Conservation strategies for African elephants would be advanced by resolution of conflicting claims that they comprise one, two, three or four taxonomic groups, and by development of genetic markers that establish more incisively the provenance of confiscated ivory. We addressed these related issues by genotyping 555 elephants from across Africa with microsatellite markers, developing a method to identify those loci most effective at geographic assignment of elephants (or their ivory), and conducting novel analyses of continent-wide datasets of mitochondrial DNA. Results showed that nuclear genetic diversity was partitioned into two clusters, corresponding to African forest elephants (99.5% Cluster-1) and African savanna elephants (99.4% Cluster-2). Hybrid individuals were rare. In a comparison of basal forest “F” and savanna “S” mtDNA clade distributions to nuclear DNA partitions, forest elephant nuclear genotypes occurred only in populations in which S clade mtDNA was absent, suggesting that nuclear partitioning corresponds to the presence or absence of S clade mtDNA. We reanalyzed African elephant mtDNA sequences from 81 locales spanning the continent and discovered that S clade mtDNA was completely absent among elephants at all 30 sampled tropical forest locales. The distribution of savanna nuclear DNA and S clade mtDNA corresponded closely to range boundaries traditionally ascribed to the savanna elephant species based on habitat and morphology. Further, a reanalysis of nuclear genetic assignment results suggested that West African elephants do not comprise a distinct third species. Finally, we show that some DNA markers will be more useful than others for determining the geographic origins of illegal ivory. These findings resolve the apparent incongruence between mtDNA and nuclear genetic patterns that has confounded the taxonomy of African elephants, affirm the limitations of using mtDNA patterns to infer elephant systematics or population structure, and strongly support the existence of two elephant species in Africa. Loxodonta) occupying tropical forest habitats were long considered to comprise a single species with elephants in other African habitats. Recent studies have renewed debate about their taxonomy, after both morphological and nuclear DNA analyses suggested that African elephants form two distinct species separated by a relatively narrow hybrid zone Central to the successful management of endangered taxa is determination of whether their populations comprise one or more species. Molecular methods can play a major role in establishing the systematics of endangered taxa. Where more than one species is revealed, a separate conservation strategy would be justified for each. African elephants  loci in African forest and savanna elephants that had previously been typed for mtDNA. Employing a Bayesian clustering approach, we sought to better quantify the degree and geographic extent of hybridization based on these STR loci. We examined the relationships between mtDNA and nuclear patterns in these elephants and determined what these patterns imply about evolutionary relationships of almost a thousand African elephants from 81 geographic locales for which mtDNA sequence had been previously generated. We determined whether geographically mapping the mtDNA and nuclear genetic partitions among the elephants of Africa might provide additional insights into resolving the differing assertions regarding their taxonomic status. We found that our novel approach to mtDNA analysis could reconcile the apparent conflict previously reported between mitochondrial and nuclear phylogenies in African elephants Based on this systematic clarification, we further sought to improve upon the other major area in which genetics can contribute to African elephant conservation: the forensic analysis of ivory samples. Although the Convention on International Trade in Endangered Species (CITES) banned the ivory trade in 1989, large numbers of elephants continue to be poached for their tusks, and the illegal trade in ivory is a major threat to their conservation Elephas maximus), using fifteen microsatellite loci, including newly developed loci  to the elephant population at each locale are shown in One of our locales, Garamba, is in a region of the Democratic Republic of Congo that historically included both forest and savanna habitats A factorial correspondence analysis (FCA) implemented using the software GENETIX Genetic relationships among different locales were also inferred from the STR data using the chord distance. The elephants from savanna and forest locales formed two distinctive phylogenetic clusters . LikewisND5 gene and detected two deep clades, designated I and II , who had sequenced individual elephants for longer mtDNA regions that overlapped with the sequences both of Debruyne (2005) and of Roca et al. (2005)   [4], , . In TURE see . By contTURE see  [4]. PreTURE see . Since tK) were considered in the STRUCTURE analysis (not shown); and this individual has previously been found to have nuclear sequences identical to that of some Central African forest elephants (though distinct from that of any savanna elephants) The Guinean forest block in West Africa is currently not contiguous with the Congolian forest block in Central Africa We therefore considered an alternative if indirect approach to assessing the genetic similarity between West and Central African elephants. There has been one previous study that examined a large number of nuclear genetic markers among elephants in both West and Central Africa, which reported on the use of these markers in geographic assignment tests of elephants Besides taxonomy and population structure, the other major area in which genetics can contribute to African elephant conservation is in the geographic assignment of ivory samples In order to quantify the ability of different genetic markers to assign the provenance of elephants, the Shannon Information Content (SIC) The ability of each of our STR loci to differentiate among elephants from different locales is shown in K = 2 (using only these two populations). However, using 4 STRs with high SIC values  in a similar analysis did begin to differentiate elephants between these two locales: using only these two populations, at K = 2 the values for STRUCTURE partitions 1 and 2 were 0.36 and 0.64, respectively, for Kruger elephants; while they were 0.61 and 0.39, respectively, for Namibia elephants. This quantitative approach comparing different STRs for their SIC can thus identify an improved set of STR loci to utilize for the purpose of assigning the geographic provenance of elephants.As has been shown in the case of human STRs Our new data and reanalyses of previous data reconciled the mtDNA phylogeographic patterns present among African elephants to the two-species division that is evident from nuclear DNA and morphological analyses. Previous studies found a deep and almost complete nuclear genetic separation of African elephants into forest and savanna species, with a few hybrids detected primarily in the regions where tropical forest and savanna habitats meet The Bayesian STRUCTURE analysis conducted for the current study used a novel set of STR loci to support and extend these conclusions. Few forest–savanna elephant hybrids were identified by STRUCTURE, and for each species the proportion of admixture from the other species was inferred to be under one percent. Previous studies have convincingly reported data supporting the “polyphyly of the forest and savannah [mtDNA] haplotypes” Our results also strongly support the observation that mtDNA patterns in elephants are incongruent with their overall population structure Since males can transmit every locus except mtDNA, the mitochondrial genome is subject to an extremely circumscribed evolutionary trajectory very different from that affecting every other genetic locus By contrast, not a single nuclear locus is “bound to the herd” like mtDNA. Males in one generation can transmit nuclear alleles from their natal herd to other herds. This occurs regardless of whether or not males traverse long distances. Males of one generation may transmit nuclear alleles to a nearby herd; their male offspring can then transmit them to even more distant herds, and thus nuclear alleles have the potential to traverse the African landscape. One notable point in this regard is that females do transmit their nuclear genetic endowment to male offspring, thus all of their nuclear alleles  thereby become easily transmitted across the landscape. Among genetic markers, only the mtDNA molecule is subject to the severe demographic constraint of being bound to the herd. All other elephant genetic markers are geographically unbound and subject to widespread and (compared to mtDNA) relatively rapid transmission across the geographic landscape We have quantified the relationship between the proportion of elephants in a population that carry S clade savanna mtDNA and the proportion of the population assigned to forest- or savanna-elephant nuclear genetic partitions . Surprisca. 100% assignment of their genotypes into the savanna elephant partition in STRUCTURE analyses  assumption that the presence of both F (forest elephant-derived) and S (savanna elephant-derived) mitochondrial DNA clades at a locale is an indication that the population of elephants at that locale must comprise a mixture of forest and savanna elephant genotypes analyses , 5. Evenanalyses . This mianalyses . One preIsolation from savanna elephant gene flow appears to be complete among forest elephants, since in the current survey not a single forest elephant was found to carry S clade mtDNA across 30 forest locales in West or Central Africa. Previous studies had failed to detect, among a smaller survey of forest elephants, even a single Y-chromosome or X-chromosome haplotype derived from savanna elephants ca. 20% of savanna elephants The situation is more complicated among savanna elephants. Forest elephant derived F clade mtDNA has a very broad geographic range that extends long distances away from current forest habitats. F clade mtDNA is carried by sensu lato, a commonly observed phenomenon detected following hybridization between distant taxa, in which the deleterious effects of hybridization tend to have greater impact on the heterogametic sex , since the mito-nuclear patterns suggest that hybrid females have been reproductively successful but hybrid males have not The historical reproductive success of female hybrids can be inferred by the presence of F clade mtDNA, derived from forest elephants, in these savanna elephant herds , 5 [1], Although cautious in their interpretation L. cyclotis” in discriminant analyses Eggert et al. (2002) LA5, the frequency of allele 191 was 1.00 in Mali and 0.46 in Waza, but 0.00 in all other savanna or forest locales sampled by Eggert et al. (2002) LA6, the three highest frequencies for allele 172 were found in Mali (1.00), Waza (0.50), and Bénoué (0.27); and for locus LafMS02, the three highest frequencies for allele 150 were also in Mali (1.00), Waza (0.50), and Bénoué (0.27). Since Waza and Bénoué elephants have been shown by all nuclear DNA analyses including the current ones to partition as savanna elephants  lephants , 2, 5, 6002 lephalephants , arrow. Given that mtDNA haplotypes among elephants are an unreliable indicator of overall genetic similarity  [4], it Along with the destruction of habitats, one of the major threats to elephant populations is illegal hunting for the ivory trade. DNA has been successfully extracted from ivory Loxodonta) primarily by biopsy darting Loxodonta cyclotis) from Sierra Leone, which had been kept at the Paris Zoo . DNA was extracted primarily using kits (Qiagen), or standard phenol-chloroform methods The study was conducted in compliance with the University of Illinois Institutional Animal Care and Use Committed (IACUC) approved protocol number 09036. Samples were obtained in full compliance with required CITES  and other permits. As previously described, samples were collected from wild African elephants (http://www.nisc.nih.gov/open_page.cgi?path=/projects/comp_seq.html). Microsatellites were identified using a repeat finder script written in Perl. PCR primers for novel STR loci were designed using Primer3 (http://fokker.wi.mit.edu/primer3/input.htm). PCR primers were tagged for fluorescence detection http://code.google.com/p/allelogram).Fifteen microsatellite loci were amplified (Supplementary http://www.fieldgenetics.com/pages/aboutCervus_Overview.jsp). Two of the novel loci developed for this study from a capture hybridization library  were excluded from some analyses because they exhibited an excess of homozygosity due to null alleles. Loci were examined for linkage using GENEPOP http://genepop.curtin.edu.au). Loci LAF30 and LAF35  were excluded because of potential linkage to the more diverse loci LAF29 (p29) and LAF37 (p37), respectively.A total of 15 loci were amplified in 555 African and 9 Asian elephants. The data was analyzed for deviation from Hardy–Weinberg equilibrium or excess of null alleles using Cervus 3.0 K between 1 and 21 genetic clusters, without any prior population information. Each analysis was run for at least 1 million Markov chain Monte Carlo generations following a burn-in of at least 100,000 steps. STRUCTURE was run using four models ad hoc statistic ΔK based on the rate of change in the log probability of the data between successive values of KWe used the program STRUCTURE 2.3.1 to apply a model-based clustering algorithm to identify subgroups of individual elephants that have distinctive allele frequencies 2, 200 uM of each dNTP (Applied Biosystems Inc. [ABI]) and 1 ug/ul final concentration of bovine serum albumin  with 0.04 units/ul final concentration of AmpliTaq Gold DNA Polymerase (ABI). PCR consisted of an initial denaturation at 95°C for 9∶45 min; cycles of denaturation for 20 sec at 94°C, annealing for 30 sec at 60° , 58°C, 56°C, 54°C, 52°C , or 50°C (last 22 cycles), and 1 min extension at 72°C; with a final extension of 7 min at 72°C. PCR amplicons were examined on a 1% agarose gel and were enzyme-purified and sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (ABI). Eight BF samples were successfully amplified and sequenced with amplicon sizes of 726 bp (681 bp excluding primers) or 375 bp (322 bp excluding primers), using the following primers: for the 726 bp amplicon, PCR primers were CBCR-F5 ATTACAATGGTCTTGTAAGCCATAAA and CBCR-R1d CTCAGACGGCCATAGCTGA; sequencing used the PCR primers and CBCR-F6 GATAAACCATAGTCTTACATAGCACAT and CBCR-R5 CTTTAATGTGCTATGTAAGACTATGG. For the 375 bp amplicon, PCR and sequencing primers consisted of CBCR-F5 and -R5. Novel mtDNA sequences have been deposited in Genbank (accession numbers: JF827273–JF827275). NCBI Blast (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) and comparison to previously sequenced elephant mtDNA were used to identify all BF elephant mtDNA sequences as F clade  was used to analyze a 2×2 contingency table for association between the presence or absence of S clade mtDNA and the type of habitat at a locale .PCR amplifications using DNA from Bili Forest (BF) elephants were performed using 0.4 uM final concentration of each primer, 1.5 mM MgClShannon Information Content (SIC) measures the amount of information contained in a message SIC has been successfully adapted to genetic studies, particularly for identifying genetic markers with frequencies highly specific for human populations of different geographic origins; which are used in admixture mapping of the human genome Figure S1CYTB sequences largely overlap the mtDNA diversity reported by Debruyne (2005) The groups identified by Johnson et al. (2007) (PDF)Click here for additional data file.Figure S2ND4-tRNAGLU sequences of Lei et al. (2008) ND5 sequences Roca et al. (2005) CYTB sequences of the same individual elephant (same “sample ID”) of Lei et al. (2008) CYTB and control region (CR) sequences of Debruyne (2005) CYTB sequences of Johnson et al. (2007) Comparison of mtDNA sequences across studies of African elephant genetics: (A) Diagram illustrating mtDNA regions sequenced by different researchers, showing overlapping and non-overlapping regions across studies. Positions follow those of the elephant reference mtDNA genome (Genbank accession number NC_000934) (PDF)Click here for additional data file.Figure S3Map and list of locations of elephants sampled and sequenced for mtDNA across genetic studies (PDF)Click here for additional data file.Table S1Fifteen elephant short tandem repeat loci amplified (PDF)Click here for additional data file.Table S2Allele range, number and frequency for 11 STR loci.(PDF)Click here for additional data file.Table S3ad hoc Evanno et al. (2005) Calculation of the (PDF)Click here for additional data file.Table S4Contingency table, formulas and other information for the Shannon Information Content.(PDF)Click here for additional data file.


The multicomponent phagocytic NADPH oxidase produces reactive oxygen species (ROS) after activation by microorganisms or inflammatory mediators . In the in vitro experiments using J774 macrophages. To obtain an alternatively activated phenotype, macrophages were stimulated with conditioned medium from apoptotic T cells (CM). By luciferase assays we figured out a 3'UTR-dependent regulation of NOX2 mRNA stability. Assuming that a protein is involved in the mRNA degradation, we performed a RNA pulldown with biotinylated NOX2-3'UTR constructs followed by mass spectrometry. We verified the role of SYNCRIP by siRNA approach. Additionally, we overexpressed NOX2 in J774 cells and analyzed the ROS production (w/wo CM treatment) by FACS.We induced polymicrobial sepsis in mice by cecal ligation and puncture. The ability of peritoneal macrophages (PMs) to produce ROS was determined by FACS via hydroethidine assay. NOX2 expression of PMs was determined by western blot and qPCR. To elucidate the mechanism causing mRNA destabilization, we performed We found an impaired expression of NOX2 at RNA and protein level along with decreased ROS production after induction of sepsis in mice as well as stimulating J774 macrophages with CM of apoptotic T cells. This is due to a time-dependent NOX2 mRNA degradation depending on SYNCRIP, a RNA-binding protein, which stabilizes NOX2 mRNA through binding to its 3'UTR under normal conditions. In line, knockdown of SYNCRIP also decreases NOX2 mRNA expression. We assume that a CM-dependent modification or degradation of SYNCRIP prevents its stabilizing function. As the overexpression of NOX2 restores ROS production of CM-treated J774 cells, we assume that NOX2 expression is crucial for maintaining NADPH activity during the hypoinflammatory phase of sepsis.Our data imply a regulatory impact of SYNCRIP on NOX2 stability during the late phase of sepsis. Therefore, further understanding of the regulation of NADPH oxidase could lead to the design of a therapy to reconstitute NADPH oxidase function, finally improving immune function in sepsis patients.


Disturbances in oxygen levels have been found to impair cardiac organogenesis. It is known that stem cells and differentiating cells may respond variably to hypoxic conditions, whereby hypoxia may enhance stem cell pluripotency, while differentiation of multiple cell types can be restricted or enhanced under hypoxia. Here we examined whether HIF-1alpha modulated Wnt signaling affected differentiation of iPS cells into beating cardiomyocytes.We investigated whether transient and sustained hypoxia affects differentiation of cardiomyocytes derived from murine induced pluripotent stem (iPS) cells, assessed the involvement of HIF-1alpha  and the canonical Wnt pathway in this process.Embryoid bodies (EBs) derived from iPS cells were differentiated into cardiomyocytes and were exposed either to 24 h normoxia or transient hypoxia followed by a further 13 days of normoxic culture.At 14 days of differentiation, 59±2% of normoxic EBs were beating, whilst transient hypoxia abolished beating at 14 days and EBs appeared immature. Hypoxia induced a significant increase in Brachyury and islet-1 mRNA expression, together with reduced troponin C expression. Collectively, these data suggest that transient and sustained hypoxia inhibits maturation of differentiating cardiomyocytes. Compared to normoxia, hypoxia increased HIF-1alpha, Wnt target and ligand genes in EBs, as well as accumulation of HIF-1alpha and beta-catenin in nuclear protein extracts, suggesting involvement of the Wnt/beta-catenin pathway.Hypoxia impairs cardiomyocyte differentiation and activates Wnt signaling in undifferentiated iPS cells. Taken together the study suggests that oxygenation levels play a critical role in cardiomyocyte differentiation and suggest that hypoxia may play a role in early cardiogenesis. Cardiomyogenesis from specific progenitor cells is critically dependent on the presence of a tightly regulated biochemical and mechanical microenvironment. An appropriate level of tissue oxygen tension is one of the most critical factors for the formation of the heart and its subsequent structural development. In this context, hypoxia is recognized to cause a range of cardiac malformations, including myocardial and valvular hypoplasia Hypoxia-inducible factors (HIFs) sense and adapt to low oxygen tension. HIFs are a family of transcription factors consisting of two subunits: the oxygen sensitive alpha subunit, and the constitutively expressed beta subunit, which binds to all HIF alpha combinations . Both HIF-1 and HIF-2 protein complexes are expressed in cardiac tissue In regard to cardiac development specifically, HIF-1alpha knockout mice are embryonic lethal by 11 days post-coitum (dpc) with diverse cardiac anomalies, including cardiac bifida, abnormal looping, abnormal remodeling of the outflow tract, blood vessel deformities and mesenchymal cell death Therefore, in this study we investigate the role of transient hypoxia on the differentiation of embryoid body (EB)-derived cardiomyocytes obtained from mouse induced pluripotent stem (iPS) cells and on the expression of genes in the Wnt pathway. The observations made in the present study may also provide insights into the causation of hypoxia induced cardiac developmental abnormalities, whilst also providing potential information related to the role of Wnt signaling during the generation of mature cardiomyocytes from iPS cells.For the present studies, iPS cells were derived from mouse skeletal muscle progenitor cells . Up-reguiPS cells were differentiated down the cardiac lineage by formation of EBs upon removal of leukemia inhibitory factor (LIF). Following 14 days of culture under normoxic conditions, 59±2% of EBs contained elements exhibiting spontaneous contractions. In parallel with the functional characterization, we evaluated the temporal expression pattern of the cardiac progenitor marker Islet-1 (Isl1), the early mesodermal transcription factor, Brachyury, and the mature cardiomyocyte markers troponin C type 1 (Tnnc1) and beta myosin heavy chain (beta-MHC) over the 14 days of normoxic culture. As shown in We next aimed to determine whether exposure to short term (24 h) periods of hypoxia influenced the capacity of iPS cells to differentiate into mature cardiomyocytes. In contrast to the development of contractile EBs under normoxic conditions as described above, EBs exposed to an initial 24 hr period of hypoxia followed by 13 days of normoxia failed to develop any evidence of contractile activity at the 14 day timepoint . To inveSince hypoxia inducible factor -1  has been previously shown to be essential for the hypoxic response in heart development Expression of members of the canonical Wnt pathway were significantly upregulated in response to transient hypoxia as shown in We also examined the temporal pattern of changes in the expression of Wnt signaling targets in response to transient hypoxia and to subsequent normoxia. As shown in Extensive data indicate that exposure to hypoxia during fetal development has the potential to cause abnormal heart morphology and function In the present study, we employed an iPS based approach to investigate the mechanism by which hypoxia influences cardiomyocyte development. In particular we wished to determine whether even short periods of hypoxia might influence cardiomyocyte differentiation from precursors, and to evaluate the potential role of altered Wnt expression. We demonstrated that a short period of hypoxia followed by a return to normoxic conditions lead to a failure of the development of a contractile phenotype in iPS derived EBs. In association this was accompanied a significant prolongation of the expression of the early developmental markers Isl1 and Brachyury, compared to EBs that had undergone differentiation in normoxic conditions.In contrast with the findings in our study, it has previously been suggested that hypoxia may exert a positive role for on cardiomyocyte differentiation albeit in mouse ES cells (mESCs), in a Cripto-1 dependent manner Our study sought to explore the possibility that hypoxia early in cardiomyocyte development may lead to activation of HIF-1α with potential subsequent effects on Wnt gene expression. This hypothesis was based on previous studies showing that distinct stem cell populations exist in hypoxic niches where HIF-1alpha directly regulates the Wnt/beta-catenin pathway to maintain pluripotency We demonstrated a complex pattern of altered Wnt expression following hypoxia, which was accompanied by downregulation of key inhibitors, together favoring a lack of differentiation. Consistent with activation of canonical Wnt signaling and possibly a direct interaction with of HIF 2+ and K+ channels.One of the striking findings of our study was that exposure to relatively short term hypoxia lead to a long term failure of development of a contractile phenotype. The prolonged appearance of immature markers would not necessarily directly explain this finding but are consistent with a failure of progressive differentiation to a contractile phenotype. We did observe a downregulation of the contractile protein beta∼MHC, which may have been a contributing factor, although this was opposite to Tnnc1 expression. We did not specifically explore the reason for the difference between beta∼MHC and Tnnc1 expression, however it is likely that this may reflect different temporal patterns of expression during development and differing expression in cardiomyocytes of varying phenotype. In addition to the modification of contractile protein expression, hypoxia also initiated the expression of key plasma membrane CaTaken together our study provides a number of important findings relating to the substantial long term alteration in cardiomyocyte development after hypoxia and the potential role of HIF-1alpha and Wnt signaling. Specifically targeted molecular or pharmacologic approaches will be required to further delineate the exact mechanism for this process.All animal experimentation was conducted with protocols and ethics approved by the Monash Medical Centre Animal Welfare Committee and the Alfred Medical Research and Education Precinct (AMREP) Animal Experimentation Ethics Committee under the guidelines of the National Health and Medical Research Council of Australia. EveryThe gastrocnemius muscle was removed from a C57/Bl6 mouse. Skeletal muscle myoblast progenitors and skeletal fibroblasts cultures were established using a modification of previously established protocols 2 and passaged using 0.05% trypsin-EDTA (Invitrogen). Phase contrast images of somatic cell lines and iPS colonies were acquired using an Olympus CKX41 microscope.Primary cultures were retrovirally reprogrammed into iPS cells via the retrovial induction of Oct4, Sox2, c-Myc and Klf4 expression as previously described Actively growing iPS cell lines were treated with 10 mM of BrdU (Sigma-Aldrich) for 14 hours followed by Karyomax Colcemid (GIBCO) for a further 4 hours to arrest cells in metaphase. The cells were then collected following trypsinization and lysed in 0.56% KCl before fixation in Methanol:Acetic Acid (3∶1). The cell nuclei were then dropped on glass slides from a height of 0.3 meters and stained with Lieshman Stain (Sigma-Aldrich) for chromosomal count analysis. At least 20 metaphase spreads were counted per cell line.6 iPS cells (at passage 3) in a 50 µl volume were injected into hind leg muscle of SCID mice using a 1 ml syringe fitted with a 26–31 needle. After 4 weeks, teratomas were dissected, fixed in 4% paraformaldehyde, embedded in paraffin, sectioned at 5.0 um and stained with haematoxylin and eosin (Sigma). Sections were examined on an Olympus BH2 microscope for cell representative of the three germ layer lineages and images acquired using ImagePro Plus software .Briefly, 1–2×10Embryoid bodies (EBs) were formed by the hanging drop method using differentiation medium containing DMEM, 15% ES-FCS (Invitrogen), 2 mM L-Glutamine, 100 uM non-essential amino acids (Invitrogen), 50 uM 2-mercaptoethanol (Sigma-Aldrich), 50 U/ml penicillin, 50 mg/ml streptomycin. Briefly, iPS cells were dissociated and 10 ul droplets (each containing 800 cells) were hung upside down for 2 days. After an additional 2 days in suspension on ultra-low-binding dishes (Corning) rounded EBs were seeded onto gelatin-coated dishes and media changed every second day for 14 days.2, balanced with N2 for 24 h) in an incubator under hypoxic or control conditions. At 24 h samples were collected as appropriate for RNA or protein extraction. A further series of samples were exposed to hypoxic conditions for 24 h and then returned to normoxia and cultured for a total of 14 days.Sixteen hours after EBs were seeded onto gelatin, cells were exposed to hypoxia . PCR arrays were performed on cDNA (SABiosciences). Real-time RT-PCR amplifications were performed with SYBR® Green PCR Master Mix (Applied Biosystems) with samples prepared in triplicate and gene expression normalized to 18S rRNA as an endogenous control . Primer sequences are detailed in Western blot analysis was performed on nuclear extracts . Nuclear fractions were concentrated then quantified using the Bradford assay. Thirty ug of protein was added to each lane and westerns performed with all samples on two separate occasions using HIF-1alpha (1∶1000 dilution) and beta-catenin (1∶2000 dilution)  for 16 h at 4°C. Nuclear loading controls were examined in separate experiments using an antibody directed to the histone associated protein RBAP46  at a dilution of 1∶1000. Secondary antibodies for anti- HIF-1alpha and beta-catenin were from BioRad (1∶2000 dilution) and Invitrogen for anti- RBAP46 (1∶10000 dilution), incubated for 1 hr at room temperature.Unpaired t-tests were used to compare individual means. Unless otherwise stated data are presented as mean ± standard deviation (SD). Statistical significance defined by P<0.05.Table S1Primer Sequences.(DOCX)Click here for additional data file.


A number of published in vitro studies also point toward a role for miRNAs in controlling chondrocyte gene expression and differentiation. However, information on miRNAs that may regulate a specific phase of chondrocyte differentiation  is lacking. To attempt to bridge this knowledge gap, we have investigated miRNA expression patterns in human embryonic cartilage tissue. Specifically, a developmental time point was selected, prior to endochondral ossification in the embryonic limb, to permit analysis of three distinct populations of chondrocytes. The location of chondroprogenitor cells, differentiated chondrocytes and hypertrophic chondrocytes in gestational day 54–56 human embryonic limb tissue sections was confirmed both histologically and by specific collagen expression patterns. Laser capture microdissection was utilized to separate the three chondrocyte populations and a miRNA profiling study was carried out using TaqMan® OpenArray® Human MicroRNA Panels (Applied Biosystems®). Here we report on abundantly expressed miRNAs in human embryonic cartilage tissue and, more importantly, we have identified miRNAs that are significantly differentially expressed between precursor, differentiated and hypertrophic chondrocytes by 2-fold or more. Some of the miRNAs identified in this study have been described in other aspects of cartilage or bone biology, while others have not yet been reported in chondrocytes. Finally, a bioinformatics approach was applied to begin to decipher developmental cellular pathways that may be regulated by groups of differentially expressed miRNAs during distinct stages of chondrogenesis. Data obtained from this work will serve as an important resource of information for the field of cartilage biology and will enhance our understanding of miRNA-driven mechanisms regulating cartilage and endochondral bone development, regeneration and repair.There is compelling  Development of cartilage tissue begins by the condensation of mesenchymal stem cells (MSCs) and subsequent differentiation of these cells toward the chondrocyte lineage. Cells within the cartilage anlagen proliferate and generate an extracellular matrix (ECM) rich in type II collagen and proteoglycans, thus permitting cartilage growth and subsequent limb formation It has been estimated that as many as 60% of coding genes are regulated by the family of small, non-coding RNAs called microRNAs (miRNAs) C. elegans in 1993, we now understand a lot about the physiological roles of miRNAs in the mammalian system with respect to controlling cell proliferation, lineage determination, differentiation, apoptosis, etc HDAC6 gene, which disrupts a miR-433 binding site, was reported to cause a dominant X-linked chondrodysplasia Since their discovery in in vivo. What is known, however, has predominantly come from studies in mice. Conditional knock-out of the pre-miRNA processing enzyme, Dicer, was shown to cause severe skeletal growth defects due to alterations in chondrocyte proliferation and hypertrophy in vitro. Among such studies, attempts have been made to define miRNA expression patterns and function during in vitro chondrogenesis of primary stem cells or precursor cell lines in vivo during growth plate or articular cartilage development, for example. To attempt to bridge this knowledge gap, we investigated differential miRNA expression patterns within human embryonic cartilage tissue. Specifically, we chose a time point of development prior to endochondral bone formation  where three populations of chondrocytes can be distinguished in tissue sections of the human embryonic limb. Utilizing laser capture microdissection, cartilage tissue containing either precursor chondrocytes, differentiated chondrocytes or hypertrophic chondrocytes was isolated and expression of miRNAs in each region was determined by TaqMan® OpenArray® analysis. Here, we report on those miRNAs that were found to be significantly differentially expressed between chondrocytes in vivo at these three specific stages of development. Data acquired from this study will be an important resource of information toward a better understanding of miRNA function in regulating cartilage and long bone development and disease. Additionally, findings from this research may aid toward the development of future miR-based tissue engineering strategies to promote articular cartilage or endochondral bone repair or regeneration.To date, there is limited information on the expression and function of specific miRNAs in cartilage development Human, normal embryonic tissue samples (limbs) at gestational day 54–56 were obtained from a tissue collection and distribution program at the Laboratory of Developmental Biology (LDB) within the Department of Pediatrics and Medicine at the University of Washington in Seattle. This service provides precisely-staged normal human embryonic tissue specimens to grant-funded researchers nationally and internationally Paraffin sections were treated with xylene, rehydrated through decreasing concentrations of ethanol, stained in Weigert's Hematoxylin for 5 min, washed in running water for 5 min and stained with 0.001% Fast Green for 3 min. Samples were then rinsed in 1% glacial acetic acid and stained in 0.1% Safranin O for 5 min. Samples were dehydrated and cleared by incubation in 95% alcohol, 100% alcohol and then xylene. For immunofluorescent antibody (Ab) staining, de-paraffinized sections were treated with 1% hyaluronidase (Sigma) for 30 min at 37°C. Sections were rinsed with 1× PBS and blocked with 10% goat serum for 1 h at room temperature and then incubated overnight at 4°C with the following primary antibodies: 1) a rabbit polyclonal “anti-IIA” antibody (Ab) that recognizes the exon 2-encoded cysteine-rich domain of the amino propeptide of type II procollagen (1/400 dilution) 2O, 0.02% Toluidine Blue (prepared in RNAse free H2O), two dH20 washes, 75% ethanol, 95% ethanol and 100% ethanol. Finally, sections were transferred to xylene for 3 min and air-dried. LCM was immediately carried out using the LMD7000 system (Leica Microsystems). Three cartilage regions of interest in each tissue section were selected using the system software resulting in a contact and contamination-free collection of tissue directly into one of the three sterile 0.5 mL Eppendorf caps containing 60 µl of lysis buffer from the RNA isolation kit of choice. This procedure was repeated for all tissue sections derived from one limb specimen  and laser-captured tissue was pooled together into the appropriate collection vial. In some cases, both limbs were obtained from the same embryo and tissue collected from sections of each limb was pooled and counted as one specimen. Subsequently, for each specimen (whether one or two limbs were received), three separate Eppendorf tubes containing tissue from the precursor chondrocyte (PC), differentiated chondrocyte (DC) or hypertrophic chondrocyte (HYP) regions were obtained. Note than PC, DC or HYP tissue was dissected from both the developing femur and tibia and pooled together. A total of nine independent specimens were processed for LCM. Total RNA, including small miRNAs, was isolated from laser capture microdissected tissue using either the Total RNA Purification Micro Kit (Norgen Biotek Corp.) or the mirVana™ miRNA Isolation Kit (Ambion®). Both assay kits were comparable with respect to the RNA yield (ranging from 15–100 ng/µL depending on the number of tissue sections pooled) as measured by spectrophotometry .Frozen blocks of human embryonic limb tissue were sectioned (20 µm) onto RNAse-free polyethylene naphthalate (PEN)-coated glass slides (Leica Microsystems) that are specifically designed for laser capture microdissection (LCM). Frozen sections were stored at minus 80°C for up to 48 h before being processed for LCM. Upon removal from cold storage, the tissue sections were exposed to 30 s incubations in cold 75% ethanol followed by 50% ethanol, 20% ethanol, dHFigure S1).The TaqMan® OpenArray® Human MicroRNA Panel enables simultaneous running of hundreds of TaqMan® MicroRNA Assays in a plate format on the Applied Biosystems OpenArray® Real-Time PCR system −ΔCt). The same list of most highly-expressed miRNAs was generated regardless of whether RNU44 or global mean normalization was used. Therefore, data on differentially-expressed miRNAs was calculated using RNU44 normalization. Using the normalized ΔCt values, the fold change between two groups of samples collected from two different cartilage regions was calculated as mean (2(−ΔCt1))/mean (2(−ΔCt2)). Significance Analysis of Microarrays (SAM) www.ncbi.nlm.nih.gov/geo/) and assigned the accession code: GSE49152.Expression data were processed using OpenArray® Real-Time qPCR Analysis Software . This software processed raw fluorescent signal and generated the cycle threshold (Ct) and cycle threshold confidence value for each assay within the array. A Ct confidence value threshold of 150 was used to identify assays with a reliable Ct value. After this filtering, Ct values were imported into the DataAssist™ software . The maximum allowable Ct value was set at 29. This Ct cut-off was chosen since OpenArray® reactions are carried out in small volumes (33 nL); a single molecule is more concentrated in a smaller reaction volume and amplifies sooner than it would from regular microplate qPCRs. Personal communication with Life Technologies technicians informed us that single copy numbers will produce, on average, a Ct value of 29 with TaqMan® assays. The correlation of Ct values between samples from the same cartilage region was examined to identify any samples with potential low data quality. For normalization using an endogenous control, the stability scores chart was used to identify which one of the three endogenous control RNAs  had the most stable expression in multiple samples. For global normalization, the average of Ct values for all the assays excluding the three endogenous controls was calculated and this value was used as the background setting. The Ct value of the selected background was subtracted from the Ct value of each assay to calculate the ΔCt values. To identify highest-expressed miRNAs, the average normalized expression level of a miRNA from all samples was calculated , potential target genes of miRNAs within each comparison group were predicted using three algorithms  COL2A1 gene Human embryonic limbs at day 54–56 of gestational development were chosen for these studies to separate three specific regions containing chondrocytes at distinct stages of differentiation. Laser capture microdissection of tissue sections from nine independent limb specimens was successfully carried out as shown in  and From the OpenArray® expression data, we identified miRNAs that were differentially expressed by 2 fold or more between: 1) PC and DC Table 2; and , , , , , , see S4.Potential target genes were identified for each miRNA listed in in vivo expression patterns of miRNAs in chondrocytes from human embryonic cartilage tissue in the developing limb. The significance of this work is further highlighted by the fact that three distinct regions of the developing limb were separated by laser capture microdissection to permit analysis of miRNA expression in precursor chondrocytes (PC), differentiated chondrocytes (DC) or hypertrophic chondrocytes (HYP) of the femur and tibia. While other studies have been carried out to identify mRNA or miRNA expression in chondrocytes from different sites of mouse or chicken cartilage tissue, either late-stage embryonic, neonatal or post-natal tissue was utilized This novel study is the first to report on the et al reported higher levels of miR-140 expression in proliferating chondrocytes of mature  murine growth plates compared to hypertrophic chondrocytes et al detected the larger primary precursor form of miR-140 (pri-miR-140) in murine growth plate sections by in situ hybridization that includes both 5p and 3p strand; different expression patterns may be obtained by specifically detecting either the mature 5p or 3p strand in vivo. We also detected expression of miR-140-3p in all three regions of developing cartilage, albeit at lower levels than miR-140-5p . However, two studies have reported higher expression of the 3p strand in mature rat or neonatal murine epiphyseal growth plate cartilage The miRNA expression database was obtained using Human MicroRNA OpenArray® Panels on the OpenArray® Real-Time PCR system  and Other highly-expressed miRNAs include those of the miR-17∼92 cluster  Table 1., , HOX gene clusters and can regulate expression of some of these patterning genes ERG transcription factors ERG is specifically localized to developing articular cartilage of the joint and functions in regulating proper cartilage development in vitro.A number of miRNAs identified in this study as significantly differentially expressed between precursor, differentiated and hypertrophic chondrocytes have also been described in other reports within the context of skeletal development or cartilage biology. miR-196b expression patterns indicate a potential role in regulating specific phases of chondrogenesis given that levels were found to be higher in precursor or differentiated chondrocytes , , 4. IHDAC6 was identified within a miR-433 binding site , , –In vitro studies showed that miR-675 indirectly affected levels of COL2A1 in differentiated human articular chondrocytes et al reported that expression of this miRNA was elevated in human OA as was the long non-coding RNA, H19, which harbors miR-675 et al also reported elevated IGF2 levels in OA cartilage while levels of miR-483 were not described in this study. However, other studies have reported increased levels of miR-483-5p in OA cartilage  and ). Also, expression levels of the 5p and 3p strands of miR-483 in PC and DC regions were similar suggesting that both mature strands are functional in chondrocytes. Whether these 5p and 3p strands have distinct functions in chondrocytes remains to be determined.Another miRNA identified as over 2-fold more highly expressed in PC compared to HYP is miR-433 Table 3. and Expression of miR-146a has also been reported in OA chondrocytes; mechanistically, miR-146a is apparently responsive to IL-1β signaling and may be involved in pain-related pathophysiology of OA  and  and  and Among those miRNAs more highly expressed in differentiated or hypertrophic chondrocytes when compared to precursor chondrocytes, it is interesting that two of these, miRs-365 and 222  and 3, have, , et almiR-335-5p and the less abundant 3p strand (miR-335*) showed significantly high fold changes in differential expression patterns in our studies , , 4. A and This study is the first to report miR-138 in cartilage and that higher expression is associated with differentiated and hypertrophic chondrocytes when compared to precursor cells . Also, the degree of miRNA repression on an individual target is usually very mild miRNA function in cartilage development and homeostasis is becoming a rapidly-growing area of research in vivo and in vitro data to be important within the context of chondrogenesis. Other significantly enriched pathways were identified from our bioinformatics approach , but making any associations with other cellular processes at this stage would be over-interpreting the data presented in this study. With respect to those developmental pathways identified  or an adjusted protocol (protocol 1) to accommodate lower concentrations of sample RNA. See Materials and Methods section for a description of what aspects of the sample preparation protocol were changed.(EPS)Click here for additional data file.Table S1Genes predicted to be targeted by one or more miRNAs identified in (XLSX)Click here for additional data file.Table S2Genes predicted to be targeted by one or more miRNAs in (XLSX)Click here for additional data file.Table S3Genes predicted to be targeted by one or more miRNAs identified in (XLSX)Click here for additional data file.Table S4Genes predicted to be targeted by one or more miRNAs identified in (XLSX)Click here for additional data file.


A large body of evidence has implicated amyloid precursor protein (APP) and itsproteolytic derivatives as key players in the physiological context of neuronalsynaptogenesis and synapse maintenance, as well as in the pathology ofAlzheimer's Disease (AD). Although APP processing and release are known tooccur in response to neuronal stimulation, the exact mechanism by which APPreaches the neuronal surface is unclear. We now demonstrate that a small butrelevant number of synaptic vesicles contain APP, which can be released duringneuronal activity, and most likely represent the major exocytic pathway of APP.This novel finding leads us to propose a revised model of presynaptic APPtrafficking that reconciles existing knowledge on APP with our presentunderstanding of vesicular release and recycling. Amyloid precursor protein (APP) is a type 1 membrane-spanning protein ofapproximately 120 kDa, which is ubiquitously expressed in mammalian cells. Theprotein has received particular attention because of its role in the nervous system.Under normal physiological conditions APP is involved in synapse formation andfunction. However, it is the involvement of this protein in the pathology ofAlzheimer's disease that has raised particular interest. Alzheimer'sdisease is the most prevalent neurodegenerative disease facing western populations.Dysregulation of APP trafficking is thought to play a central role in theprogression of this condition, because formation of the senile plaquescharacteristic of the disease is intimately linked to APP metabolism As such there is considerable interest in understanding both the molecular processingand trafficking pathways of this protein in neurons. Indeed over the last twentyyears, these problems have formed the basis of intensive investigations by a largenumber of groups. Unfortunately, while the molecular events surrounding APPprocessing have been comprehensively elucidated, the cellular mechanisms regulatingits intracellular trafficking in neurons remain unclear.in vivo microdialysis experiments with knowledge of APPtransport in tissue culture cells to present a model of synaptic traffickingconsistent with the known molecular processing of this protein. In this model,full-length APP is constitutively transported from the ER-Golgi network to the cellsurface, where cleavage by α-secretase results in the release of a 100–110kDa soluble fragment (sAPPα), which plays a crucial role in synapse formationand maintenance To date, the most widely accepted model of APP trafficking in the presynapticterminal is one put forward by Cirrito and colleagues Unfortunately, investigating the trafficking of APP in neurons, in particular itsrole within the secretory apparatus, has always been complicated by two majorfactors. First, endogenous APP is expressed at very low levels, typically at thelimit of assay sensitivity, in both rat and mouse neurons Recently, however, Frykman and colleagues found that γ-secretase is highlyenriched in a crude synaptic vesicle fraction, together with APP cleavage productsFull details of all procedures (including antibodies) can be found in Synaptic vesicles were purified according to standard protocols Approximately 10–20 µg of synaptic vesicle proteins wereseparated by 1D SDS-PAGE using a tricine mini-gel Proteins were separated using a tricine based gel system (as above). Proteinswere then transferred to nitrocellulose membrane using standard semi-drytechniques Electron microscopy was performed as previously described with minormodifications in vitro (DIV) using amodified calcium phosphate transfection procedure. Microscopy was performedat 14–21 DIV Hippocampal neurons of the CA3/CA1 region from 1- to 3 day old Wistar ratswere prepared in a sparse culture ,and transfected after 3–4 days Cells were covered with 20 µl of buffer and sealed with a secondcoverslip coated with sub-resolution red fluorescent beads for focaladjustments . Thespace between the two coverslips was always less than 30 µm. Imageswere obtained with a commercial 4Pi microscope .5′-cctgaggcggatcttccactcgcacac-3′; thereverse primer sequence was 5′-gcggccgcgtcaaaagccgagggtgagtaaat-3′. Theintegrity of the pHluorin-APP construct was verified by sequencing.A pHluorin-synaptotagmin-1 vector construct 2, 2.5 CaCl2, 10 glucose, 18 NaHCO3;pH 7.4 was maintained using 5% atmospheric CO2. The cellswere then washed twice and placed in a perfusion chamber containing Tyrodesolution for imaging.Antibody labeling was performed with antibodies against synaptotagminconjugated to cypHer5E. Labeling was performed on pHluorin-APP transfectedhippocampal neurons, by incubating the neurons with antibody for 3–4hours at 37° in a bicarbonate buffer containing (in mM) 120 NaCl, 5 KCl,1 MgCl2, 2 CaCl2, 10glucose, 10 HEPES buffer, pH 7.4 NaOH) was used for all experiments unlessotherwise indicated. Synaptic boutons were stimulated by electric fieldstimulation . 10 µM CNQX and 50 µM AP5 were added tothe bath solution to prevent recurrent synaptic activity as a result of AMPAreceptor activation. Fast solution exchanges were achieved using apiezo-controlled stepper device, with a three-barrel glass tubing. Theperfusion rate during the experiments was kept at a constant 1 ml/min. Toblock reacidification of freshly recycled synaptic vesicles, 65 nM folimycinwas applied to the neuronal culture before the experiment. For dequenchingof vesicular pHAPP, ammonium chloride solution (pH 7.4) was prepared byequimolar substitution of 50 mM NH4Cl for NaCl in the Tyrodesolution. All other components remained unchanged.Imaging was performed as described previously Imaging was performed using a cooled CCD camera mounted on a Zeiss Axiovert135TV microscope equipped with a 60×, 1.2 NA water-immersion objectiveand a FITC/Cy5 dual-band filter set. Excitation wavelengths of 480 nm(pHAPP) and 640 nm (cypHer) were produced by a computer-controlledmonochromator.All tissues used in this study were obtained from Wistar rats , bred and kept for experimental purposes at the Max Planck Institute forBiophysical Chemistry by authorization of the federal state of Lower Saxony,Germany (licence Az. 32.22/Vo). The killing of rats for tissue preparations isnot an animal experiment under the terms of federal animal protections laws, soapproval by an ethics committee was not necessary. However, all experimentsinvolving the use of animals were carried out following the stringent guidelinesissued by the Max Planck Institute, to ensure the highest standards of animalwelfare.Although our laboratories recently completed a detailed proteomic analysis of thesynaptic vesicle, several known (membrane) proteins were not identified,including the chloride channels ClC3 and ClC7, as well as the vesicularneurotransmitter transporters responsible for monoamine (VMAT) and acetylcholine(VAChT) uptake into vesicles Given that APP is a large protein, containing a large number of potential trypsincleavage sites and is reasonably hydrophilic (as judged by its GRAVY score), wehypothesized that APP is difficult to detect in synaptic vesicles bymass-spectrometry because it is present in low amounts, consistent with previousstudies, which found low levels of APP in rodent brain 1/2 1 hr). ImmatureAPP is localized exclusively to the ER, and only mature APP that has been N- andO-glycosylated ever leaves the ER/Golgi compartment(s), via sorting at the TGNThe predominant fraction of intracellular APP is thought to be localized to theendoplasmic reticulum, Golgi apparatus and early endosomes; the localization ofAPP to these biosynthetic organelles being partly explained by the very highrate of synthesis and turnover of this protein   . The APPAlthough our biochemical and nanoscopy data are in agreement with previous workwhole ratbrain, it is possible that our results actually under-represent the number ofAPP containing vesicles in distinct neuronal regions, such as the hippocampusWhen considering that the vesicle fraction we used for our biochemical analysisrepresents an ensemble of vesicles isolated from Although immunoblotting and mass spectrometry indicate that neuronal clathrinlight chain is absent from our synaptic vesicle preparation , we coulbona fide synaptic vesicles should undergostimulation-dependent secretion from neurons. Therefore, we were interested toknow how APP-positive vesicles behave under physiological conditions; forinstance, do these vesicles undergo exocytosis, or are they refractory torelease? Answering this question required the use of an assay that alloweddirect visualization of exocytosis in the nerve terminal. For this reason, weagain turned to cultured hippocampal neurons, which have been extensively usedfor the study of synaptic transmission using optical methods We reasoned that Hippocampal neurons in culture that express pHAPP show very little surfacefluorescence ,althougUsing this dual labeling strategy, active boutons could be unequivocallyidentified by the uptake of the cypHer 5 labeled antibody against synaptotagmin1(αSyt1-cypHer) during vesicle recycling. We thus defined active synapticboutons as αSyt1-cypHer positive regions and analyzed the corresponding lociin the pHAPP channel. In resting neurons, APP surface fluorescence wasco-localized to synaptic boutons labeled with αSyt1-cypHer in only a limitednumber of regions . However4’) While the above data proves that at least a fraction of full-length pHAPPrecycles with synaptic vesicles it cannot be discounted that some pHAPP iscleaved during vesicle cycling, particularly considering that the processingenzymes BACE and Presenilin 1 co-purify with synaptic vesicles . If APP In this work we reinvestigated the subcellular localization of APP in neurons.Using techniques that allowed direct access to presynaptic mechanisms, weconsistently found that small amounts of APP are present in synaptic vesicles,which undergo activity dependent secretion.bonafide functional components functionalpools of vesicles in the synaptic terminal .At first glance, our findings might seem controversial; over the past decade ithas become dogma that APP is absent from synaptic vesicles, and this has heavilyinfluenced the prevailing view of APP trafficking in neurons. However, when werevisited the original literature, we found our results to be entirelyconsistent with earlier work. In these studies, immunoblotting showed a smallfraction of APP was found in isolated synaptic vesicles, and partialco-localization of APP and synaptophysin was demonstrated at the lightmicroscopy level An important remaining question concerns how much APP is actually released byexocytosis of synaptic vesicles. Previously, it was thought that theAβ-peptides formed from APP processing were secreted from exosomes in theneuron. However, this form of release was found to account for only 1% ofthe total peptide secretion Identifying the synaptic vesicle as a secretory organelle for APP closes thetrafficking cycle for this protein at the synapse. In Our schematic attempts to illustrate more clearly the similarities andinterconnectedness of the trafficking pathways. In particular, we propose thatthe presynaptic endosome plays a crucial role in linking synaptic vesicle andAPP cycling.2+ mediated fusion withthe plasma membrane, in response to neuronal stimulation. Following exocytosis,vesicles are retrieved from the plasma membrane by endocytosis. Underphysiological conditions this is thought to occur via a clathrin-mediated Synaptic vesicles may recycle through one of several pathways in the presynapticterminal. Synaptic vesicle precursors are brought to the presynaptic terminal intransport vesicles. It is thought that these transport vesicles undergo a roundof fusion with the plasma membrane followed by retrieval and sorting, possiblyin an endosomal intermediate to form fully functional synaptic vesicles bona fidesynaptic vesicle components such as Rab3a and synaptobrevin 2 bona fide synaptic vesicle proteinsAPP is also thought to be trafficked to the presynaptic terminal in transportvesicles, which interestingly seem to contain Obviously, APP needs to be proteolytically processed at some point duringtrafficking. In this respect, it is interesting that our data shows pHAPPfluorescence decreased significantly after neuronal stimulation, consistent withproteolytic cleavage of the construct .One possible explanation is cleavage of APP at the plasma membrane surface byα-secretase. An alternative explanation, however, is that APP is processedto Aβ in synaptic vesicles, a proposal which is supported by complementarydata from Frkyman and colleagues who recently found Aβ using biochemicalmethods in a somewhat cruder preparation of isolated synaptic vesicles 2+ influx into thedendritic spine, with subsequent spine shrinkage and retraction leading to anoverall reduction in neuronal spine density. These effects promote long-termdepression (LTD), inhibiting the induction of long-term potentiation (LTP) byNMDA receptor-dependent signaling. As LTP is thought to be the neural correlateof learning and memory, this would explain why Aβ can produce memoryimpairment when overproduced Conceptually, exocytic release of APP (derivatives) from synaptic vesicles is anattractive possibility; modulation of synaptic function, particularly by Aβ,would be possible over a time course of seconds to minutes, in direct responseto alterations in neuronal activity. Furthermore, it appears that APP does nothave to be specifically enriched in synaptic vesicles to achieve such effects.There is increasing evidence that even picomolar concentrations of low n-numberoligomers (particularly trimers) of Aβ rapidly and effectively inhibit NMDAreceptor activity, leading to reduced CaThe synaptic depressing effects of Aβ are interesting when coupled tofindings indicating that increased neural activity can drive the processing ofAPP to Aβ In the future, it will be interesting to delineate the exact mechanisms whichcouple synaptic vesicle recycling with APP trafficking, processing and secretion– with a view to developing more effective therapeutic strategies. Indeed,pharmacologic modulation of synaptic transmission Figure S1APP is localized to synaptic vesicles as shown by immunogold electronmicroscopy. Top; synaptic vesicles isolated from rat brain wereimmunolabeled for synaptophysin, APP or double labeled , before viewing by negative stain electron microscopy.The low magnification images show the synaptic vesicle preparation toconsist solely of small, homogeneously shaped vesicles, with diameters inthe range of 40–50 nm. The insets show higher magnification images ofvesicles from the same field of view. Single labeling resulted in 99%of all vesicles immunopositive for synaptophysin (as previously reported)and 10% of all vesicles immunopositive for APP. Similar results wereobtained with double labeling. Bottom; negative control experiments in whichthe primary antibody was omitted. For single labeling experimentsn = 3; for double labeling experimentn = 2. Scale bars, low magnification 100 nm; highmagnification 50 nm.(TIF)Click here for additional data file.Figure S2The use of pH sensitive probes to monitor exo- and endocytosis.A) Schematic illustrating the design of the pHAPP construct and its use inmonitoring synaptic activity. pHluorins are pH sensitive variants of GFPwhich can be tagged to specific synaptic vesicle proteins and used toquantify synaptic vesicle exo- and endocytosis. When pHluorin is attachedN-terminally to APP, it is directed towards the lumen of the synapticterminal, which is acidic under resting conditions. Thus the pHluorin willbe quenched . During neuronal stimulation,synaptic vesicles undergo fusion with the plasma membrane, and the luminalsurface becomes exposed to the more alkaline pH of the external culturemedia, and the fluorescence of the pHluorin increases(‘Exocytosis’). Following exocytosis the fluorescence signal isreduced, either due to compensatory endocytosis , or from loss of the N-terminaltag into the culture media as a result of proteolytic processing(‘Cleavage’). Ammonium chloride is membrane permeable and willneutralize the pH of the vesicle lumen; hence, ammonium chloride can be usedto report the entire pHluorin content of the synaptic terminal(‘NH4’). B) Schematic illustrating the use ofαSyt1-cypHer antibodies to monitor synaptic activity. Followingexocytosis, the intravesicular domain of synaptotagmin 1 is exposed to theexternal culture media and can be labeled with an antibody, which isinternalized when the vesicles are retrieved. This antibody is directlyconjugated to the dye cypHer 5. CypHer fluorescence shows an inverse profileto that of pHluorin, being fluorescent only in the acidic environment of thevesicle . Following exocytosis, cypHerfluorescence is quenched in the alkaline pH of the culture media(‘Exocytosis’). Following endocytosis, cypHer fluorescenceincreases as the reformed vesicle is reacidified.(TIF)Click here for additional data file.Methods S1(DOC)Click here for additional data file.


To develop a method for generating high spatial resolution (10 µm) matrix-assisted laser desorption ionization (MALDI) images of lipids in rodent optic nerve tissue.Ice-embedded optic nerve tissue from rats and mice were cryosectioned across the coronal and sagittal axes of the nerve fiber. Sections were thaw mounted on gold-coated MALDI plates and were washed with ammonium acetate to remove biologic salts before being coated in 2,5-dihydroxybenzoic acid by sublimation. MALDI images were generated in positive and negative ion modes at 10 µm spatial resolution. Lipid identification was performed with a high mass resolution Fourier transform ion cyclotron resonance mass spectrometer.Several lipid species were observed with high signal intensity in MALDI images of optic nerve tissue. Several lipids were localized to specific structures including in the meninges surrounding the optic nerve and in the central neuronal tissue. Specifically, phosphatidylcholine species were observed throughout the nerve tissue in positive ion mode while sulfatide species were observed in high abundance in the meninges surrounding the optic nerve in negative ion mode. Accurate mass measurements and fragmentation using sustained off-resonance irradiation with a high mass resolution Fourier transform ion cyclotron resonance mass spectrometer instrument allowed for identification of lipid species present in the small structure of the optic nerve directly from tissue sections.An optimized sample preparation method provides excellent sensitivity for lipid species present within optic nerve tissue. This allowed the laser spot size and fluence to be reduced to obtain a high spatial resolution of 10 µm. This new imaging modality can now be applied to determine spatial and molecular changes in optic nerve tissue with disease. The optic nerve is the conduit for transmitting visual signals along retinal ganglion cell axons to multiple sub-cortical nuclei involved in various vision-related tasks . These aMatrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) is a novel imaging modality capable of providing molecular ion maps across tissue surfaces including maps of lipids, proteins, and metabolites -12. In aSeveral recent publications have demonstrated high spatial resolution mass spectrometry imaging of fatty acids, lipids, and proteins with varying sample preparation methods ,22-25. MThis study was conducted in accordance with regulations set forth in the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Animal protocols were approved by the Institutional Animal Care and Use Committee of the Vanderbilt University Medical Center in accordance with guidelines published in the US Public Health Service Policy on Humane Care and Use of Laboratory Animals.Whole eyes with the optic nerve attached were removed from Brown Norway rats and C57 mice following cervical dislocation and rapidly frozen in deionized water by placing the eye tissue into a cup fashioned from parafilm that was floated in a weighing boat on top of liquid nitrogen in a polystyrene container. Care was taken to orient the optic nerve so that it could be sectioned along the sagittal axis of the nerve. The ice-embedded sample was then mounted using Tissue-Tek OCT compound . Twelve-micron sections were cut with a Leica CM3050S cryostat  and thaw mounted onto AB Sciex  gold-coated MALDI target plates. The sections were dehydrated in a vacuum desiccator overnight and then washed three times for 30 s each with 100 mM ammonium acetate  before being dried again for another 2 h. 2,5-Dihydroxybenzoic acid  was deposited by sublimation onto the samples using a custom sublimation apparatus for 20 min at 54 mTorr pressure at 120 °C. The region containing the optic nerve was imaged in positive and negative ion modes for mouse tissue and positive ion mode for rat tissue using a MALDI-TOF (time-of-flight) mass spectrometer  equipped with a Smartbeam II 1 kHz Nd:YAG frequency tripled to 355 nm wavelength. The laser was set to the minimum spot size with 8% laser power. External calibration was performed with a series of phosphorus clusters before imaging data were acquired , and intn, n=5–65) resulting in mass accuracies of better than 2 ppm for all analytes. Tandem mass spectrometry experiments were performed using a combination of continuous accumulation of selected ions (CASI) and sustained off-resonance irradiation collision-induced dissociation (SORI-CID) [Lipid identification from the mouse optic nerve tissue was performed using information from two experiments: 1) accurate mass profile scans and 2) tandem mass spectrometry. Both experiments were conducted on tissue sections taken adjacent to those imaged. These data were collected using a MALDI FT-ICR mass spectrometer  equipped with an Apollo II dual ion source and the same 355 nm solid-state laser as described above. External calibration was done using a series of phosphorus clusters  . CASI (5ORI-CID) ,32, and Intense signals were obtained from the meninges surrounding the optic nerve and from the internal nerve tissue using the sublimated DHB matrix. +), ethylene phosphate , and lithium ethylene phosphate  are characteristic peaks associated with all glycerophosphocholines [sn-1 position is known to be preferentially lost over the sn-2 fatty acyl chain during fragmentation [sn-1 position.cholines ,34. Addientation ,34. The 48H92NO11S, sulfatide ; To image coronal sections, a slightly larger rat optic nerve was examined. Using high spatial resolution (10×10 μm) MALDI imaging mass spectrometry, we demonstrated localized and identified lipid species and their relative abundances within small structures of mouse and rat optic nerve tissue of 200–600 μm diameter. The sample preparation method described allowed images to be acquired at 10 μm spatial resolution. Images acquired in positive ion mode provided the distribution of several phosphatidylcholine species and one sphingomyelin associated with the internal axon fibers and surrounding meninges of the optic nerve  laser spot to be used. This method allows for reliable imaging analysis and identification of endogenous lipids within small tissue structures. Selective ion accumulation in the hexapole region of the FT-ICR using the CASI method before fragmentation made lipid identification easier as the signal intensity for the fragments were significantly improved. In conclusion, the MALDI-IMS technology shows great promise for generating spatial and molecular information in diseases affecting optic nerve structure and function, leading to information that can be used to understand molecular mechanisms of disease and to develop new therapeutic strategies.


E. coli K 12 MG1655 grown on LB medium supplemented with glycerol. Global gene expression analysis indicated that the expressions of hundred genes were significantly altered in simulated microgravity conditions compared to that of normal gravity conditions. Under these conditions genes coding for adaptation to stress are up regulated (sufE and ssrA) and simultaneously genes coding for membrane transporters  and carbohydrate catabolic processes  are down regulated. The enhanced growth in simulated gravity conditions may be because of the adequate supply of energy/reducing equivalents and up regulation of genes involved in DNA replication (srmB) and repression of the genes encoding for nucleoside metabolism . In addition, E. coli cultured in LB medium supplemented with glycerol (so as to protect the cells from freezing temperatures) do not exhibit multiple stress responses that are normally observed when cells are exposed to microgravity in LB medium without glycerol.This study demonstrates the effects of simulated microgravity on  Microbes have the ability to sense and respond to environmental changes occurring in their vicinity. This adaptability confers on them the capacity to thrive under various extreme environmental niches including microgravity. When exposed to microgravity cells experience reduced gravity resulting in a relative lack of sedimentation, low shear stress and low turbulence Escherichia coli strains as a model organism have provided insights in to the effects of microgravity at the genome level. Tucker et al., E. coli under microgravity conditions varied depending on whether the culture was grown on minimal medium or rich medium like Luria-Bertani (LB) broth E. coli K 12 identified a number of genes that were significantly altered in expression under microgravity conditions Escherichia coli it was clearly demonstrated that microgravity induced expression of multiple stress genes depends on the nutritional status and nutrient resources modulate the responses Salmonella typhimurium microgravity decreased the virulence of the bacteria if LB medium was supplemented with phosphate ions Functional genomic technologies E. coli K12 MG1655 grown in LB medium supplemented with glycerol to monitor the effects on growth and global gene expression. The need for supplementing LB with glycerol was crucial because the incubator in which the bacteria is proposed to be grown at 30°C in space, under microgravity conditions, in a space recovery capsule due to unforeseen reasons may fail and the incubation temperature may decrease to below 0°C. Such unforeseen failures are not very uncommon in space flights. Haga and Saleh In this study, a clinostat that mimics microgravity conditions was used to investigate the effects of microgravity on The present study differs from the earlier studies in that the LB medium used in the present study is supplemented with glycerol. There is a reasonable chance to assume that the response of the bacterium to microgravity may vary with respect to growth and expression of genes. These assumptions are based on the earlier findings that media components influence the growth and expression of genes of bacteria exposed to microgravity E. coli exhibits enhanced growth rate and that several genes are differentially expressed when exposed to microgravity The results of the present study is in accordance with earlier studies that had indicated that E. coli under microgravity may be because of adequate supply of energy/reducing equivalents and increase in transcripts for DNA replication.Further, the genes that facilitate the formation of the nutrient depletion zone in cells grown under microgravity conditions such as up regulation of genes coding for nutrient transport and metabolic enzymes and simultaneously down regulation of genes associated with translation apparatus, DNA replication and cell division yfjD, ydcQ, ynfA, ybdJ, yniB, ygaQ and hokE were assigned to various functional processes in response to microgravity conditions in E. coli.This study also for the first time assigns the differentially regulated genes to sixteen different functional pathways such as several metabolic pathways , ABC transporter systems, two component systems etc., by KEGG (Kyoto Encyclopedia of Genes and Genomes). In addition, DAVID  was used for geneontology annotations and the analysis yielded up regulated genes involved in DNA transcription and regulation of transcription, genes coding for integral membrane proteins, ion binding and DNA binding etc. The 21 down regulated genes were implicated in nitrogen and amine compound biosynthetic process, ion transport, carbohydrate catabolic process and nucleoside metabolic process. DAVID analysis also annotated genes involved in cellular components such as cell wall, cell membrane and organelle membrane and seven hypothetical protein coding genes namely Escherichia coli K12 MG1655 was grown in LB broth  which was supplemented with 10% (v/v) glycerol (hereafter LB) at 30°C. To develop the inoculums, a single colony of E. coli K 12 MG1655 from the LB agar plate was inoculated into LB broth and incubated for growth under shaking at 150 rpm and at 30°C. E. coli growth was monitored by measuring absorbance at 600 nm. E. coli grown to a OD of 0.8 (106 CFU/ml) was used as inoculum for all the experiments unless otherwise mentioned.In this study, 2, R = radius from the centre of rotation, ω = constant angular velocity (ω) where angular velocity is equal to angular displacement in radians/time taken (θ/τ). The angular velocity obtained using the clinostat was 2 rads/s. At this angular velocity, the simulated microgravity is 1×10−3−3.A three-dimensional (3D) clinostat is an apparatus that nullifies the effect of gravity, and it has been used to evaluate the effects of microgravity on cells E. coli culture (0.8 OD) was added and 15 ml of this inoculum was distributed into 15 ml separate vessels and grown in clinostat at 30°C. Under these conditions the g was 1×10−3g. After every one hour interval one vessel was detached from the instrument and OD600nm of the culture was determined and used to construct the growth curves. E. coli culture that was setup as above and grown in an incubator shaker at 30°C under normal gravity conditions served as control. Data of three experiments that were conducted under similar conditions separately was considered in plotting the growth curves.To 300 ml of LB medium 3.0 ml of logarithmically growing E. coli culture and grown in a 50 ml vessel in a clinostat at 30°C. A control was set up as above and grown under normal gravity conditions in an incubator shaker at 30°C. Three separate vessels were maintained under each condition. When the OD600nm reached 0.8, 10 ml of the culture collected from each of these vessels was suspended in 3 volumes of RNAlater solution  in their respective sample bottles and stored at 4°C. From the stored culture 3 ml was used for the isolation of RNA.For simulated microgravity studies, 50 ml of the LB broth was inoculated with 500 µl of logarithmically growing E. coli culture. The culture was pelleted, suspended in 200 µl Tris-lysozymemixture at room temperature for 5 min and then subjected to lysis with 700 µl of lysis buffer containing 1% β-mercaptoethanol. Lysis was facilitated by vigorous vortexing the suspension of cells for 5 min. Absolute ethanol (500 µl) was added and the mixture was transferred to a mini-spin column placed in a 2 ml centrifuge tube and centrifuged at 13,000 rpm for 30 s. At this stage, 10 µl of DNase prepared in 90 µl of buffer was added to the columns and the columns were kept at room temperature for 10 min. The columns were then washed at 13,000 rpm for 30 s with 500 µl of wash buffers RW1 and purified RNA was collected in 20 µl of RNase-free water after centrifugation at 13,000 rpm for 2 min. Quality of the purified RNA was assessed following gel electrophoresis and the OD 260/280 ratio of the purified RNA ranged from 1.8–2.0. Quantification of the RNA was done using a Nanodrop spectrophotometer .Qiagen RNeasy mini-prep kit was used for the extraction of RNA from about 3 ml of RNA (5–10 µg) extracted as above was used for cDNA synthesis using the first strand cDNA synthesis kit from Invitrogen . Annealing of the primers with the purified RNA was accomplished by incubating the RNA and primer reaction mix at 70°C for 10 min followed by incubation at 25°C for 10 min. cDNA was then synthesized by adding superscript III and incubating the reaction mixture at 25°C for 10 min, 37°C for 60 min, 42°C for 60 min and 70°C for 10 min. The cDNA was fragmented with DNAse 1  and then labelled with biotin at the 3′ end using the labelling reagent from Affymetrix  and Terminal transferase enzyme (Promega).E.coli Genome 2.0 gene chip arrays were purchased from Affymetrix. The chip contained the complete genome of four E. coli strains . The gene chip consists of approximately 10,000 probe sets for the 20,366 genes of all the four strains of E. coli. DNA micro array chips were hybridized with the labelled cDNA using the Affymetrix protocol (www.affymetrix.com). Microarray slides were then scanned using Affymetrix 428 Array Scanner and GCOS software to obtain images of the chips and further processed to get intensity cell files for the probe sets. The intensity cell files were then imported normalized for background correction and data analysed using Gene Spring 11.5 software. Genes that exhibited ≥1.5 fold increase or decrease (treated versus control) in expression and P≤0.05 were considered as differentially regulated due to microgravity.The microarray data was submitted to Gene Expression Omnibus (GEO) web deposit of National Centre for Biotechnology Information (NCBI) with an accession number GSE40648.T method which is based on product cycle threshold (CT). Expression of 16S rRNA gene was used as an internal standard for RT-PCR. All values reported represent the mean of at least three independent experiments.The RT-PCR reactions (10 µl) were performed in triplicate with 2.5 pM primer  and SYBEGenes identified by the microarray analysis were analyzed to identify relevant functional pathways by KEGG (Kyoto Encyclopedia of Genes and Genomes) and DAVID . A cutoff p value of 0.05 was used for enriched KEGG pathways and geneontology functions by DAVID. Out of 100 genes that were differentially regulated only 57 genes were considered for analysis using KEGG and DAVID. The remaining 43 genes that coded for unknown or intergenic regions were not included in the analysis.E. coli was monitored under both simulated microgravity conditions and normal gravity conditions. Cultures grown in the clinostat exhibited enhanced growth rate and reached the stationary phase earlier than the culture grown at normal gravity . Three chips were used for control and experimental conditions respectively. Fifty three genes were up regulated while forty seven genes were down regulated under simulated microgravity conditions (srmB), multidrug efflux system protein (mdtD/yegB), cysteine desulfuration protein involved in oxidative stress (sufE/ynhA), periplasmic proteins involved in metal detoxification (cusF), chaperone for hydrogenase isoenzyme (hyaE), a DNA binding transcriptional regulator (IlvY), non coding RNA genes (ryjA and ssrA), one pseudo gene (intK), twelve hypothetical proteins , thirty one intergenic region genes and one gene coding for an unknown protein (In nditions . The up  protein .ompC), his operon genes , insertion element repressor protein (insA), lysine decarboxylase (ldcC), transporter proteins , 3′-phosphoadenosine 5′-phosphosulphate reductase , metal transport proteins (mgtA and nikB), fused predicted PTS system enzyme (ptsA), glutathionine S-transferase (gst), rhamulose-1-phosphate aldolase (rhaD), transcriptional activator (rhaS), putative transcriptional regulatory protein (ydcR and yeaM), transthyretin-like protein (hiuH), a bifunctional synthase (dfp), bifunctional (p)ppGpp synthetase II/guanosine-3′,5′-bis pyrophosphate 3,-pyrophosphohydrolase (spoT), dihydro-orotate oxidase (pyrD), lysophospholipase L2 (pldB), agmatinase (speB) and xylosidase (yicL). The other down regulated genes include two pseudo genes , seven hypothetical protein coding genes , one unknown gene and ten intergenic region coding genes  and the results indicated that, pyrD and rhaD (P<0.001), yicL, (P<0.05), hyaE, ldcC, mdtD and srmB (P<0.1) were either significantly up regulated or down regulated in accordance with the DNA microarray results (E. coli exposed to simulated microgravity (T) value. The data was analysed using ANOVA (Prism 3.0 software).RT-PCR was carried out on seven genes , the two-component system (cusF and ompC), purine metabolism (hiuH and spoT), microbial metabolism in diverse environments , biosynthesis of secondary metabolites (hisA and ldcC), ABC transporters (cysW and nikB), glycerophospholipid metabolism (pldB), phosphotransferase system (ptsA), pantothenate and CoA biosynthesis (dfp), pyrimidine metabolism (pyrD), fructose and mannose metabolism (rhaD), pentose and glucuronate interconversion (rhaD), arginine and proline metabolism (speB), histidine metabolism (hisA), lysine degradation (ldcC) and sulfur metabolism (cysH). The KEGG pathway analysis also indicated that certain genes like cysH, hisA, hiuH, ldcC, pyrD, speB, and rhaD were associated with more than one pathway.Out of 100 genes that were differentially regulated only 57 genes were considered for analysis using KEGG and DAVID. The remaining 43 genes that coded for 2 unknown and 41 Intergenic regions were not included in the analysis. Out of 57 genes that were differentially regulated only 14 down regulated and one up regulated gene could be associated with a particular pathway when analysed by KEGG pathways. Hyperlinks have been provided to these genes. These fifteen genes belonged to sixteen different pathways like metabolic pathways (ilvY and ydcQ), one cellular component which included a number of genes coding for integral membrane proteins  and three molecular functioning terms including ion binding (cusF and ygaQ) and DNA binding (ilvY and ydcQ). The 21 down regulated genes were implicated in four biological processes , ion transport , carbohydrate catabolic process  and nucleoside metabolic process . DAVID analysis also annotated genes involved in cellular components such as cell wall, cell membrane and organelle membrane . Similarly GO terms enriched for molecular functioning included genes coding for proteins involved in metal-ion binding, cation-binding and ion-binding .DAVID was also used for geneontology annotations and term enrichments for various biological processes, molecular functioning and cellular components. Out of 57 genes that were differentially regulated and analysed by DAVID only 21 down regulated and 9 up regulated genes yielded term enrichments. Hyperlinks have been provided to these genes. The analysis yielded that the 9 upregulated genes were involved in two biological processes namely DNA transcription and regulation of transcription , genes associated with multiple stress responses , genes involved in biofilm formation , curli biosynthesis (csgDEF), and lipid biosynthesis (yfbEFG). None of these genes were up regulated in the current study. The only difference between the present study and that of Vukanti et al. E. coli K12 MG1655 in the present study was grown in LB containing glycerol. Therefore the effect of glycerol on gene expression was studied (GEO accession number is GSE34275). It was observed that in E. coli K12 MG1655 grown in the presence of glycerol 103 genes were up regulated whereas 209 were down regulated , genes associated with multiple stress responses , genes involved in curli biosynthesis , and lipid biosynthesis (ybjP) as observed by Vukanti et al. Wilson et al. E. coli grown in the presence of glycerol exhibits up regulation of genes related to acid stress  were also down regulated. Gene livG that belongs to livFGHMK operon that is involved in high-affinity branched-chain amino acid transport system is down regulated. While ilvGMEDA operon an amino acid biosynthetic operon required for the synthesis of the branched chain aminoacids isoleucine, leucine and valine is down regulated in previous studies Comparison of genes identified in the present study to genes reported in previous studies cysW involved in sulfate/thiosulfate transport, cysH involved in cystein biosysnthesis and gene actP that is involved in acetate transport are down regulated, so as to possibly maintain pH homeostasis in the acidified media conditions. This is in contrast to the previous observations that genes pertaining to acetate metabolism acs and actP and cysAV involved in sulfate/thiosulfate transport as well as genes involved in cysteine biosynthesis cysCDIJKN were upregulated In the present study E. coli was cultured under microgravity conditions. Analysis based on the DAVID geneontology (GO) term enrichments included the following up regulated genes belonging to DNA transcription (srmB) and those involved in regulation of transcription (ilVY and ydcQ). Gene srmB encodes for DNA helicase. Upregulation of helicases is interesting as these plays a vital role in DNA repair, recombination and RNA transcription dfp, pyrD, spoT. Gene dfp is an essential gene required for DNA synthesis pyrD involved in pyrimidine biosynthesis ydcR that belongs to one of the transcriptional regulator family GntR is also repressed. The low abundance of these genes may be because of the active growth of E. coli culture under microgravity conditions. The spoT gene which codes for ppGpp in response to nutrients limitation rhaS, rhaD, ldcC and ptsA) are also down regulated suggesting that energy/reducing equivalents are adequately present. Gene ilvY is involved in the transcriptional regulation of the ilvA gene of the ilvGMEDA operon encoding for biosynthesis of branched chain aminoacids leucine, isoleucine and valine genes hisA, speB, aroP, pyrD, livG, mgtG and cysH. These results imply that cells growing under microgravity conditions are not deprived of nitrogenous compounds.In the present study 100 genes were either up or down regulated when cusF and ygaQ) and DNA-binding (ydcQ). Tucker et al., cusF when E. coli was grown in MOPS medium under simulated microgravity conditions. cusF is a periplasmic chaperone and exports copper and silver ions both from cytoplasm and periplasm to the external environment to maintain cell homeostasis. Enrichment of DAVID GO terms for ion transport, metal ion transport and cation transport for the down regulated genes  is also indicated. These transport systems assist in uptake of essential nutrients, regulate the flow of physiologically relevant chemicals, and release substances such as signalling molecules. Gene ompC coding for major outer membrane protein functions as a porin, allows free diffusion of small hydrophilic molecules across the membrane gst encoding for glutathione transferase that protects cells by detoxification of metals Hg2+ and Cd2+The other up regulated genes enriched include those coding for cation-binding .(DOCX)Click here for additional data file.Table S2Glycerol-induced down regulation of genes in Escherichia coli. DNA microarray analysis of E. coli grown in the presence of 10% glycerol showed down regulation of 209 genes with a fold change >1.5 (P<0.05).(DOCX)Click here for additional data file.