Variation In Protein Expression Research Articles

Dynamic variation of kidney injury molecule-1 mRNA and protein expression in blood and urine of renal transplant recipients: a cohort study.

Acute renal dysfunction still constitutes a highly significant obstacle to renal transplantation outcome. Kidney injury molecule-1 is highly upregulated in proximal tubular cells and shed into the urine and blood circulation following kidney injury. The aim of current cohort study was to evaluate the urine KIM-1 (uKIM-1) mRNA expression level and its protein concentration in blood and urine samples to determine whether sequential monitoring of KIM-1 in renal allograft recipients is a reliable biomarker for predicting the clinical status and outcome. Both uKIM-1 mRNA expression level and the level of serum and uKIM-1 protein concentration in the 52 renal transplant recipients were respectively quantified using real-time PCR and ELISA methods at 2, 90 and 180days after transplantation. KIM-1 mRNA and protein expression level in the blood and urine samples of patients with graft dysfunction was significantly higher than patients with well-functioning graft on days 2, 90 and 180 after transplantation. Receiver-operating characteristic curve analysis of mRNA and protein expression levels showed that urinary and blood KIM-1 at months 3 and 6 could predict acute renal dysfunction at 6months and 1year after transplantation. Sequential monitoring of uKIM-1 mRNA expression level and its protein concentration in the serum and urine samples of renal transplant patients suggests that KIM-1 could be a sensitive and specific biomarker for early diagnosis and prognosis of kidney allograft injury.

The mRNA-bound Proteome of Leishmania mexicana: Novel Genetic Insight into an Ancient Parasite

Leishmania parasite infections, termed the leishmaniases, cause significant global infectious disease burden. The lifecycle of the parasite embodies three main stages that require precise coordination of gene regulation to survive environmental shifts between sandfly and mammalian hosts. Constitutive transcription in kinetoplastid parasites means that gene regulation is overwhelmingly reliant on post-transcriptional mechanisms, yet strikingly few Leishmania trans-regulators are known. Using optimized crosslinking and deep, quantified mass spectrometry, we present a comprehensive analysis of 1400 mRNA binding proteins (mRBPs) and whole cell proteomes from the three main Leishmania lifecycle stages. Supporting the validity, although the crosslinked RBPome is magnitudes more enriched, the protein identities of the crosslinked and non-crosslinked RBPomes were nearly identical. Moreover, multiple candidate RBPs were endogenously tagged and found to associate with discrete mRNA target pools in a stage-specific manner. Results indicate that in L. mexicana parasites, mRNA levels are not a strong predictor of the whole cell expression or RNA binding potential of encoded proteins. Evidence includes a low correlation between transcript and corresponding protein expression and stage-specific variation in protein expression versus RNA binding potential. Unsurprisingly, RNA binding protein enrichment correlates strongly with relative replication efficiency of the specific lifecycle stage. Our study is the first to quantitatively define and compare the mRBPome of multiple stages in kinetoplastid parasites. It provides novel, in-depth insight into the trans-regulatory mRNA:Protein (mRNP) complexes that drive Leishmania parasite lifecycle progression.

Abstract 1568: Clinical implications of tmprss2-erg fusion and tumor location in prostate cancer

Abstract Introduction: There is limited data on the impact of race on tumor location and TMPRSS2-ERG (ERG) gene fusion status in Prostate Cancer (PCa). African American men (AAM) often lack the ERG fusion and are likely to experience poor outcomes. Nonetheless, the extent to which ERG status, race, and tumor location impacts PCa outcomes remains unclear. Methods: This multi-institutional study included a retrospective cohort of 495 AAM and 422 European American men (EAM) who received prostatectomy at Moffitt Cancer Center (MCC) and University of Pennsylvania Health system (UPHS). To assess the race-dependent variation in ERG protein expression and its association with tumor location and PCa pathology, ERG antibody staining on 4 micron sections of FFPE blocks were carried out on all samples. ERG expression was categorized as ERGpos and ERGneg by two pathologists. Additionally, the staining pattern of ERG was analyzed within ERGpos tumors. A review of anatomic location of the dominant tumor lesions was performed and analyzed using methods of categorical analysis and logistic regression. Alpha value of < 0.05 was considered as statistically significant. Results: In all samples, analysis of ERG immunohistochemistry (IHC) yielded 39.7% ERGpos and 60.6% ERGneg. Variability in histopathologic expression of ERG was significant between races with AAM being more likely to lack TMPRSS2-ERG fusion (ERGneg) compared to EAM, (62% vs 38%, p < 0.001). Lack of ERG fusion was also associated with anatomically distinct tumor location with 72.1% of anteriorly located tumors having ERGneg staining, while only 27.9% were ERGpos, (p < 0.001). Among ERGpos patients, AAM (65.2%) showed significant heterogeneous expression of ERG compared to EAM (31.8%). Furthermore, ERGneg status was associated with higher pretreatment PSA. In a multivariate logistic regression model adjusted for tumor location and race, ERGneg tumors were more likely to have Gleason Score 4+3=7 (Grade Group 3) or higher compared with ERGpos tumors, Odds ratio 2.07, 95% CI 1.20 - 3.56, p = 0.008. Conclusions: The absence of TMPRSS2-ERG fusion is associated with higher grade disease and is a strong predictor for anatomic tumor location within the prostate gland. The clinical implications of ERGneg tumors among AAM and its association with anteriorly located tumors can be vital in addressing disparities in PCa. Citation Format: Kosj Yamoah, Shivanshu Awasthi, Priti Lal, Arash O. Naghavi, Shelly Mahajan, Jong Y. Park, Rob Rounbehler, Travis Gerke, Anders Berglund, Julio PowSang, Jasreman Dhillon, Timothy R. Rebbeck. Clinical implications of tmprss2-erg fusion and tumor location in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1568.

Protein profiling of metal-resistant Bacillus cereus VITSH1.

The aim of the study was to investigate the proteomic changes and antioxidant enzyme activity in chromium-resistant Bacillus cereus VITSH1 in response to heavy metal toxicity. The variation in protein expression and antioxidant enzyme activity in the presence and absence of metal was studied in B. cereus VITSH1. The differentially expressed proteins in chromium-treated conditions were determined by SDS PAGE. The proteins involved in metal binding, protein biosynthesis, protein folding, energy metabolism and motility were identified by mass spectrometry coupled with bioinformatics search tools. The in gel assays for antioxidant enzymes indicated a change in their activity under metal stress conditions. The findings of this study suggest that the organism combats metal stress probably by restricting the entry of metal inside the cell. The role of the differentially expressed proteins clearly indicates that the first line of defence is to avoid the entry of metal into the cell either by possessing a modified outer membrane or by moving away from the toxicant. Further work on the identification of other proteins playing a role in resistance would help in integrating the available knowledge on the resistance mechanisms the organisms employ to combat toxicity. The proteomic changes in the metal-exposed bacteria would give an insight into the proteins involved in metal resistance mechanisms and thereby aid in the development of biosensor-based technology for heavy metal detection.

Dynamic expression of glial fibrillary acidic protein and ionized calcium binding adaptor molecule 1 in the mouse spinal cord dorsal horn under pathological pain states

ABSTRACTObjective: Animal models of chronic pain have demonstrated that glial cells are promising target for development of analgesic drugs. However, preclinical studies on glial response under chronic pain conditions vary depending on the cellular markers, the species used, the experimental design and model. Therefore, we investigate the expression profile of GFAP and Iba-1 during the behavioral manifestation of sensory disorder in inflammatory and neuropathic pain models.Methods: the expression profile of fibrillary acidic protein (GFAP) and ionized calcium binding adaptor molecule-1 (Iba-1) were quantitated in the spinal dorsal horn of Balb/C mice submitted to six models of chronic pain. Protein analysis was performed by western blot and the results colligated with pain-related behavior.Results: Using the same method to quantitate proteins we observed that while GFAP is upregulated after axotomy, partial nerve injury and cutaneous inflammation, its expression is not changed during muscle inflammation, non-inflammatory muscle pain, and in a viral-associated pain. Differently, Iba-1 is downregulated after axotomy but upregulated after partial lesion of peripheral nerve as well as after virus inoculation and during non-inflammatory muscle pain. Cutaneous and muscle inflammation induced no change in Iba-1 expression in the dorsal horn.In spite of a marked time-dependent variation in protein expression, mechanical allodynia was present at any time of all the models investigated.Discussion: Under distinct pain conditions, GFAP and Iba-1 expression is dependent on the origin of the stimulus, disease progression and tissue affected. Moreover, their expression and is not necessarily associated to the behavior manifestation of pain.

Alterations of protein expression of phospholamban, ZASP and plakoglobin in human atria in subgroups of seniors

The mature mammalian myocardium contains composite junctions (areae compositae) that comprise proteins of adherens junctions as well as desmosomes. Mutations or deficiency of many of these proteins are linked to heart failure and/or arrhythmogenic cardiomyopathy in patients. We firstly wanted to address the question whether the expression of these proteins shows an age-dependent alteration in the atrium of the human heart. Right atrial biopsies, obtained from patients undergoing routine bypass surgery for coronary heart disease were subjected to immunohistology and/or western blotting for the plaque proteins plakoglobin (γ-catenin) and plakophilin 2. Moreover, the Z-band protein cypher 1 (Cypher/ZASP) and calcium handling proteins of the sarcoplasmic reticulum (SR) like phospholamban, SERCA and calsequestrin were analyzed. We noted expression of plakoglobin, plakophilin 2 and Cypher/ZASP in these atrial preparations on western blotting and/or immunohistochemistry. There was an increase of Cypher/ZASP expression with age. The present data extend our knowledge on the expression of anchoring proteins and SR regulatory proteins in the atrium of the human heart and indicate an age-dependent variation in protein expression. It is tempting to speculate that increased expression of Cypher/ZASP may contribute to mechanical changes in the aging human myocardium.

Imbalanced sphingolipid signaling is maintained as a core proponent of a cancerous phenotype in spite of metabolic pressure and epigenetic drift.

Tumor heterogeneity may arise through genetic drift and environmentally driven clonal selection for metabolic fitness. This would promote subpopulations derived from single cancer cells that exhibit distinct phenotypes while conserving vital pro-survival pathways. We aimed to identify significant drivers of cell fitness in pancreatic adenocarcinoma (PDAC) creating subclones in different nutrient formulations to encourage differential metabolic reprogramming. The genetic and phenotypic expression profiles of each subclone were analyzed relative to a healthy control cell line (hTert-HPNE). The subclones exhibited distinct variations in protein expression and lipid metabolism. Relative to hTert-HPNE, PSN-1 subclones uniformly maintained modified sphingolipid signaling and specifically retained elevated sphingosine-1-phosphate (S1P) relative to C16 ceramide (C16 Cer) ratios. Each clone utilized a different perturbation to this pathway, but maintained this modified signaling to preserve cancerous phenotypes, such as rapid proliferation and defense against mitochondria-mediated apoptosis. Although the subclones were unique in their sensitivity, inhibition of S1P synthesis significantly reduced the ratio of S1P/C16 Cer, slowed cell proliferation, and enhanced sensitivity to apoptotic signals. This reliance on S1P signaling identifies this pathway as a promising drug-sensitizing target that may be used to eliminate cancerous cells consistently across uniquely reprogrammed PDAC clones.

Microbial diversity and metaproteomic analysis of activated sludge responses to naphthalene and anthracene exposure.

The activated sludge process can effectively remove polycyclic aromatic hydrocarbons (PAHs) from wastewater via biodegradation. However, the degradable microorganisms and functional enzymes involved in this process remain unclear. In this study, we successfully employed a laboratory-scale sequential batch reactor to investigate variations in microbial community and protein expression in response to the addition of different PAHs and process time. The analysis of bacterial community structure by 454 pyrosequencing of the 16S rRNA gene indicated that bacteria from Burkholderiales order were dominant in PAHs treated sludge. Mass spectrometry performed with 2D protein profiles of all sludge samples demonstrated that most proteins exhibiting differential expression profiles during the process were derived from Burkholderiales populations; these proteins are involved in DNA replication, fatty acid and glucose metabolism, stress response, protein synthesis, and aromatic hydrocarbon metabolism. Nevertheless, the protein expression profiles indicated that naphthalene, but not anthracene, can induce the expression of PAH-degrading proteins and accelerate its elimination from sludge. Though only naphthalene and anthracene were added into our experimental groups, the differentially expressed enzymes involved in other PAHs (especially biphenyl) metabolism were also detected. This study provides apparent evidence linking the metabolic activities of Burkholderiales populations with the degradation of PAHs in activated sludge processes. Overall, our findings highlighted the successful application of metaproteomics integrated with microbial diversity analysis by high-throughput sequencing technique on the analysis of environmental samples, which could provide a convenience to monitor the changes in proteins expression profiles and their correlation with microbial diversity.

High-resolution antibody array analysis of proteins from primary human keratinocytes and leukocytes.

Antibody array analysis of labeled proteomes has high throughput and is simple to perform, but validation remains challenging. Here, we used differential detergent fractionation and size exclusion chromatography in sequence for high-resolution separation of biotinylated proteins from human primary keratinocytes and leukocytes. Ninety-six sample fractions from each cell type were analyzed with microsphere-based antibody arrays and flow cytometry (microsphere affinity proteomics; MAP). Monomeric proteins and multi-molecular complexes in the cytosol, cytoplasmic organelles, membranes and nuclei were resolved as discrete peaks of antibody reactivity across the fractions. The fractionation also provided a two-dimensional matrix for assessment of specificity. Thus, antibody reactivity peaks were considered to represent specific binding if the position in the matrix was in agreement with published information about i) subcellular location, ii) size of the intended target, and iii) cell type-dependent variation in protein expression. Similarities in the reactivity patterns of either different antibodies to the same protein or antibodies to similar proteins were used as additional supporting evidence. This approach provided validation of several hundred proteins and identification of monomeric proteins and protein complexes. High-resolution MAP solves many of the problems associated with obtaining specificity with immobilized antibodies and a protein label. Thus, laboratories with access to chromatography and flow cytometry can perform large-scale protein analysis on a daily basis. This opens new possibilities for cell biology research in dermatology and validation of antibodies.

Genetic and environmental variation in transcriptional expression of seminal fluid proteins.

Seminal fluid proteins (SFPs) are crucial mediators of sexual selection and sexual conflict. Recent studies have chiefly focused on environmentally induced plasticity as one source of variation in SFP expression, particularly in response to differing sperm competition levels. However, understanding the evolution of a trait in heterogenous environments requires estimates of both environmental and genetic sources of variation, as well as their interaction. Therefore, we investigated how environment (specifically mating group size, a good predictor of sperm competition intensity), genotype and genotype-by-environment interactions affect seminal fluid expression. To do so, we reared 12 inbred lines of a simultaneously hermaphroditic flatworm Macrostomum lignano in groups of either two or eight worms and measured the expression levels of 58 putative SFP transcripts. We then examined the source of variation in the expression of each transcript individually and for multivariate axes extracted from a principal component analysis. We found that mating group size did not affect expression levels according to the single transcript analyses, nor did it affect the first principal component (presumably representing overall investment in seminal fluid production). However, mating group size did affect the relative expression of different transcripts captured by the second principal component (presumably reflecting variation in seminal fluid composition). Most transcripts were genetically variable in their expression level and several exhibited genotype-by-environment interactions; relative composition also showed high genetic variation. Collectively, our results reveal the tightly integrated nature of the seminal fluid transcriptome and provide new insights into the quantitative genetic basis of seminal fluid investment and composition.

Epigenetic Enzymes, Age, and Ancestry Regulate the Efficiency of Human iPSC Reprogramming.

Epigenetic enzymes regulate higher-order chromatin architecture and cell-type specific gene expression. The ATPase BRG1 and the SWI/SNF chromatin remodeling complex are epigenetic enzymes that regulate chromatin accessibility during steady and transitional cell states. Experiments in mice show that the loss of BRG1 inhibits cellular reprogramming, while studies using human cells demonstrate that the overexpression of BRG1 enhances reprogramming. We hypothesized that the variation of SWI/SNF subunit expression in the human population would contribute to variability in the efficiency of induced pluripotent stem cells (iPSC) reprogramming. To examine the impact of an individual's sex, ancestry, and age on iPSC reprogramming, we created a novel sex and ancestry balanced cohort of 240 iPSC lines derived from human dermal fibroblasts (DF) from 80 heathy donors. We methodically assessed the reprogramming efficiency of each DF line and then quantified the individual and demographic-specific variations in SWI/SNF chromatin remodeling proteins and mRNA expression. We identified BRG1, BAF155, and BAF60a expression as strongly correlating with iPSC reprogramming efficiency. Additionally, we discovered that high efficiency iPSC reprograming is negatively correlated with donor age, positively correlated with African American descent, and uncorrelated with donor sex. These results show the variations in chromatin remodeling protein expression have a strong impact on iPSC reprogramming. Additionally, our cohort is unique in its large size, diversity, and focus on healthy donors. Consequently, this cohort can be a vital tool for researchers seeking to validate observational results from human population studies and perform detailed mechanistic studies in a controlled cell culture environment. Stem Cells 2018;36:1697-1708.

Domestic Fowl Breed Variation in Egg White Protein Expression: Application of Proteomics and Transcriptomics.

Avian egg white is essential for protecting and nourishing bird embryos during their development. Being produced in the female magnum, variability in hen oviduct gene expression may affect egg white composition in domestic chickens. Since traditional poultry breeds may represent a source of variation, in the present study we describe the egg white proteome (mass spectrometry) and corresponding magnum transcriptome (high-throughput sequencing) for 20 hens from five domestic fowl breeds (large breeds: Araucana, Czech golden pencilled, Minorca; and small breeds: Booted bantam, Rosecomb bantam). In total, we identified 189 egg white proteins and 16391 magnum-expressed genes. The majority of egg white protein content comprised proteins with an antimicrobial function. Despite general similarity, Between-class Principal Component Analysis revealed significant breed-specific variability in protein abundances, differentiating especially small and large breeds. Though we found strong association between magnum mRNA expression and egg white protein abundance across genes, coinertia analysis revealed no transcriptome/proteome costructure at the individual level. Our study is the first to show variation in protein abundances in egg white across chicken breeds with potential effects on egg quality, biosafety, and chick development. The observed interindividual variation probably results from post-transcriptional regulation creating a discrepancy between proteomic and transcriptomic data.

Variable cardiac myosin binding protein-C expression in the myofilaments due to MYBPC3 mutations in hypertrophic cardiomyopathy

BackgroundMutations in MYBPC3 are the most common cause of hypertrophic cardiomyopathy (HCM). These mutations produce dysfunctional protein that is quickly degraded and not incorporated in the myofilaments. Most patients are heterozygous and allelic expression differs between cells. We hypothesized that this would lead to cell-to-cell variation in cardiac myosin binding protein-C (cMyBP-C, encoded by MYBPC3 gene) protein levels. MethodsTwelve HCM patients were included (six had no sarcomere mutations (HCMsmn) and served as the control group and six harbored mutations in the MYBPC3 gene (MYBPC3mut). Western blot and RNA sequencing analysis of cardiac tissue lysates were performed to detect overall cMyBP-C protein and mRNA levels. Cellular expression of cMyBP-C and α-actin was obtained by immunofluorescence staining. Quantification of cell-to-cell variation of cMyBP-C expression between cardiomyocytes was measured by determining the ratio of cMyBP-C:α-actin stained area of each cell. ResultsProtein and mRNA analysis revealed significantly reduced cMyBP-C levels in MYBPC3mut patients compared with HCMsmn patients (0.73 ± 0.09 vs. 1.0 ± 0.15, p < .05; 162.3 ± 16.4 vs. 326.2 ± 41.9 RPKM, p = .002), without any sign of truncated proteins. Immunofluorescence staining of individual cardiomyocytes in HCMsmn patients demonstrated homogenous and equal cMyBP-C:α-actin staining ratio. In contrast, MYBPC3mut patients demonstrated inhomogeneous staining patterns with a large intercellular variability per patient. Coefficient of variance for cMyBP-C/α-actin staining for each patient showed a significant difference between both groups (17.30 ± 4.08 vs. 5.18 ± 0.65% in MYBPC3mut vs. HCMsmn, p = .02). ConclusionThis is the first study to demonstrate intercellular variation of myofilament cMyBP-C protein expression within the myocardium from HCM patients with heterozygous MYBPC3 mutations.

Utility of Two-Dimensional Difference Gel Electrophoresis in Diagnosis of Multiple Sclerosis.

Two-dimensional difference gel electrophoresis (2D-DIGE) has been used for identification of possible biomarkers in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients. However, in different studies inconsistent results have been obtained. We wanted to analyze the diagnostic value of 2D-DIGE in early MS patients by comparing protein patterns between single and pooled samples of MS patients and controls. CSF samples of 20 MS patients and 10 control subjects were processed with 2D-DIGE. The so obtained protein patterns were analyzed with DeCyder 6.5 software, whereby we described variation of patterns presented in one gel as well as between different gels. Even when running single samples of patients of the same group in one gel, variation of protein patterns was high. The number of identified spots with different protein level varied between 4 and 30, depending on which sample batches were compared. We did not find a consistent pattern throughout all possible batch combinations. The inter-individual variation of protein expression as well as the susceptibility of 2D-DIGE for methodological variations makes use of 2D-DIGE as a diagnostic tool for MS and for detection of possible candidate biomarkers difficult, since detected proteins vary depending on which samples are compared.

Analyses of the genetic diversity and protein expression variation of the acyl: CoA medium-chain ligases, ACSM2A and ACSM2B.

Benzoate (found in milk and widely used as preservative), salicylate (present in fruits and the active component of aspirin), dietary polyphenols produced by gut microbiota, metabolites from organic acidemias, and medium-chain fatty acids (MCFAs) are all metabolised/detoxified by the glycine conjugation pathway. Xenobiotics are first activated to an acyl-CoA by the mitochondrial xenobiotic/medium-chain fatty acid: CoA ligases (ACSMs) and subsequently conjugated to glycine by glycine N-acyltransferase (GLYAT). The MCFAs are activated to acyl-CoA by the ACSMs before entering mitochondrial β-oxidation. This two-step enzymatic pathway has, however, not been thoroughly investigated and the biggest gap in the literature remains the fact that studies continuously characterise the pathway as a one-step reaction. There are no studies available on the interaction/competition of the various substrates involved in the pathway, whilst very little research has been done on the ACSM ligases. To identify variants/haplotypes that should be characterised in future detoxification association studies, this study assessed the naturally observed sequence diversity and protein expression variation of ACSM2A and ACSM2B. The allelic variation, haplotype diversity, Tajima's D values, and phylogenetic analyses indicated that ACSM2A and ACSM2B are highly conserved. This confirmed an earlier hypothesis that the glycine conjugation pathway is highly conserved and essential for life as it maintains the CoA and glycine homeostasis in the liver mitochondria. The protein expression analyses showed that ACSM2A is the predominant transcript in liver. Future studies should investigate the effect of the variants identified in this study on the substrate specificity of these proteins.

Chemical Constituents from Cimicifuga dahurica and Their Anti-Proliferative Effects on MCF-7 Breast Cancer Cells

This study was designed to search for novel anti-cancer compounds from natural plants. The 70% ethanolic extract from the rizhomes of Cimicifuga dahurica (Turcz.) Maxim. (Ranunculaceae) was found to possess significant in vitro anti-proliferative effects on MCF-7 breast cancer cells. A phytochemical investigation using assay-guided fractionation of the ethanolic extract of C. dahurica resulted in the isolation of one new phenolic amide glycoside 3, two new lignan glycosides 4 and 7, one new 9,19-cycloartane triterpenoid glycoside 6, and thirteen known constituents 1, 2, 5, and 8–17. The structures of 3, 4, 6, and 7 were established using contemporary NMR methods and from their HRESIMS data. The anti-proliferative effects of isolated compounds were evaluated using the BrdU-proliferation kit. Five among the 17 isolated compounds showed significant anti-proliferative effects (p ≤ 0.05), wherein compound 7 showed the most significant anti-proliferative and cell cycle arresting effect (p ≤ 0.05) which followed a dose dependent manner. Western blot protein expression analysis showed a down expression of c-Myc and cyclin D1 which further elucidated the anti-proliferation mechanism of compound 7 while apoptotic effects were found in association with Bcl-2 family protein expression variations. Conclusively this study reports the isolation and identification of 17 compounds from C. dahurica, including four novel molecules, in addition to the fact that compound 7 possesses significant anti-proliferative and apoptotic effects in vitro that may require further exploration.

No Evidence of Copy Number Variation in Acidic Mammalian Chitinase Genes (CHIA) in New World and Old World Monkeys

Copy number variation may be the most common form of structural genetic variation in the genome. Numerous studies have shown that gene copy number variation can correlate with phenotypic variation, where higher copy numbers correspond to increased expression of the protein and vice versa. Examples include some digestive enzyme genes, where variation in copy numbers and protein expression may be related to dietary differences. Increasing the expression of a digestive enzyme through higher gene copy numbers may thus be a potential mechanism for altering an organism’s digestive capabilities. I investigated copy number variation in genes coding for acidic mammalian chitinase, a chitinolytic digestive enzyme that may be used for the digestion of insect exoskeletons, in nonhuman primates with varying levels of insect consumption. I hypothesized that CHIA copy number correlates positively with level of insectivory, predicting higher copy numbers in more insectivorous primates. I assessed copy number variation with the QuantStudio 3D digital PCR platform, in a comparative sample of Old World and New World primate species (N = 10 species, one or two individuals each). Contrary to my prediction, no evidence of copy number variation was found and all species tested had two gene copies per diploid genome. These findings suggest that if acidic mammalian chitinase expression varies according to insect consumption in primates, it may be up- or downregulated through another mechanism.

Cand1-Mediated Adaptive Exchange Mechanism Enables Variation in F-Box Protein Expression

Skp1⋅Cul1⋅F-box (SCF) ubiquitin ligase assembly isregulated by the interplay of substrate binding, reversible Nedd8 conjugation on Cul1, and the F-box protein (FBP) exchange factors Cand1 and Cand2. Detailed investigations into SCF assembly and function in reconstituted systems and Cand1/2 knockout cells informed the development of a mathematical model for how dynamical assembly of SCF complexes is controlled and how this cycle is coupled to degradation of an SCF substrate. Simulations predicted an unanticipated hypersensitivity of Cand1/2-deficient cells to FBP expression levels, which was experimentally validated. Together, these and prior observations lead us to propose the adaptive exchange hypothesis, which posits that regulation of the koff of an FBP from SCF by the actions of substrate, Nedd8, and Cand1 molds the cellular repertoire of SCF complexes and that the plasticity afforded by this exchange mechanism may enable large variations in FBP expression during development and in FBP gene number during evolution.

Sensing of Vimentin mRNA in 2D and 3D Models of Wounded Skin Using DNA-Coated Gold Nanoparticles.

Wound healing is a highly complex biological process, which is accompanied by changes in cell phenotype, variations in protein expression, and the production of active biomolecules. Currently, the detection of proteins in cells is done by immunostaining where the proteins in fixed cells are detected by labeled antibodies. However, immunostaining cannot provide information about dynamic processes in living cells, within the whole tissue. Here, an easy method is presented to detect the transition of epithelial to mesenchymal cells during wound healing. The method employs DNA-coated gold nanoparticle fluorescent nanoprobes to sense the production of Vimentin mRNA expressed in mesenchymal cells. Fluorescence microscopy is used to achieve temporal detection of Vimentin mRNA in wounds. 3D light-sheet microscopy is utilized to observe the dynamic expression of Vimentin mRNA spatially around the wounded site in skin tissue. The use of DNA-gold nanoprobes to detect mRNA expression during wound healing opens up new possibilities for the study of real-time mechanisms in complex biological processes.

Time-dependent alterations in mRNA, protein and microRNA during in vitro adipogenesis.

Adipogenesis is a complex biological process involving synchronised interplay of different nuclear receptors. Aberration in the process leads to obesity and associated disorders. Addressing the complexity of molecular mechanisms, we worked on characterising the changes in NR1C3/PPARγ-, NR1H3/LXRα- and NCoAs/SRCs-associated microRNA, genes and proteins during different time points of adipogenesis. Glucose uptake of differentiating cells was checked at selected time points with FACS. Observations on gene expression pattern pointed a correlation in adipogenic-related genes and increased expression of PPARγ, but not LXRα. Western blot experiments also supported the gene expression pattern. MicroRNAs that vary during adipogenesis was selected using bioinformatics tools and database. Real-time PCR-based experiments showed a change in the expression of mmu-mir-23a-3p, 206-3p, 17-3p, 126a-3p and 1a-3p. Mmu-mir-23a-3p showed a gradual decrease in expression corresponding to the progression of adipogenesis. MicroRNA 23a-3p and 1a-3p showed positive association to the mRNA levels of NCoA1 and 3. Overall, the study elaborates time-dependent variations in nucleic acid and protein expression during adipogenesis in accordance to fatty acid and glucose metabolism.