5 complex. We also provide a structural rationale for the meiotic defects observed in the msh4/5 point mutations. The mutations are likely to affect stability of the Msh4/5 proteins and/or interactions with DNA. The Msh4–Msh5 model will facilitate the design and interpretation of new mutational data as well as structural studies of this important complex involved in meiotic chromosome segregation.The Msh4–Msh5 protein complex in eukaryotes is involved in stabilizing Holliday junctions and its progenitors to facilitate crossing over during Meiosis I. These functions of the Msh4–Msh5 complex are essential for proper chromosomal segregation during the first meiotic division. The Msh4/5 proteins are homologous to the bacterial mismatch repair protein MutS and other MutS homologs .  The MutS homodimer in bacteria is involved in the repair of mismatches that occur during DNA replication The MutS homodimer has the shape of an oval disk with two channels of dimensions ∼30×20 and ∼40×20 Å with DNA passing through the larger channel Saccharomyces cerevisiae meiotic crossovers are initiated by the programmed introduction of ∼140–170 DNA double strand breaks (DSBs) by the Spo11 protein in combination with accessory factors S. cerevisiae have provided molecular details into the sequence of events during repair of DSBs into crossover products In S. cerevisiae msh4Δ, msh5Δ mutants have strong defects in meiotic crossing over (2.5 fold decrease), spore viability (30–40%) and disjunction of homologous chromosomes Consistent with the physical studies, genetic and biochemical data support the role of the Msh4–Msh5 complex in meiotic crossover formation. S. cerevisiae and mammals. A smaller subset of crossovers in these organisms is made through the Mus81-Mms4 pathway msh5 mutants showed more severe phenotypes compared to msh4 mutants. msh4/5 mutations were classified into three types based on crossover frequency and spore viability. These include a) mutations with spore viability and crossover frequency similar to that of msh4/5 “null” mutations, b) mutations with intermediate defects in spore viability and crossing over and c) mutations with only crossover defects. Interestingly, mutations in equivalent positions in Msh4 and Msh5 ATPase and DNA binding domains were observed that had asymmetric effects on crossover frequency and spore viability.The Msh4–Msh5, Mlh1–Mlh3 complexes are part of the major crossover pathway in msh4/5 mutations. As no crystal structure is available for the Msh4–Msh5 complex, homology modeling was used to generate a structural model for this complex using the hMSH2–hMSH6 crystal structure as the template. Homology modeling has proved to be useful in a number of cases where crystal structures are not available for a protein msh4/5 mutations result in meiotic defects by two mechanisms: by affecting stability of the Msh4/5 proteins or interaction of the Msh4–Msh5 complex with the DNA. The model has not only been used to explain the structural basis of the meiotic defects observed in the mutations but also to propose further mutations that may be analyzed. These include residues at the putative interface of the Msh4–Msh5 complex, residues that may be involved in DNA binding and double mutations that may serve as compensatory mutations. Such information is useful to predict incompatibilities between segregating polymorphisms in MSH4 and MSH5 genes in populations. More generally the availability of a model for Msh4–Msh5 structure will facilitate the design of new mutational studies of the complex, interpretation of MSH4/5 polymorphism data in populations and a mechanistic understanding of Msh4–Msh5 function in meiotic crossing over.The aim of this study is to provide a structural basis for understanding Msh4–Msh5 function as well as molecular explanation for each of these S. cerevisiae Msh4–Msh5 based on alignment with the hMSH2–hMSH6 complex (PDB code 2o8b) as the template. Alignments between the templates and targets obtained from automatic programs are considered unsatisfactory. This is because, automatic programs tend to introduce breaks in the middle of regular secondary structural elements, or align hydrophobic residues in the target with solvent exposed residues in the template among other reasons. Extensive manual intervention was therefore required to arrive at a high quality alignment suitable for the comparative modeling. Reasons for choosing hMSH2–hMSH6 complex as the template are discussed below.Crystal structures of bacterial MutS, and eukaryotic MutSα and MutSβ complexes are available The choice of using hMSH2–hMSH6 as the template was intentional based on the rigorous analysis of the quality of alignment between Msh4 & hMSH2, Msh4 & hMSH6, Msh5 & hMSH2, Msh5 & hMSH6 and Msh4 & hMSH3 pairs. Poor sequence identity of the order of 20% amongst all pairs meant that templates could not be decided purely based on sequence identity. Therefore all possible alignments were assessed to decide the template. The curated alignment obtained has been provided in It was observed that there existed more cases of in/dels within regular secondary structures in case of hMSH2 as template for Msh4 and hMSH6 as template for Msh5. The assessment of the alignment involved the use of structural environments around the sequence. Also, features such as solvent accessibility and secondary structure of residues were considered. For example, buried apolar residues replaced by buried polar residues and exposed polar residues being replaced by exposed apolar residues were commonly seen when Msh4 was aligned with hMSH2 and Msh5 with hMSH6 as the template. So, if the templates are swapped in modeling Msh4 and Msh5, not only was the quality of alignment poor, the modeled structure had large number of short contacts and collapsed during the energy-minimization steps. Two non-bonded atoms are said to be in short contact if their inter-atomic distance is too short in comparison to the classic contact criteria proposed by Ramachandran and coworkers The Msh4–Msh5 model was used to address asymmetry of the Msh4 and Msh5 subunits within the complex, to map interface residues between Msh4 and Msh5 and to analyze the interaction of the complex with Holliday junction DNA. These are discussed in further detail below.msh4 G639A and msh5 G648A have spore viability similar to msh4/5Δ mutants , Y480A (67%), V488A (40%), and L548A (50%) respectively. Reasons for their asymmetric phenotypes are outlined below. The ATP binding mutations msh4 G639A and msh5 G648A are poorly tolerated in both Msh4 and Msh5 because of structural constraint. The Glycine residues have positive φ values which is not comfortably adopted by non-Glycine residues. This is also indicated by the high conservation of these residues. The ATP hydrolysis residue, Msh5 R685 is involved in main chain hydrogen bonding to stabilize the β sheet as shown in msh5 R685W mutation is therefore poorly tolerated compared to the equivalent mutation msh4 R676W. In the DNA binding domain, Msh5 D527 is a solvent exposed residue and hence leads to instability when mutated to a hydrophobic residue such as Alanine. The Msh5 Y480 in the DNA binding domain is involved in aromatic interactions as shown in Subunits of the MutS and MutSα complexes show asymmetry for mismatch binding and ATP hydrolysis  mutants  [6]. But3. The images of the cavity are shown in 3. The Holliday junction is known to take up stacked conformations in the presence of metal ions Volume of the cavity in the Msh4–Msh5 modeled structure was calculated to be 16676 ÅMost probable Msh4–Msh5 interface residues were identified as discussed in The Msh4–Msh5 model structure can be used to predict mutational changes that are compensatory. For example, in the putative interface region, Msh4 K819, D283 and K284 are involved in ionic interactions with Msh5 D732, H264 and D269 respectively. In principle if these residues are mutated such that the overall interaction is retained, for example K819D and D732K, the phenotype is expected to be close to the wild type. The ongoing efforts are directed towards design and generation of such mutants which will further our understanding of sequence-structure-function relationship of Msh4–Msh5 complex.msh4/5 mutations are likely to cause meiotic defects by three main modes. The mutation may disrupt the structural integrity of local regions and hence affect the overall stability of the Msh4–Msh5 complex. The mutation may disrupt the interaction between the Msh4 and Msh5 proteins and prevent complex formation. Finally, the mutation may affect DNA binding by the Msh4/5 proteins.The msh4/5 mutations cause significant meiotic defects  could not be explained with the Msh4–Msh5 modeled structure. Yeast-two-hybrid analysis suggests sixteen mutations disrupt the Msh4–Msh5 complex  have meiotic defects similar to msh4/5Δ. In our model these involve residues stabilizing α-helical regions, residues part of the left handed α helical region of the Ramachandran map, residues involved in aromatic-aromatic interactions, cation-pi interactions, ionic interactions, hydrogen bonding and buried or solvent exposed residues. A significant proportion of interactions involve hydrogen bonding of main chain and side chain atoms or side chain and side chain atoms. A detailed information on the residues that constitute this network has been indicated in Nine mutations, two in Residues stabilizing α-helical regionsMsh4 D139 and Msh5 D433 are involved in stabilizing α-helical regions. The Msh4 D139 serves as an N-cap residue to stabilize a helix four residues downstream. In the case of Msh5, N430 is not a good initiator of the α-helix and hence the D433 stabilizes the structure by means of a hydrogen bond between side chain of D433 and main chain amide of N430 as shown in Residues with conformations in left handed α-helical region of the Ramachandran mapMsh5 G648 and Msh4 G639 are two residues with a positive ø dihedral angle. These angles are accommodated only in the case of Glycine due to the lack of side chain. When these residues are mutated to Alanine with this combination of ø and Ψ angles the residues experience short contacts involving their side chains and hence destabilize the structure.Residue involved in aromatic-aromatic and cation-pi interactionsmsh5 W298A, Alanine has an aliphatic side chain which cannot participate in such an interaction and hence affects stability. The Msh5 W298 is also involved in a cation pi interaction with Msh5 R312 . These residues are involved in aromatic-aromatic interactions, cation-pi interactions and ionic interactions. A few residues are involved in stabilizing the DNA binding region. However, in two cases msh4 L493A and msh4 D532A no explanation could be provided on the basis of the modeled structure. This is mainly due to the high sequence variation between the hMSH2–hMSH6 template and the Msh4–Msh5 model in this region.In terms of severity of phenotypes, these set of mutations second the null mutations. There are nine such mutations, four in msh5 D680A mutant.Msh4 Y143, F194 and Msh5 Y480 are involved in aromatic-aromatic interactions with surrounding aromatic residues within a distance of 6 Å as shown for Y480 in msh4/5-t mutations. There are 9 such mutations distributed in both MSH4 and MSH5 . These residues are involved in aromatic-aromatic interactions (Msh5 Y486), cation-pi interactions (Msh4 F491) and ionic interactions  with surrounding residues in 6 Å radius. Msh4 E276 and N532 are involved in a tight packing which may be disturbed upon mutation to Alanine which has a smaller side chain. The Msh5 S416 is a residue proximal to DNA and hence may affect the stability of binding. Structural explanation could not be provided for the meiotic defects observed in mutations msh5 D76A and D539A.These mutations are deviant from the wild type only with respect to recombination frequency and have been described previously as S. cerevisiae Msh4–Msh5 complex. The modeling studies suggest that Msh4 is most likely functionally similar to hMSH6 of the hMSH2–hMSH6 complex and likewise Msh5 is similar to hMSH2. The model also explains the functional asymmetry between Msh4 and Msh5 with respect to ATP and DNA binding mutations S. cerevisiae Msh4 and Msh5 proteins have been analyzed by mutational studies msh4/5 mutations affecting crossover frequency and spore viability. The model can also facilitate the design of new mutational studies, design of structure based inhibitors of the Msh4–Msh5 complex as well as predict the functional impact of polymorphisms in the MSH4, MSH5 genes. Such studies will be useful for understanding the mechanism of crossover formation by the Msh4–Msh5, Mlh1–Mlh3 pathway The Msh4–Msh5 complex plays an important role in different stages of the meiotic recombination pathway. In the absence of a crystal structure for this complex, we built a homology model of the −10 suggesting high confidence and that both complexes are likely to adopt the same fold. In addition, structural alignments of the S. cerevisiae Msh4, Msh5 protein sequences were also obtained from Bioinfo metaserver or 3D-Jury which uses the alignment information that is predicted consistently by other reliable servers S. cerevisiae Msh2–Msh6 complex is also known to bind Holliday junction structures which further justifies its use as a template Two possible templates (hMSH2–hMSH6 and hMSH2–hMSH3) are available for the modeling of Msh4–Msh5 complex The model of Msh4–Msh5 complex was built using MODELLER (version 9.10) auto-model program with added energy optimization steps Intra and inter protein interactions were identified using the PIC  server Homologues of Msh4 and Msh5 of not necessarily known 3-D structure were obtained by use of PSI-BLAST queried against the UNIPROT-SPROT database Volume measurements were made using the 3V server, which is known to use a robust algorithm to determine the size of cavities. We used 3V to calculate the size of the cavity formed by the Msh4–Msh5 heterodimer and also to calculate the volume of Holliday junction DNA Figure S1Alignment of hMSH6, hMSH3 and Msh4 sequences. 2o8bB corresponds to the hMSH6 from the hMSH2–hMSH6 complex and 3THY corresponds to hMSH3 from the hMSH2–hMSH3 complex. Highlighted in yellow are the deletions that are unique to hMSH3.(PDF)Click here for additional data file.Figure S2Alignment of Msh4 and Msh5 amino acid sequences with the hMSH2–hMSH6 complex (PDB code 2o8b). The alignment was used to model the Msh4–Msh5 complex structure. Msh4/5 residues whose mutations cause null phenotype, intermediate defects in crossing over and viability or only crossover defects are shown in blue, green and red boxes respectively.(PDF)Click here for additional data file.Figure S3Structure based sequence alignment of Msh1–6 and hMSH2, hMSH3, hMSH6. Highly conserved positions are highlighted in red, positions with conservative substitutions are highlighted in a blue box with residues marked in red. DNA binding residues identified from literature survey are highlighted in a yellow box. Residues predicted by Multi-VORFFIP (MV) server to have high probability of DNA binding are highlighted in a black box.(PDF)Click here for additional data file.Figure S4Protocol used to model Msh4–Msh5 structure. The sequences were submitted to three structure prediction servers and the alignment obtained from all these were compiled and manually curated to get final alignment which was provided to MODELER to build the structure. The structure was then energy minimized and checked for stereo-chemical quality .(PDF)Click here for additional data file.Table S1Sequence to structure mapping for Msh4.(XLSX)Click here for additional data file.Table S2Sequence to structure mapping for Msh5.(XLSX)Click here for additional data file.Table S3Explanation for meiotic defects observed in msh4/5 mutants based on the homology model.(DOCX)Click here for additional data file.Table S4Probability of each residue to bind/stabilize DNA.(XLSX)Click here for additional data file.


Much of Figure 1 was removed due to a cropping error. Additionally, the color of each figure was incorrectly modified which may affect the readability. Please see the corrected figures here:Figure 1: Figure 2: Figure 3: Figure 4:


In studying the epidemiology of parvovirus 4 (PARV4) in Taiwan, we detected DNA in plasma of 3 mothers and their newborns with hydrops. In 1 additional case, only the mother had PARV4 DNA. Our findings demonstrate that PARV4 can be transmitted through the placenta. Parvoviridae family  LPGYNYVGPGNPL  and YKYPYVLGQGQDTL  YDYPYVLGHNQDTL .Transmission routes of human parvovirus 4 (PARV4), a recently discovered member of the To exclude the possibility of antibody cross-reaction between PARV4 and B19V, we tested plasma samples sent to our laboratory for confirmation of B19V infection with PARV4 immunoblot. Unexpectedly, we detected PARV4 DNA in plasma from a mother and her newborn with hydrops. Therefore, we examined samples from 5 additional infants with hydrops.During 2000–2009, our laboratory received blood samples from 6 infants with nonimmune idiopathic hydrops . Paired Antibodies to PARV4 and B19V were tested by immunoblots. DNA of PARV4 and B19V was detected by seminested and nested PCR, respectively. PARV4 immunoblot and PCR were performed according to the methods in our previous report (Four of the 5 mothers had immunoglobulin (Ig) M against PARV4 . Two of Only the mother and newborn of case A had detectable B19V DNA (genotype 1). By contrast, PARV4 DNA (genotype 2) was found in plasma of all but 1 of the 6 case-patients. The newborn negative for PARV4 DNA received a whole-blood exchange before sampling.,,,The first serologic study (Contrary to the epidemiology of PARV4 in Europe, studies in Africa found different transmission routes and a higher seropositive rate in blood donors and the general population. In Ghana, 8.6% of infants had PARV4 viremia (,PARV4 can be transmitted through nonparenteral routes (Maternal PARV4 infections were diagnosed by detection of PARV4 DNA in all 5 mothers; 4 of whom had IgM against PARV4. Using IgM against PARV4 as evidence of recent infection must be done cautiously because of persistent IgM against PARV4 (Persons with past B19V infection are expected to have IgG against B19V VP1 but not VP2 in immunoblot (In conclusion, PARV4 can be transmitted parenterally and placentally. Other transmission routes might exist and remain to be discovered. Prospective studies of PARV4 infection during pregnancy are needed to clarify the effect of PARV4 infection on fetal outcome.Infants with nonimmune idiopathic hydrops fetalis are not routinely checked for parvovirus B19 infection.


Heat shock transcription factor 1 (HSF1) plays an important role in the cellular response to proteotoxic stresses. Under normal growth conditions HSF1 is repressed as an inactive monomer in part through post-translation modifications that include protein acetylation, sumoylation and phosphorylation. Upon exposure to stress HSF1 homotrimerizes, accumulates in nucleus, binds DNA, becomes hyper-phosphorylated and activates the expression of stress response genes. While HSF1 and the mechanisms that regulate its activity have been studied for over two decades, our understanding of HSF1 regulation remains incomplete. As previous studies have shown that HSF1 and the heat shock response promoter element (HSE) are generally structurally conserved from yeast to metazoans, we have made use of the genetically tractable budding yeast as a facile assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. We show that when human HSF1 is expressed in yeast its phosphorylation at S303 is promoted by the MAP-kinase Slt2 independent of a priming event at S307 previously believed to be a prerequisite. Furthermore, we show that phosphorylation at S303 in yeast and mammalian cells occurs independent of GSK3, the kinase primarily thought to be responsible for S303 phosphorylation. Lastly, while previous studies have suggested that S303 phosphorylation represses HSF1-dependent transactivation, we now show that S303 phosphorylation also represses HSF1 multimerization in both yeast and mammalian cells. Taken together, these studies suggest that yeast cells will be a powerful experimental tool for deciphering aspects of human HSF1 regulation by post-translational modifications. S. cerevisiae HSF directly activates the expression of genes whose protein products are involved in protein folding and degradation, ion transport, signal transduction, energy generation, carbohydrate metabolism, vesicular transport, cytoskeleton formation and other cellular functions All organisms are exposed to proteotoxic stresses that result in the accumulation of misfolded proteins. In response to these stresses cells have evolved adaptive responses to protect and stabilize cellular proteins until more favorable conditions for cell proliferation are encountered While mammalian cells express four distinct HSF proteins encoded by separate genes, HSF1 is the primary factor responsible for stress responsive gene transcription in vitro phosphorylation experiments and in vivo studies using either lexA or Gal4-HSF1 fusion proteins lacking the native HSF1 DNA binding domain. As such, many of the earlier studies exploring S303 and S307-dependet regulation of HSF1 activity have resulted in conflicting results. For example, previous phosphorylation experiments suggested that S307 was phosphorylated by ERK which, in turn, acted as an essential priming step for GSK3-dependent phosphorylation of S303 in vitro studies suggested that S303 could also be phosphorylated by a variety of mitogen activated protein kinases (MAPK) including the stress responsive MAPK p38 in vivo data suggested S303 phosphorylation could occur independently of S307 phosphorylation The activity of HSF1 is also thought to be negatively regulated through a number of post-translational modifications including phosphorylation, sumoylation and acetylation While the specific mechanism by which S303 and S307 phosphorylation repress HSF1 activity remains unclear, evidence has suggested that S303 and S307 phosphorylation represses the transactivation potential of HSF1 S. cerevisiae is unable to complement for the loss of the essential yeast HSF protein While HSF1 and the cognate HSEs are quite well conserved from yeast to humans, our previous results demonstrated that human HSF1 expressed in Here we report the use of the yeast assay system to further understand the mechanisms that regulate human HSF1 through phosphorylation of serine 303. Our results suggest that S303 phosphorylation blocks human HSF1 homotrimerization thereby preventing human HSF1 activation and complementation of the loss of yeast HSF. Furthermore, we demonstrate that S303 phosphorylation also blocks HSF1 homotrimerization in mammalian cells. We show that phosphorylation of HSF1 S303 in yeast occurs via the action of the MAPK Slt2 and not via the action of GSK3 and we extend these findings to show that S303 phosphorylation also occurs independent of GSK3 in mammalian cells.When human HSF1 is expressed in yeast it is unable to homotrimerize, promote gene expression and complement for the loss of the essential yeast HSF protein To ascertain whether HSF1 is being phosphorylated in yeast, we employed a commercially available antibody specific for phospho-S303 (pS303). Because this antibody has not previously been characterized in the literature, we tested its specificity in human HeLa cells where S303 is known to be constitutively phosphorylated Consistent with a contribution to HSF1 repression  S303 is Human HSF1 S303 phosphorylation is known to promote sumoylation of lysine 298, which also contributes to the repression of HSF1 activity −/− mouse embryonic fibroblasts (MEF) Previous reports have suggested that phosphorylation of S303 represses the ability of HSF1 to transactivate gene expression In addition to post-translational modifications, HSF1 activity is also thought to be repressed through intramolecular interactions between carboxyl- and amino-terminal coiled-coil domains and mutations in these domains render HSF1 constitutively trimerized, nuclear localized and bound to DNA in mammalian cells in vitro phosphorylation experiments have suggested that HSF1 is phosphorylated at S303 by glycogen synthase kinase 3 (GSK3) in vivo. To test if GSK3 contributes to HSF1 repression, we assayed human HSF1-dependent yeast growth in a strain also lacking the yeast GSK3 homolog Rim11. Supporting the notion that yeast GSK3 can repress human HSF1 activity in yeast we observed human HSF1-dependent yeast growth as well as HSF1 multimerization in the rim11Δ strain  in yeast phosphorylate human HSF1 at S303 to promote HSF1 repression, we assayed S303 phosphorylation in several previously generated protein kinase deletion strains obtained from the yeast gene deletion collection e strain . Homotriin yeast . In mammΔ strain  also supin vitro phosphorylation experiments in vivo using lexA/Gal4-HSF1 fusion proteins lacking the native HSF1 DNA binding domain −/− MEFs which lack endogenous HSF1. When we expressed S303A, S307A or S303/307A HSF1 mutants in hsf1−/− MEFs we observed a modest elevation of Hsp70 expression under normal growth conditions  supplemented with protease inhibitors (Roche) and Halt phosphate inhibitor cocktail (Thermo Scientific Pierce). Proteins extracts were generated from mammalian cell culture using cell lysis buffer supplemented with protease and phosphatase inhibitors. Protein concentrations were quantified using the BCA assay and 80–100 µg of total protein was resolved by SDS-PAGE and transferred to a nitrocellulose membrane. HSF1 oligomerization was assessed using the amine-specific cross-linker ethylene glycol bis-succinimidyl succinate (EGS) (Pierce). Crosslinking analysis were carried out as described previously


Chronic hepatitis C virus infection and its sequela are major health problems facing the Egyptian community. The high prevalence and spread rates of the disease require serious actions to stop or decrease these rates. Determination of HCV genotypes and subgenotypes adds significant knowledge about the epidemiology of the disease, and provides an added value in the decision making process of what strategy to follow and what therapy response to expect. The molecular epidemiology and genetic variability of HCV variants circulating in Egypt still need further analysis.The study was held to evaluate the genotype and subgenotype of the hepatitis c virus circulating in Sharkia as one of the large governorates of Egypt, which was not included in any study for genotyping of the virus before.The HCV molecular epidemiology in Sharkia governorate was studied using direct sequencing and further phylogenetic analysis of a partial NS5B region of the HCV genome from 63 patients. HCV genotype and subtype were successfully determined in 62 out of 63 patients.The highest prevalent genotype was genotype 4a, which was found in 57 patients (92%) followed by 2 isolates (3%) with genotype 4o, 2 strains (3%) with genotype 1g and one isolate (2%) with genotype 4n.This molecular epidemiology study revealed high prevalence of HCV genotype 4, subtype 4a among Egyptian patients residing in Sharkia governorate, Egypt. Hepatitis C virus (HCV) infectionis estimated to affect 130–170 million people worldwide (1). Although the clinical manifestation of HCV acute infection is generally mild or asymptomatic, it can lead to chronic liver disease, cirrhosis, and hepatocellular carcinoma (HCC). The main indication for liver transplantation in the industrialized world is related to HCV infection .HCV is a positive-sensed single-stranded RNA virus belonging to the Flaviviridae family. The HCV genome is an RNA molecule of approximately 9600 nucleotides, structured in a coding region that contains one large open reading frame (ORF), flanked by non-translated regions (NTR) at the 5’ and 3’ ends encoding a polyprotein precursor of about 3,000 amino acids. The precursor is cleaved into at least 10 different proteins comprising the structural proteins, core, E1, E2, and p7 as well as the non-structural proteins, NS2, NS3, NS4A, NS4B, NS5A, and NS5B , following the manufacturer’s instructions.HCV genotypes were identified via direct sequencing of non-structural (NS5B) viral genes with universal primers described by Murphy et al. 2007 , and sequenced using the dideoxynucleotide chain termination method with the ABI PRISM®BigDye Terminator Cycle Sequencing Reaction kit  Sequence analysis was performed with the ABITM3130 Genetic Analyzer . The sense and antisense primers described above were used as sequencing primers. The chromatogram sequencing files were inspected using Chromas 2.3 . The genotype of each sample was determined by comparing its sequence with those of HCV prototypes obtained from GenBank, followed by further genetic analysis. The DNA alignments were generated with ClustalX version2.0 software .HCV genoThe NS5B region corresponds to a 389 nt fragment covering the middle section of the NS5B gene of the HCV genome. The direct sequencing and phylogenetic analysis of the HCV NS5B gene was successful in 62out of 63 positive samples. Samples number Eg 21 and Eg 22 had been sequenced two times and both of them have exactly the same sequence in each run. They were expressed in the tree as one sample (Eg 21). The result of the study revealed that subtype 4a was the most prevalent in this population with a total number of 57 (92%).Subtype 1g and 4o were each identified in 2 samples (3%), and 4n was identified in 1 sample only (2%). Preliminary phylogenetic analysis containing 56 sequences (data not shown) was performed and allowed a selection of 37 additional GenBank sequences corresponding to the most related ones to the study isolates. These reference sequences were named after their accession numbers, subgenotype and country of origin. Partial NS5B coding sequences obtained from Egyptian samples were aligned with the reference sequences. The phylogenetic tree obtained by performing neighbor-joining (NJ) analysis of the alignment of sequences is shown in Three samples clustered in a different branch with reference sequences from genotype 4. HCV isolates Eg-10 and Eg-48 clustered with reference sequences of HCV genotype 4o from Egypt and from Canada with a high bootstrap value of 98%. The Eg-16 isolate clustered with three reference sequences of genotype 4n from Egypt, Canada and an Egyptian immigrant from the Netherlands, with a bootstrap value of 95%.Another 2 samples (Eg-40 and Eg-62) branched with reference sequences of HCV genotype 1g from Canada and from Egypt with a bootstrap value of 94%.Sequences were submitted to GenBank (accession numbers: JN203143 to JN203205).According to different reports, the prevalence of HCV in Egypt ranges from 11% to higher than 14% of the general population, 5–7 million people with active infection having detectable HCV RNA and over 500,000 new infections each year -17. In cIt is known that genotype and even subtypes differences of HCV havea high impact on interferon responsiveness and give an idea about the natural history of the disease . In EgypIn a study on 206 samples collected from the Kasr El-Aini School of Medicine and the National Cancer Institute, Cairo, Egypt, HCV genotype 4 was detected in 186 samples (90.3%). Five other subtypes could be also identified: subtypes 4a, 4d, 4m, 4n, and 4o. They also found that 9.7% of the patient’swereinfected with HCV genotype 1g and 1a . In the Another study, from the Alexandria district, showed that 78% of the isolates were HCV genotype 4a variants, whereas the remaining identified variants were 4m (11%), 4o (5.5%), 4n (2.7%) and 4p (2.7%) . In IsmaIn conclusion, this study shows that HCV genotype 4ais predominant in the governorate of Sharkia, and it confirms that the Egyptian HCV epidemic is composed of multiple lineages of genotypes 4 and 1.