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Advances in single-cell level profiling of the proteome require quantitative and versatile platforms, especially for rare cell analyses such as circulating tumor cell (CTC) profiling. Here we demonstrate an integrated microfluidic chip that uses magnetic nanoparticles to capture single tumor cells with high efficiency, permits on-chip incubation, and facilitates in situ cell-surface protein expression analysis. Combined with phage-based barcoding and next-generation sequencing technology, we were able to monitor changes in the expression of multiple surface markers stimulated in response to CTC adherence. Interestingly, we found fluctuations in the expression of Frizzled2 (FZD2) that reflected the microenvironment of the single cells. This platform has a high potential for in-depth screening of multiple surface antigens simultaneously in rare cells with single-cell resolution, which will provide further insights regarding biological heterogeneity and human disease.

MRP4 (gene ABCC4) is a polymorphic efflux transporter that has been implicated in drug-induced toxicity. We selected ten commonly observed MRP4 coding variants among Europeans for experimental characterization including nine variants predicted to be deleterious or functional (combined annotation-dependent depletion score >15). We assessed protein localization and activity by quantifying intracellular accumulation of two prototypic substrates, taurocholic acid (TCA) and estradiol 17-β-glucuronide (E217βG), in HEK293T over-expressing MRP4 wildtype or variant where cellular substrate loading was optimized through co-transfection with an uptake transporter. V458M, a novel variant not previously studied, and T1142M, showed reduced activity compared to MRP4 wildtype for E217βG and TCA (P

Cerium oxide (CeO2) has potential applications in medicine and various consumer products. This study investigated the effect of CeO2 on the expression of genes associated with apoptosis and testicular development in mouse embryos. The experimental groups of pregnant mice were injected intraperitoneally with CeO2 at a concentration of 10 mg/kg on days 7 and 14 of pregnancy. Six days after birth, the testicles of neonatal male mice were collected for mRNA expression determination using real-time PCR, protein expression analysis by immunohistochemistry, and apoptotic cell population determination using the TUNEL assay. The results showed that the mRNA expression of the Bax, Caspase-3, and Gsk3-β genes, unlike the Bcl2 gene, decreased significantly in the experimental group compared to the control group. The expression ratio of Bax/Bcl2 in the experimental group was lower than in the control group. A similar trend was observed in the population of apoptotic cells. In the experimental group, the expression levels of, Gata4, Sox8, and Rad54 at both the mRNA and protein levels increased significantly compared to the control group. Based on the results of this study, CeO2 at a concentration of 10 mg/kg, in addition to producing anti-apoptotic effects on the testicular cells of neonatal mice, can increase the expression of genes involved in testicular development and performance. The current experimental study proved the protective effects of 10 mg/kg CeO2 in developmental and apoptosis genes of testicular tissue in 6-day-old NMRI mice fetuses; however, more experiments are required to evaluate the possible side effects and interactions.

The current World Health Organization (WHO) classification defines a new disease entity of high-grade B-cell lymphoma with MYC and BCL2 and/or BCL6 rearrangements, making fluorescence in situ hybridization (FISH) screening for these genes mandatory. In addition, the prognostic significance of MYC expression was reported, with a cut-off value of 40%. However, interobserver discrepancies arise due to the heterogeneous intensity of MYC expression by immunohistochemistry. Moreover, a cut-off value of positivity for MYC protein in diffuse large B-cell lymphoma (DLBCL) varies among studies at present. Here, we applied a high-sensitivity semiquantitative immunohistochemical technique using fluorescent nanoparticles called phosphor-integrated dots (PID) to evaluate the MYC expression in 50 de novo DLBCL cases, and compared it with the conventional diaminobenzidine (DAB)-developing system. The high MYC expression detected by the PID-mediated system predicted poor overall survival in DLBCL patients. However, we found no prognostic value of MYC protein expression for any cut-off value by the DAB-developing system, even if the intensity was considered. These results indicate that the precise evaluation of MYC protein expression can clarify the prognostic values in DLBCL, irrespective of MYC rearrangement.

Transient receptor potential (TRP) channels are broadly required in animals for sensory physiology. To provide insights into regulatory mechanisms, the structures of many TRPs have been solved. This has led to new models, some of which have been tested in vitro. Here, using the classical TRP required for Drosophila visual transduction, we uncovered structural requirements for channel function in photoreceptor cells. Using a combination of molecular genetics, field recordings, protein expression analysis, and molecular modeling, we interrogated roles for the S4–S5 linker and the TRP domain, and revealed mutations in the S4–S5 linker that impair channel opening or closing. We also uncovered differential requirements for the two highly conserved motifs in the TRP domain for activation and protein stability. By performing genetic complementation, we found an intrasubunit interaction between the S4–S5 linker and the S5 segment that contributes to activation. This analysis highlights key structural requirements for TRP channel opening, closing, folding, and for intrasubunit interactions in a native context—Drosophila photoreceptor cells. SIGNIFICANCE STATEMENT The importance of TRP channels for sensory biology and human health has motivated tremendous effort in trying to understand the roles of the structural motifs essential for their activation, inactivation, and protein folding. In the current work, we have exploited the unique advantages of the Drosophila visual system to reveal mechanistic insights into TRP channel function in a native system–photoreceptor cells. Using a combination of electrophysiology (field recordings), cell biology, and molecular modeling, we have revealed roles of key motifs for activation, inactivation and protein folding of TRP in vivo.

Objectives For many years, tumor development has been viewed as a cell-autonomous process; however, today we know that the tumor microenvironment (TME) and especially cancer-associated fibroblasts (CAFs) significantly contribute to tumor progression. Caveolin-1 (Cav-1) is a scaffolding protein which is involved in several cancer-associated processes as important component of the caveolae. Our goal was to shed light on the expression of the two different isoforms of Cav-1 in normal fibroblasts (NFs) and CAFs of patients with oral squamous cell carcinoma (OSCC). Materials and methods Fibroblasts from normal mucosa and CAFs were isolated and propagated in vitro. Gene expression of the different Cav-1 isoforms was assessed via quantitative real-time PCR (qPCR) and supplemented by protein expression analysis. Results We could show that the Cav-1β isoform is more highly expressed in NFs and CAFs compared to Cav-1α. Furthermore, the different Cav-1 isoforms tended to be differently expressed in different tumor stages. However, this trend could not be seen consistently, which is in line with the ambiguous role of Cav-1 in tumor progression described in literature. Western blotting furthermore revealed that NFs and CAFs might differ in the oligomerization profile of the Cav-1 protein. Conclusion These differences in expression of Cav-1 between NFs and CAFs of patients with OSCC confirm that the protein might play a role in tumor progression and is of interest for further analyses. Clinical relevance Our findings support a possible role of the two isoforms of Cav-1 in the malignant transformation of OSCC.

Purpose AMD genetic studies have revealed various genetic loci as causal to AMD pathology. We have described the genetic complexity of Indian AMD by describing the interaction of genotypes and subsequent changes in protein expression under the influence of environmental factors. This can be utilized to enhance the diagnostic and therapeutic efficacy in AMD patients. Design Genotype association was studied in 464 participants (AMD =277 & controls = 187) for eight genetic variants and their corresponding protein expression Methods SNP analysis and protein expression analysis was carried out in AMD and controls in tandem with longitudinal assessment of protein levels during the course of AMD pathology. ANCOVA and contrast analysis were used to examine the genotypic interactions and corresponding alterations in protein levels. In order to identify the important genetic variants Logistic Regression (LR) modeling was carried out and to authenticate the model Area under the Receiver Operating Characteristic curve (AUROC) were also computed. Results We have found genetic variants of rs5749482 (TIMP-3), rs11200638 (HTRA1), rs769449 (APOE) and rs6795735 (ADAMTS9) to be associated with AMD, concomitant with significant alterations of studied proteins levels. Analysis also revealed that the genetic interaction between APOE-HTRA1 genotypes and changes in LIPC levels (>6 pg/ug) by one unit change in SNP, play a crucial role in AMD. LR model suggested that the seven factors (including both genetic and environmental) can be utilized to predict the AMD cases with 88% efficacy and 95.6% AUROC. Conclusion Results suggest that diagnostic and therapeutic strategy for Indian AMD must include estimation of genetic interaction and concomitant changes in expression levels of proteins under influence of environmental factors.

Genetic studies including the quest, cloning and expression of genes encoding proteins responsible for various vital physiological processes and beneficial characteristics of economic perspective have made the biotechnology research progressively auspicious. Due to its great zootechnical and industrial importance somatotropin gene have been cloned from various animal species. Current study was designed to clone mature ovine growth hormone complementary DNA (oGH cDNA) of a sheep breed, Kajli and carry out over expression studies of cloned GH cDNA in a suitable prokaryotic expression system. Sheep GH cDNA was cloned in T/A (thymine / adenine) vector with signal peptide and confirmed by nested polymerase chain reaction (PCR) and restriction digestion. The gene was then ligated in pLEX expression vector and restricted plasmids showed a fragment insert of ~ 600 bps. Restriction analysis confirmed positive clones, were induced for protein expression analysis. The pET vectors (plasmid for expression by T7 RNA polymerase) have an isopropylthio-β-galactoside (IPTG) inducible strong T7 promoter and Escherichia coli expression strain of BL21 (DE3) pLysS contains DNA fragment from T7 phage which harbors RNA polymerase. Therefore, for expressing recombinant proteins, cells were induced with various IPTG concentrations to optimize expression levels. Cells were induced with different IPTG concentrations (0.1 to 0.8 mM) followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Results indicated maximum expression level of oGH at 5 hrs after induction of cells with 0.3 mM IPTG concentration with a molecular weight of 22 kDa. As for as cellular localization of protein is concerned accumulation of expressed oGH is observed in inclusion bodies. The successful expression of the cloned GH cDNA of sheep confirmed the functional viability of the clone. The above mentioned technique of genetic engineering has provided to boost the dairy industry by the production of large quantities of recombinant bovine somatotropin (rbST).

Bacillus thuringiensis (Bt) strains may express several insecticidal proteins with synergistic features, achieving high insecticidal toxicity and delaying development of resistance in insect pests. Previous work showed that Cry9Aa and Vip3Aa proteins present synergistic activity against Chilo suppressalis. In this study, genome-wide analysis of 489 Bt genomes revealed that cry9A was associated with the vip3A gene in seven Bt strains. Among all Bt genomes analyzed, not a single strain was found to have the cry9A gene alone without the presence of the vip3A gene. The complete genome sequencing of two Bt strains, 4AP1 and 4AO1, revealed that cry9A and vip3A genes were located in the same plasmid in both strains. The genome context analysis suggested a recombination mechanism responsible for the insertion of the cry9A gene into the plasmid containing vip3A. The coexistence of Cry9A with Vip3A proteins in strain 4AP1 was confirmed by liquid chromatography-tandem mass spectrometry and western blot analyses. Furthermore, another Cry9 protein codified by the gene in the identical plasmid also showed synergistic activity with the Vip3A protein. Overall, our results support that cry9 genes coexisted with vip3A and that complete genome sequencing combined with protein expression analysis may be used to identify associations of insecticidal proteins with potential synergistic toxicity.

Background CTCF encodes 11-zinc finger protein which is implicated in multiple tumors including the carcinoma of the breast. The Present study investigates the association of CTCF mutations and their expression in breast cancer cases. Methods A total of 155 breast cancer and an equal number of adjacent normal tissue samples from 155 breast cancer patients were examined for CTCF mutation(s) by PCR-SSCP and automated DNA sequencing. Immunohistochemistry (IHC) method was used to analyze CTCF expression. Molecular findings were statistically analyzed with various clinicopathological features to identify associations of clinical relevance. Results Of the total, 16.1% (25/155) cases exhibited mutation in the CTCF gene. Missense mutations Gln > His (G > T) in exon 1 and silent mutations Ser > Ser (C > T) in exon 4 of CTCF gene were analyzed. A significant association was observed between CTCF mutations and some clinicopathological parameters namely menopausal status (p = 0.02) tumor stage (p = 0.03) nodal status (p = 0.03) and ER expression (p = 0.04). Protein expression analysis showed 42.58% samples having low or no expression (+), 38.0% with moderate (++) expression and 19.35% having high (+++) expression for CTCF. A significant association was found between CTCF protein expression and clinicopathological parameters include histological grade (p = 0.04), tumor stage (p = 0.04), nodal status (p = 0.03) and ER status (p = 0.04). Conclusions The data suggest that CTCF mutations leading to its inactivation significantly contribute to the progression of breast cancer.

We developed a normalization method utilizing the expression levels of a panel of endogenous proteins as normalization standards (EPNS herein). We tested the validity of the method using two sets of tandem mass tag (TMT)-labeled data and found that this normalization method effectively reduced global intensity bias at the protein level. The coefficient of variation (CV) of the overall median was reduced by 55% and 82% on average, compared to the reduction by 72% and 86% after normalization using the upper quartile. Furthermore, we used differential protein expression analysis and statistical learning to identify biomarkers for colorectal cancer from a CPTAC data set. The expression changes of a panel of proteins, including NUP205, GTPBP4, CNN2, GNL3, and S100A11, all of which highly correlate with colorectal cancer. Applying these five proteins as model features, random forest modeling obtained prediction results with the maximum AUC of 0.9998 using EPNS-normalized data, comparing favorably to the AUC of 0.9739 using the raw data. Thus, the normalization method based on EPNS reduced the global intensity bias and is applicable for quantitative proteomic analysis.

Receptor-ligand complex mediated signaling significantly contributesin cellular activities such as growth, proliferation, differentiation, and survival. However, augmented expression of signal transducing receptors and ligands is the most frequent molecular event and major hallmark of oral carcinogenesis. Among these receptors, Epidermal Growth Factor Receptor (EGFR) with intracellular tyrosine kinase activity is the most frequently overexpressed molecule by Squamous epithelial cells of oral cavity. Aberrated EGFR mediated signaling has laid the foundation of targeted therapy thus providing rationale for the conducted study. We have selected EGFR pathway as targeted intracellular signaling cascade inOral squamous cell carcinoma (OSCC). Deactivating EGFR by blocking the binding sites is likely to result in prevention of intracellular downstream signaling. In this context, Tyrosine Kinase Inhibitors (TKIs) have come into play. Quinazolines (aromatic heterocyclic compounds) and their derivatives have shown promising clinical outcomes. Present study focused to investigate anti-EGFR potential of quinazoline derivative, varlitinib-a pan-EGFR inhibitor on oral squamous epithelial cell lines. We performed proteomic analyses to identify differential expression pattern of proteins in SCC-25 cells in response to varlitinib treatment. Identified proteins include Binding Immunoglobulin Protein (BiP), Heat Shock Protein 7C (HSP7C), Protein Disulfide Isomerase 1 A (PDIA1), Vimentin (VIME), Keratin type I Cytoskeletal 14 (K1C14), and β-Actin (ACTB). Among these, five proteinswere found to be downregulated upon varlitinib treatment whereas only Keratin type I Cytoskeletal 14 was upregulated. Differential expression of proteins and possible role of varlitinib as potential antitumor drug in oral carcinoma is discussed.

SUMMARY We conducted PrediXcan analysis of hydrocephalus risk in ten neurological tissues and whole blood. Decreased expression of MAEL in the brain was significantly associated (Bonferroni-adjusted p < 0.05) with hydrocephalus. PrediXcan analysis of brain imaging and genomics data in the independent UK Biobank (N = 8,428) revealed that MAEL expression in the frontal cortex is associated with white matter and total brain volumes. Among the top differentially expressed genes in brain, we observed a significant enrichment for gene-level associations with these structural phenotypes, suggesting an effect on disease risk through regulation of brain structure and integrity. We found additional support for these genes through analysis of the choroid plexus transcriptome of a murine model of hydrocephalus. Finally, differential protein expression analysis in patient cerebrospinal fluid recapitulated disease-associated expression changes in neurological tissues, but not in whole blood. Our findings provide convergent evidence highlighting the importance of tissue-specific pathways and mechanisms in the pathophysiology of hydrocephalus., Graphical abstract, In brief Hale et al. present an integrated omics approach to characterize the genetic basis of hydrocephalus. They reveal tissue-specific genetic associations and enrichment of genes associated with human brain structure phenotypes. Validation of hydrocephalus-associated genes in mouse choroid plexus and human cerebrospinal fluid supports polygenic contributions to hydrocephalus risk.

Triple negative breast cancer (TNBC) harbors a heterogeneous group of carcinomas with poor prognosis and high genetic variability. As a potential aim for targeted therapy, genetic mutations leading to an activation of the phosphoinositide 3-kinase pathway in a catalytic subunit (PIK3CA) in breast cancer have been analyzed currently. Little is known about the clinical impact and prognostic or predictive value of this marker in TNBC subtypes. Samples from 119 TNBC cases were submitted to immunohistochemical PIK3CA protein expression analysis and scored semi-quantitatively as negative, weak (1 +), or strongly expressed (2 +). Expression scores were correlated to patient’s characteristics, imaging features, and TNBC subtypes. TNBC subtypes were categorized into four subtypes: basal like, mesenchymal like, luminal androgen receptor (LAR), and immunomodulatory. We did not observe differences in clinical aspects and imaging features between TNBC with and without PIK3CA expression. PIK3CA expression was in general higher in the LAR subtype. The disease-free survival and overall survival were significantly better in TNBC with PIK3CA protein expression, independent of TNBC subtypes. Despite conflicting results in the literature, our study clearly shows a better outcome of PIK3CA-expressing TNBC, independent of TNBC subtypes. PIK3CA expression in TNBC is not associated with specific clinical or diagnostic features. Further molecular studies and meta-analysis are warranted to clarify the prognostic and predictive role of PIK3CA protein expression.

Inhibitory compounds liberated from lignocellulose pretreatment are representative toxic chemicals that repress microbial growth and metabolism. A tolerant strain of the industrial yeast Saccharomyces cerevisiae is able to detoxify a major class of toxic compounds while producing ethanol. Knowledge on the yeast tolerance was mostly obtained by gene expression analysis and limited protein expression evidence is yet available underlying the yeast adaptation. Here we report a comparative protein expression profiling study on Y-50049, a tolerant strain compared with its parental industrial type strain Y-12632. We found a distinctive protein expression of glucose-6-phosphate dehydrogenase (Zwf1) in Y-50049 but not in Y-12632, in the relatively conserved glycolysis and pentose phosphate pathway (PPP) in response to a combinational challenge of 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF). A group of proteins with aldehyde reduction activity was uniquely induced expressed in Y-50049 but not in Y-12632. Such evidence allowed fine-tuning a mechanism of the renovated in situ detoxification by Y-50049. As the key protein, Zwf1 drove the glucose metabolism in favor of the oxidative branch of the PPP facilitating in situ detoxification of the toxic chemicals by Y-50049. The activated expression of Zwf1 generated the essential cofactor nicotinamide adenine dinucleotide phosphate (NADPH) enabling reduction of furfural and HMF through a group of aldehyde reduction enzymes. In return, the activate aldehyde reductions released desirable feedbacks of NADP+ stimulating continued oxidative activity of Zwf1. Thus, a well-maintained cofactor regeneration cycle was established to restore the cofactor imbalance caused by furfural-HMF. Challenges and perspectives on adaptation of significantly differential expressions of ribosomal proteins and other unique proteins are also discussed.

Uterine cervical carcinoma (CACX) is one of the leading causes of deaths in Indian women. Chromosomal alterations including 11p15.5 locus were reported in CACX. Consequently, we strived for the first time to understand the molecular status of the candidate gene Insulin-like growth factor 2, IGF2 (11p15.5) in Indian CACX patients (n = 128). DNA copy number (CN) analysis using CGH-SNP analysis showed no genetic alteration and it was further validated by comparison with publicly available CN datasets. But promoter hypo-methylation during the progression of CACX was observed and also found to be concordant with publicly available DNA methylation datasets. Interestingly, we found diverse expression of IGF2 transcript in both normal cervical epithelium (NCE) and CACX tumors. Similar heterogeneous expression pattern was seen in publicly available expression datasets as well. Finally, protein expression analysis in NCE showed concordance with transcript expression but tumors showed frequent low expression. Log-rank test showed a difference (p-value = 0.057) in overall survival between cases with and without alteration for IGF2 in Indian CACX patients. Collectively, our study proposes that regulation of IGF2 expression in NCE appeared to be multifaceted and deregulation during the development of CACX resulted in the differential expression.

Purpose5-aminolevulinic acid (5-ALA) fluorescence-guided surgery (FGS) appears to be a promising treatment for glioma. However, 5-ALA-mediated fluorescence cannot always be detected in grade II/III gliomas. We hypothesized that gene expression patterns in the Protoporphyrin IX (PpIX) synthesis pathway may be associated with intraoperative fluorescence status of grade II/III gliomas, and then attempted to identify the key molecule of 5-ALA-mediated fluorescence.MethodsUsing 50 surgically obtained specimens, which were diagnosed as grade II and III gliomas, we analyzed gene expression within the PpIX synthesis pathway to identify candidate molecules according to intraoperative 5-ALA-mediated fluorescence status. The most likely candidate gene was selected and confirmed by protein expression analysis. To evaluate the biological function of the molecule in PpIX synthesis, functional analysis was performed using specific, small interference (si)RNA in the SW-1783human grade III glioma cell line.ResultsAmong the genes involved in the porphyrin synthesis pathway, the mRNA expression of Peptide transporter 2 (PEPT2) in FGS fluorescence-positive gliomas was significantly higher than that in fluorescence-negative gliomas. Protein expression of PEPT2 was also significantly higher in the fluorescence-positive gliomas, which was confirmed by western blot analysis and immunofluorescence analysis. The siRNA-mediated downregulation of the mRNA and protein expression of PEPT2 led to decreased PpIX fluorescence intensity, as confirmed by fluorescence spectrum analysis.ConclusionsThe results suggest PEPT2 is an important candidate molecule in 5-ALA-mediated FGS in grade II/III gliomas. As the overexpression of PEPT2 was associated with higher PpIX fluorescence intensity, PEPT2 may improve fluorescence-guided resection in grade II/III gliomas.

Background: Glycogen storage disease type IV (GSD IV), caused by GBE1 mutations, has a quite wide phenotypic variation. While the classic hepatic form and the perinatal/neonatal neuromuscular forms result in early mortality, milder manifestations include non-progressive form (NP-GSD IV) and adult polyglucosan body disease (APBD). Thus far, only one clinical case of a patient with compound heterozygous mutations has been reported for the molecular analysis of NP-GSD IV. This study aimed to elucidate the molecular basis in a NP-GSD IV patient via protein expression analysis and to obtain a clearer genotype-phenotype relationship in GSD IV. Case presentation: A Japanese boy presented hepatosplenomegaly at 2 years of age. Developmental delay, neurological symptoms, and cardiac dysfunction were not apparent. Observation of hepatocytes with periodic acid-Schiff-positive materials resistant to diastase, coupled with resolution of hepatosplenomegaly at 8 years of age, yielded a diagnosis of NP-GSD IV. Glycogen branching enzyme activity was decreased in erythrocytes. At 13 years of age, he developed epilepsy, which was successfully controlled by carbamazepine. Molecular analysis: In this study, we identified compound heterozygous GBE1 mutations (p.Gln46Pro and p.Glu609Lys). The branching activities of the mutant proteins expressed using E. coli were examined in a reaction with starch. The result showed that both mutants had approximately 50% activity of the wild type protein. Conclusion: This is the second clinical report of a NP-GSD IV patient with a definite molecular elucidation. Based on the clinical and genotypic overlapping between NP-GSD IV and APBD, we suggest both are in a continuum. Keywords: Epilepsy, Functional analysis, Glycogen storage disease type IV, Glycogenosis, glycogen branching enzyme 1, GSD

p300/CBP Associated Factor (PCAF), a GNAT family member protein, represent a valid target for therapeutic interventions since its dysfunction has implicated in variety of diseases like cancer, diabetes, inflammatory diseases, etc. Despite its potential for therapeutics, only a small number of PCAF inhibitors were reported. Hence, in this study, the catalytic domain of PCAF was explored to screen novel, potent and cell permeable inhibitor from three small molecule databases like Life Chemical, Maybridge and Chembridge by using Structure Based Virtual Screening (SBVS) method. Further, Induced Fit Docking, Binding Free Energy calculation, Single Point Energy calculation and Molecular Dynamics Simulation were performed on selected hits. In silico results revealed that F2209-0381 has higher binding energy of −109.722 and have greater cell permeability (QPPCaco = 1456.764; QPPMDCK = 742.941) than rest of hits. Cytotoxicity effect and protein expression analysis of F2209-0381 on A549 cells reveals that it exhibited strong inhibition with IC50 value of 58.31 μg/ml and significantly reduced the expression of PCAF after 72 h time point. Thus, this study warrants that F2209-0381 could become a novel, potent and cell permeable drug of PCAF thereby it could combat its mediated diseases.

ObjectiveThe phenotypic and pathological features of small cell cervical carcinoma (SMCC) and small small cell lung cancer (SCLC) are very similar; thus, the chemotherapy regimens used for the rare SMCC have been routinely based on regimens used for common SCLC. We set out to explore the protein expression profile similarities between these 2 cancers to prove that linking their therapeutic regimens is justified, with a secondary aim of finding tumor-specific proteins to use as additional biomarkers for more accurate diagnosis of SMCC, and potentially to use as therapeutic targets.MethodsProtein expression analysis was performed for 3 cases of SMCC and 1 example each of SCLC, mucinous adenocarcinoma of the cervix (MACC), lung mucinous adenocarcinoma (MACL), and squamous cell carcinoma of the cervix (SCC). We used cancer tissue–originated spheroids (CTOS) and isobaric tags for relative and absolute quantitation (iTRAQ)–based comprehensive and quantitative protein expression profile analysis. Expression in corresponding clinical samples was verified by immunohistochemistry.ResultsRather than organ of origin–specific patterns, the SMCC and SCLC samples revealed remarkably similar protein expression profiles—in agreement with their matching tumor pathology phenotypes. Sixteen proteins were expressed at least 2-fold higher in both small cell carcinomas (SMCC and SCLC) than in MACC or SCC. Immunohistochemical analysis confirmed higher expression of creatine kinase B-type in SMCC, compared with MACC and SCC.ConclusionsWe demonstrate a significant overlapping similarity of protein expression profiles of lung and cervical small cell carcinomas despite the significant differences in their organs of origin.

Administration of a compound can induce drug-metabolizing enzymes (DMEs) in the liver. DME induction can affect various parameters in toxicology studies. Therefore, evaluation of DME induction is important for interpreting test compound-induced biological responses. Several methods such as measurement of hepatic microsomal DME activity using substrates, electron microscopy, or immunohistochemistry have been used; however, these methods are limited in throughput and specificity or are not quantitative. Liquid chromatography mass spectrometry (LC/MS)-based protein analysis can detect and quantify multiple proteins simultaneously per assay. Studies have shown that formalin-fixed paraffin-embedded (FFPE) samples, which are routinely collected in toxicology studies, can be used for LC/MS-based protein analysis. To validate the utility of LC/MS using FFPE samples for quantitative evaluation of DME induction, we treated rats with a DME inducer, phenobarbital, and compared the protein expression levels of 13 phase-I and 11 phase-II DMEs between FFPE and fresh frozen hepatic samples using LC/MS. A good correlation between data from FFPE and frozen samples was obtained after analysis. In FFPE and frozen samples, the expression of 6 phase-I and 8 phase-II DMEs showed a similar significant increase and a prominent rise in Cyp2b2 and Cyp3a1 levels. In addition, LC/MS data were consistent with the measurement of microsomal DME activities. These results suggest that LC/MS-based protein expression analysis using FFPE samples is as effective as that using frozen samples for detecting DME induction.

Dissection of gene function requires sophisticated tools to monitor gene expression. Gene tagging with epitope peptides and fluorescent protein tags is a routine method to investigate protein expression using tag-specific antibodies and western blotting with tedious blotting and immunodetection steps. Nanoluciferase (NanoLuc) exhibits extremely bright bioluminescence and is engineered as a sensitive genetic reporter. Due to its small size and high bioluminescent activity, NanoLuc could be engineered to function as a novel protein tag that permits direct detection of tagged protein in the gel matrix (in-gel detection). In this study, we developed Gateway compatible vectors to tag proteins with NanoLuc in plants. We also tailored the in-gel detection conditions which can detect NanoLuc-tagged MPK3 from as low as 200 pg of total protein extracts. Compared to FLAG tag and western blotting-based detection, NanoLuc tag and optimized in-gel detection exhibit increased detection sensitivity but omit the blotting and immunodetection steps. We also demonstrated versatile applications of the NanoLuc-based in-gel detection method for protein expression analysis, probing protein-protein interactions by coimmunoprecipitation, and in vivo protein phosphorylation detection with Phos-tag gel electrophoresis. Finally, NanoLuc was used to tag the gene at its endogenous locus using the wheat dwarf virus replicon and CRISPR/Cas9-mediated gene targeting. Our data suggest that NanoLuc tag and in-gel detection permit fast detection of tagged protein with high sensitivity. The versatile NanoLuc toolkit and convenient in-gel detection method are expected to facilitate in vitro and in vivo protein analysis for plant functional genomics. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s11032-021-01210-7.

Aim: This study aims to perform differential protein expression analysis of serum samples from Oral Squamous Cell Carcinoma (OSCC) patients and healthy controls in search of potential diagnostic and/or prognostic biomarker(s). Objective: OSCC is usually diagnosed late, which results in poor survival and high mortality. Identification of non-invasive prognostic biomarkers is of utmost importance for early diagnosis and proper management of the disease; hence we used a proteomic approach to identify potential biomarkers from serum. Methods: Serum samples (OSCC n=45 and control n=30) were depleted, and proteins were separated using 2-D gel electrophoresis followed by identification by mass spectrometric analysis. Gene expression analysis of identified proteins in malignant and normal tissue was also performed to complement proteomics studies. Results: Among differentially expressed proteins, up-regulation of heat shock protein alpha (HSP90α) from the serum of oral cancer patients was observed. We also observed elevated levels of Haptoglobin (HP) along with downregulation of Type II keratin cytoskeletal 1(KRT1) and serum albumin (ALB) in oral cancer patients. Gene expression studies on identified proteins in malignant and normal tissue revealed a similar pattern with the exception of KRT1. We believe that elevated levels of serum HSP90 alpha might be used as a potential biomarker. Conclusion: Our findings suggest a contribution of HSP90 alpha and other identified proteins in oral pathology as pro/anti-apoptotic modulators, thus considering their potential as predictive biomarkers.

Nanos3, a zinc finger RNA-binding protein, suppresses the apoptosis in primordial germ cells (PGCs) during migration to gonads and maintains the PGC population. The genetic variations and expression of NANOS3 in patients with non-obstructive azoospermia (NOA) were evaluated in this study. The study included 100 idiopathic infertile men with NOA and 100 fertile men as the as the case and control groups, respectively. NANOS3 gene variations were analyzed using the standard polymerase chain reaction (PCR) and sequencing. For mRNA and protein expression analysis, testicular biopsy specimens from 27 patients including 9 obstructive azoospermia (OA), 9 maturation arrest (MA), and 9 Sertoli cell-only syndromes (SCOS) were collected and evaluated using the real-time PCR technique and immunohistochemistry. Although the evaluation of the 5`UTR regulatory region has shown the significant difference in the numbers of TG repeats in rs11182456 between groups, the odd ratio was not strong enough to consider that as a certain risk factor lead to azoospermia and infertility. Meanwhile, NANOS3 expression at mRNA level had a significant difference among OA, SCOS, and MA groups.

Background and Objective. Glutamate-rich WD repeat-containing protein 1 (GRWD1), as a novel CDT1-binding protein, plays a critical role in the initial stage of DNA replication. To date, few studies have shown that GRWD1 is a driving factor for lung cancer, bladder cancer, and colorectal cancer. However, no systematic pancancer analysis has been carried out. This study was aimed at exploring its expression signature, prognostic value, immune infiltration pattern, and biological functions using bioinformatics tools. Methods. The expression level of GRWD1 in human cancer tissues was analyzed using the Tumor Immune Evaluation Resource (ver. 2.0, TIMER2), Gene Expression Profiling Interactive Analysis (ver. 2, GEPIA2), and UALCAN databases. The Kaplan-Meier plotter was utilized to analyze the survival data. Spearman’s correlation analysis was used to find out the correlation between the expression level of GRWD1 and predictive biomarkers, such as tumor mutation burden (TMB) and microsatellite instability (MSI). Furthermore, the MEXPRESS website was used to study the potential relationship between DNA methylation level of GRWD1 and pathological staging. We utilized the “immune” module provided on the TIMER2 website to explore the relationship between the expression level of GRWD1 and immune infiltration in all types of cancer in TCGA. Pearson’s correlation analysis was used to investigate the correlation between the expression level of GRWD1 and the expression levels of immune checkpoint-related genes. For protein expression analysis, we used the CPTAC module provided by the UALCAN portal to compare the total protein and phosphorylated protein level of GRWD1 in adjacent normal and tumor tissues. Results. GRWD1 was overexpressed in tissues of most types of cancer, in which the expression levels of GRWD1 in the kidney chromophobe (KICH), kidney renal papillary cell carcinoma (KIRP), and kidney renal clear cell carcinoma (KIRC) tissues showed an opposite trend, and the expression level of GRWD1 was correlated to only the KIRC tumor stage. The results of survival analysis showed that the expression level of GRWD1 was significantly associated with overall survival in six types of cancer and disease-free survival (DFS) in three types of cancer. Importantly, the increased expression level of GRWD1 was strongly correlated with prognosis of KIRC patients. There was a positive relationship between the expression level of GRWD1 and immune cell infiltration in several types of cancer, and the expression level of GRWD1 was also positively correlated with TMB, MSI, and DNA methylation in some types of cancer. The results of Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that “ubiquitin mediated proteolysis,” “spliceosome,” and “nucleotide excision repair” were involved in the effect of GRWD1 expression on tumor pathogenesis. Conclusion. This pancancer analysis provided a comprehensive overview of the carcinogenic effects of GRWD1 on a variety of human cancers. The results of bioinformatics analysis indicated GRWD1 as a promising biomarker for detection, prognosis, and therapeutic assessment of diverse types of cancer, and GRWD1 could act as a tumor suppressor in KIRC.

The importance of two-dimensional electrophoresis (2DE) in the life sciences, more precisely protein studies, is large. Although the 2DE technology has shortcomings, however, due to significant 2DE possibilities, this technology still is irreplaceable. Thus we focus on the analysis of 2DE gels (2DEG) and its automation capabilities. In Chapter 6 are formulated the efficiency and reliability of 2DEG analysis evaluation criteria and based on them proposed automatic 2DEG image analysis strategy. The strategy consists of two essential steps: image matching and protein expression analysis. In order to eliminate geometric distortions in gel images, new 2DEG image matching solutions are proposed and investigated. The most problematic protein expression analysis part—2DEG image segmentation, is approached presenting novel image segmentation into meaningful areas algorithms and protein spot modeling studies involving protein spots reconstruction and parameterization.

Summary Proteomics is a powerful tool for protein expression analysis and is becoming more readily available to researchers through core facilities or specialized collaborations. However, one major bottleneck for routine implementation and accessibility of this technology to the wider scientific community is the complexity of data analysis. To this end, we have created ProVision, a free open-source web-based analytics platform that allows users to analyze data from two common proteomics relative quantification workflows, namely label-free and tandem mass tag-based experiments. Furthermore, ProVision allows the freedom to interface with the data analysis pipeline while maintaining a user-friendly environment and providing default parameters for fast statistical and exploratory data analysis. Finally, multiple customizable quality control, differential expression plots as well as enrichments and protein–protein interaction prediction can be generated online in one platform. Availability and implementation Quick start and step-by-step tutorials as well as tutorial data are fully incorporated in the web application. This application is available online at https://provision.shinyapps.io/provision/ for free use. The source code is available at https://github.com/JamesGallant/ProVision under the GPL version 3.0 license.

The ability to reprogram human somatic cells into human induced pluripotent stem cells (hiPSCs) has enabled researchers to generate cell types in vitro that have the potential to faithfully recapitulate patient-specific disease processes and phenotypes. hiPSC-derived cardiomyocytes (hiPSC-CMs) offer the promise of in vitro patient- and disease-specific models for drug testing and the discovery of novel therapeutic approaches for treating cardiovascular diseases. While methods to differentiate hiPSCs into cardiomyocytes have been demonstrated, the heterogeneity and immaturity of these differentiated populations have restricted their potential in reproducing human disease and the associated target cell phenotypes. These barriers may be overcome through comprehensive single-cell characterization to dissect the rich heterogeneity of hiPSC-CMs and to study the source of varying cell fates. In this study, we optimized and validated a new Single-Cell Western method to assess protein expression in hiPSC-CMs. To better understand distinct subpopulations generated from cardiomyocyte differentiations and to track populations at single-cell resolution over time, we measured and quantified the expression of cardiomyocyte subtype-specific proteins (MLC2V and MLC2A) using Single-Cell Westerns. By understanding their heterogeneity through single-cell protein expression and quantification, we may improve upon current cardiomyocyte differentiation protocols, generate hiPSC-CMs that are more representative of in vivo derived cardiomyocytes for disease modeling, and utilize hiPSC-CMs for regenerative medicine purposes. Single-Cell Westerns provide a robust platform for protein expression analysis at single-cell resolution.

Down Syndrome (DS) is one of the most common disorders caused by the presence of an extra copy of chromosome 21. It has been shown that the expression of various genes located on chromosomes other than the extra 21 chromosomes is affected in DS. Given the practical and ethical difficulties in human tissue studies, the Ts65Dn mouse model has been widely used in DS research. In this study, we propose a pipeline composed of a supervised learning approach, modularity analysis of a bipartite network, and multivariate analysis of variance (MANOVA), for identification of differentially expressed proteins (DEP) among different classes of mice models. The proposed pipeline is tested using the expression levels of 77 proteins in eight different classes of mice models. The data includes the protein expression measurements for 34 trisomic Ts65Dn and 38 control mice. Each group is broken up into four classes based on either being stimulated for learning or not, each injected with memantine or saline. The previously proposed approaches have been unable to identify DEP among all of the eight classes simultaneously. Here, we show that our proposed pipeline can successfully identify the set of proteins expressed differently among all the eight classes. The findings of this study can inform the study of learning responses to different treatments and protein-treatment associations in DS. Also, the proposed pipeline can be adopted to identify DEP in DS or other diseases and health conditions, which can consequently inform the development of improved personalized treatment and management strategies.

Background: Prostate cancer (PC) is the most frequently diagnosed cancer among men worldwide. The poor prognosis of PC is largely due to late diagnosis of the disease when it has progressed to advanced stages marked by androgen-independence. We interrogated proteomic signatures that embody the transition of PC from an androgen-dependent (AD) to an androgen-independent (AI) state. Methods: We have previously established AD and AI murine PC cell lines, PLum-AD and PLum-AI, respectively, which recapitulate primary and progressive PC at phenotypic and subcellular levels. We statistically surveyed global protein expression profiles in these cell lines. Differential profiles were functionally interrogated by pathways and protein&ndash, protein interaction network analyses. Results: Protein expression pattern analysis revealed a total of 683 proteins, among which 99 were significantly differentially altered in PLum-AI cells as compared to PLum-AD cells (45 increased and 54 decreased). Principal component analysis (PCA) revealed that the two different cell lines clearly separated apart, indicating a significant proteome expression difference between them. Four of the proteins (vimentin, catalase, EpCAM, and caspase 3) that were differentially expressed in PLum-AI cells compared to PLum-AD cells were subjected to biochemical validation by Western blotting. Biological process gene ontology (GO) analysis of the differentially expressed proteins demonstrated enrichment of biological functions and pathways in PLum-AI cells that are central to PI3 kinase and androgen receptor pathways. Besides, other relevant biological processes that are enriched in PLum-AI cells included cell adhesion and cell migration processes, cell and DNA damage, apoptosis, and cell cycle regulation. Conclusions: Our protein expression analysis of a murine in vitro model of PC progression identified differential protein spots that denote this progression and that comprise high-potential targets for early treatment of PC with a personalized patient-specific approach. Efforts are underway to functionally assess the potential roles of these proteins as therapeutic targets for PC progression.

Isolated brain capillaries are essential for analyzing the changes of protein expressions at the blood–brain barrier (BBB) under pathological conditions. The standard brain capillary isolation methods require the use of at least five mouse brains in order to obtain a sufficient amount and purity of brain capillaries. The purpose of this study was to establish a brain capillary isolation method from a single mouse brain for protein expression analysis. We successfully isolated brain capillaries from a single frozen mouse brain by using a bead homogenizer in the brain homogenization step and combination of cell strainers and glass beads in the purification step. Western blot and proteomic analysis showed that proteins expressed at the BBB in mouse brain capillaries isolated by the developed method were more enriched than those isolated from a pool of five mouse brains by the standard method. By using the developed method, we further verified the changes in expression of BBB proteins in Glut1-deficient mouse. The developed method is useful for the analysis of various mice models with low numbers and enables us to understand, in more detail, the physiology and pathology of BBB.

Next generation sequencing (NGS) together with protein expression analysis is back bone of molecularly targeted therapy in precision medicine. Our retrospective study shows our experience with NGS of 324 genes in combination with protein expression in patients with advanced breast cancer (aBC). The primary purpose was to analyze the prevalence of individual genetic alterations combined with protein expression to define potential targets for an individualized therapy. Between April 2018 and September 2019, 41 patients with aBC were offered a NGS test. The test was used to detect clinically relevant genomic alterations and to support further targeted therapy decisions. Hormone receptors, ERBB2 of tumors and PD-L1 was stained by immunohistochemistry. The data was recorded up to September 2019. After prior consent 41 results were available for further analysis. The most common BC subtypes were triple-negative (n = 16), HR+/ERBB2− (n = 15), and ERBB2+ (n = 9), with one missing data of the primary tumor. 27 patients had more than one genetic alteration. The most common alterations were PIK3CA (n = 14) and ERBB2 alterations (n = 11). Followed by ESR1 (n = 10), FGFR1 (n = 7) and PTEN (n = 7). 68% of the alterations were clinically relevant (tier I and II of ESCAT classification). The most common treatment recommendation was ERBB2-directed therapy (single or double blockade, trastuzumab emtansine and lapatinib) followed by alpelisib in combination with fulvestrant. Comprehensive genomic profiling combined with protein expression analysis in aBC allowed a guided personalized therapy for half of our patients. So far there are no well-defined tools allowing interpretations of genomic alterations detected by NGS in combination with protein expression and other factors.

Mismatch repair (MMR) pathway is one of the underlying mechanisms of predisposition to breast cancer (BC). The present study explored the association of MSH2 exonic deletions, respective survival analysis, protein structure prediction, transcription profiling, and expression analysis with BC risk. Genotyping analysis of 493 BC cases and 387 controls confirmed the association of two MSH2 exonic deletions, i.e., exon 3 (OR:6.4, CI = 3.4-12.1) and 9 (OR:7.8, CI = 4.1-14.8) with BC risk. In order to confirm the phenotypic-genotypic relationship, we have performed MSH2 transcriptomic (p < 0.05) and protein expression analysis (OR:30, CI = 4-230) which further confirmed its downregulation/loss in BC biopsy samples highlighting potential role in the onset of breast carcinogenesis. Additionally, we have presented that MSH2 mutations can alter the expression profile of other BC associated biomarkers like ER, PR, CK-7, GATA-3, and E-cadherin. Subsequently, the effect of exonic deletions on secondary structure of protein has shown missing of beta and alpha helices in their protein products via in-silico analysis. However, loss of exon 3 results in the altered core protein structure leading to dysfunction protein, possible cause of BC development. No association of MSH2 exonic deletions with survival statistics was observed conceivably due to the shorter follow-up time. Thus, our results at genetic, transcriptomic, and proteomic levels confirmed the downregulated MSH2, emphasizing its potential contribution in MMR mechanisms for breast tumorigenesis. In conclusion, MSH2 deficiency may cause breast cancer development and progression.

Differential analysis of MS-data to identify biomarkers or to understand biology is a cornerstone in proteomics. DEqMS is a robust method for analysis of both labelled and label-free MS-data. The method takes into account the inherent dependence of protein variance on the number of PSMs or peptides used for quantification, thereby providing a more accurate variance estimation. Compared to current methods, DEqMS achieves better accuracy and statistical power in quantitative proteomics. The tool is available as user-friendly R package., Graphical Abstract Highlights DEqMS is a method for statistical analysis of quantitative MS-data. Variance estimates based on the actual MS-data structure. Improved statistical power and accuracy in protein differential analysis. DEqMS is available as a user-friendly R package in Bioconductor., Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all data sets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several data sets including E. coli proteome spike-in data, using both label-free and TMT-labeled quantification. Compared with previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared with other statistical methods in both label-free and labeled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.

Cancer immunotherapy, including vaccination, is considered a major scientific and medical breakthrough. However, cancer immunotherapy does not result in durable objective responses against colorectal cancer (CRC). To improve the efficacy of immunotherapy, the present study investigated several biomarkers for selecting patients who were expected to respond well to immunotherapy. Firstly, a comprehensive proteomic analysis was performed using tumor tissue lysates from patients enrolled in a phase II study, in which five human leukocyte antigen (HLA)-A*24:02-restricted peptides were administered. Sialic acid-binding immunoglobulin type lectin (Siglec)-7 was identified as a potential predictive biomarker. Subsequently, this biomarker was validated using western blot analysis, and immunofluorescence using tissue samples from the patients enrolled in the phase II study. The expression levels of Siglec-7 detected by immunofluorescence were quantified and their association with overall survival (OS) in patients treated with the peptide vaccine was examined. Furthermore, considering the important role of tumor-infiltrating lymphocytes (TILs) for CRC prognosis, the densities of CD3+, CD4+, CD8+ and forkhead box P3 (FOXP3)+ T cells in CRC tissues were examined and compared with Siglec-7 expression. The mean expression levels of Siglec-7 were significantly higher in patients with poor prognosis, with an OS of ≤2 years, as shown in comprehensive proteomic analysis (P=0.016) and western blot analysis (P=0.025). Immunofluorescence analysis demonstrated that Siglec-7 was expressed in intratumoral macrophages. The OS in patients with high Siglec-7 expression was significantly shorter than in that in patients with low Siglec-7 expression (P=0.017) in the HLA-A*24:02-matched patients. However, this difference was not observed in the HLA-unmatched patients. There was no significant difference in OS between patients according to the numbers of TILs, nor significant correlation between TILs and Siglec-7 expression. In conclusion, Siglec-7 expression in macrophages in tumor tissue may be a novel predictive biomarker for the efficacy of immunotherapy against metastatic CRC.

Purpose Temporal lobe epilepsy (TLE) is the most common type of epilepsy. Hippocampal sclerosis is the most distinctive pathological feature of TLE; however, its role in the pathogenesis of TLE remains to be clarified. We performed global protein expression analysis of hippocampus from TLE patients and controls, aiming to reveal the molecular signaling pathways related to TLE. Method Proteomic and bioinformatic analyses of the hippocampus were performed on 4 TLE and 4 control samples. High-resolution liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS), in combination with TMT-6plex quantification, was applied for global protein expression analysis. The proteomics results were validated by Western blot with 25 TLE and 25 control individuals and Immunohistochemistry analysis with 33 TLE and 10 control individuals. Results Bioinformatics analysis demonstrated differentially expressed proteins in the synaptic vesicle pathway, the prostaglandin synthesis and regulation pathway and endocannabinoids and retrograde modulation of synaptic transmission pathway. Among these, excitatory amino acid transporter 1 (EAAT1) and Vesicular glutamate transporter 1 (VGLUT1) are critical for TLE and dysregulated expression might be closely related to the uptake of extracellular glutamate and contribute to the pathophysiology of TLE. Ras-related protein Rab-3A (RAB3A) downregulation might indicate the TLE-induced compensatory deficit in glutamate release. Conclusion Our study indicates that expression of some proteins involved in the packaging of vesicular neurotransmitters is altered in TLE. In addition, upregulated expression of annexin family proteins, which are also related to TLE, might play an important role in protection against TLE.

Bcl-2 protein (B-cell lymphoma 2) is one of the proteins in the Bcl-2 protein family that are anti apoptotic and encoded by the BCL-2 gene. This protein controls the permeability of the outer membrane of the mitochondria, which plays a role in the release of cytochrome c and several other proteins that contribute to cell death. Bcl-2 protein plays a role in regulating cell death and regulating the balance between new and old cells. Several studies showed that changes in the structure and function of Bcl-2 play a role in cell apoptosis disorders, producing cancer cells. High expression of Bcl-2 protein is seen in lung, breast, lymph node and brain cancers. Analysis of Bcl-2 protein expression at the molecular level in cancer cell is needed to determine the expression level of this protein. This study aims to produce the Bcl-2 primer for qPCR method that specifically identifies the target gene, and can be used to see the BCL-2 gene expression level in cells. A BCL-2 sequence was obtained from the National Center for Biotechnology Information (NCBI). The next process is further analysis with Primer-BLAST (NCBI). Some primer candidates are produced and undergo another selection based on several criteria. Finally, one pair of potential primers was obtained for the reaction of qPCR to BCL-2. Primer specificity was tested with Basic Local Alignment Tools (BLAST) tools and result in specific primer for human BCL-2 gene. Annealing temperature optimization shows that the primer can react optimally at 59∘C. Keywords: Bcl-2 protein, BCL-2 gene, qPCR, primer, gene expression, apoptosis

Gegenwärtig besteht die Hauptproblematik der Krebstherapeutika darin, dass (neben rascher Resistenzentwicklung) Zytostatika zahlreiche unerwünschte Nebenwirkungen und Toxizität hervorrufen. Einer der Gründe hierfür ist die fehlender Tumorspezifität, welche dazu führt, dass nicht nur Krebsgeschwüre, sondern auch gesundes Gewebe von den Therapeutika angegriffen werden. Einen vielversprechenden Lösungsansatz stellen daher Prodrugsyteme dar, welche die spezifischen Eigenschaften des malignen Gewebes für eine tumorspezifische Arzneimittelfreisetzung ausnutzt. Dazu zählen unter anderem neuartige Albumin-bindenden Krebsmedikamente, welche spezifisch im Tumorgewebe akkumulieren und so an Tumorspezifität gewinnen. Allerdings fehlen für diese neue Medikamentenklasse Biomarker, welche für die Vorhersage des Ansprechens einer Krebserkrankung herangezogen werden können. Daher ist das Ziel dieser Arbeit, diverse Parameter der Albuminhomöostase in mehreren Kolon-, Lungen- und Ovarialkarzinomzelllinien zu analysieren und mit der bereits bekannten Wirksamkeit eines neuen Albumin-bindenden Krebsmedikaments zu korrelieren. Mithilfe von Fluoresceinisothiocyanat (FITC)-markiertem bovinen und humanen Albumin wurde sowohl durchflusszytometrisch als auch mikroskopisch die Albuminhomöostase in den Zelllinien untersucht. Zudem wurden Vergleichsuntersuchungen der Albuminaufnahmeergebnisse mit Proliferationsraten, KRAS-Mutationsstatus sowie Expressionsanalysen verschiedener Proteine (unter anderem FcRn, SPARC oder m-Tor) durchgeführt. Außerdem wurde die Albuminverteilung im Tumorgewebe in bereits verfügbaren histologischen Präparaten immunhistochemisch analysiert. Die Auswertungen der Durchflusszytometrieversuche erbrachte starke Unterschiede zwischen Gewebeherkunft und Albuminaufnahme. Während die Lungenkarzinomlinien geringere Aufnahmekinetiken zeigten, war diese bei den Ovarialkarzinomzellen erhöht. Als weiterer Parameter wurde die Wachstumsrate der verschiedenen Zelllinien evaluiert für Charakterisierung von schnell beziehungsweise langsam wachsend. Zusammenfassend konnte eine Korrelation einiger Zellen bezüglich Albuminhomöostase und Zellproliferationsrate festgestellt werden, deren weitere Analyse noch aussteht. Die Proteinexpressionen erbrachten zum Teil interessante Wechselbeziehungen zwischen Serumentzug und Expression, wie besonders an den Veränderungen der mTOR Aktivität in bestimmten Zellen entnommen werden kann. Eine Verbindung zwischen KRAS-Mutationsstatus und einer erhöhten Albuminhomöostase konnte nicht eindeutig nachgewiesen werden. Besonders interessant ist die (unerwartete) inverse Korrelation zwischen Albuminaufnahme und Tumoransprechen, welche in den Lungenkrebsmodellen beobachtet wurde. The main problem of current cancer therapeutics is that (in addition to rapid development of drug resistance) cytostatic drugs cause numerous undesired side effects and dose-dependent toxicity. One of the reasons for this is the lack of tumour specificity, which leads to the fact that not only cancerous tumors, but also healthy tissue is affected by the therapeutics. Therefore, prodrug systems, which exploit the specific properties of the malignant tissue for tumour-specific drug release, represent a promising approach. These include novel albumin-binding cancer drugs that accumulate specifically in the tumour tissue and by this become more tumour-specific. However, this new class of drugs lacks biomarkers that can be used to predict the response to cancer. The focus of this work is to analyse various parameters of albumin homeostasis in several colon, lung and ovarian cancer cell lines and to correlate them with the already known efficacy of a new albumin-binding cancer drug. Using fluorescein isothiocyanate (FITC)-labelled bovine and human albumin, the albumin homeostasis in the cell lines was investigated both, flow cytometrically and microscopically. In addition, comparative investigations of albumin uptake results with proliferation rates, KRAS mutation status and expression analyses of different proteins (including FcRn, SPARC or m-Tor) were performed. Furthermore, the albumin distribution in tumor tissue was analyzed immunohistochemically in already available histological preparations. The evaluation of the flow cytometry experiments revealed strong differences between tissue origin and albumin uptake. While the lung carcinoma lines showed lower uptake kinetics, it was increased in the ovarian carcinoma cells. As a further parameter, the growth rate of the different cell lines was evaluated for the characterization of fast and slow growing cells. In summary, a correlation of some cells with regard to albumin homeostasis and cell proliferation rate was found, which has to analyzed in detail in course of subsequent studies. The protein expressions revealed some interesting correlations between serum deprivation and protein expression, as can be seen especially in the changes in mTOR activity in certain cells. A link between KRAS mutation status and increased albumin homeostasis could not be clearly demonstrated. Of particular interest is the (unexpected) inverse correlation between albumin uptake and tumor response observed in lung cancer models.

Expression levels of cellular proteins can be affected by various perturbations, such as genetic knockout of interactors, drug treatments or cell stress. To specifically measure the effects on protein levels post-synthesis under different experimental conditions, it is important to compensate for transcriptional and other upstream changes. Here, we provide a protocol for a dual-fluorescence flowcytometry-based assay to determine protein levels. The protein of interest is genetically linked to enhanced GFP (eGFP) followed by a viral 2A self-cleaving peptide sequence and mCherry. As a result, translation of the reporter construct leads to two fluorescent protein products from the same mRNA template, which enables unambiguous protein expression analysis with normalization across samples.

Liquid chromatography using a tandem mass spectrometer (LC-MS/MS) is a method of proteomic analysis. A shotgun analysis by LC-MS/MS comprehensively identifies proteins from tissues and cells with high resolution. The hepatic function of mice with acute hepatitis following the intraperitoneal administration of CCL4 was improved by the tail vein administration of the culture conditional medium (CM) of human mesenchymal stem cells from adipose tissue (hMSC-AT). In this study, a secreted protein expression analysis of hMSC-AT was performed using LC-MS/MS; 128 proteins were identified. LC-MS/MS showed that 106 new functional proteins and 22 proteins (FINC, PAI1, POSTN, PGS2, TIMP1, AMPN, CFAH, VIME, PEDF, SPRC, LEG1, ITGBL, ENOA, CSPG2, CLUS, IBP4, IBP7, PGS1, IBP2, STC2, CTHR1, CD9) were previously reported in hMSC-AT-CMs. In addition, various proteins associated with growth (SAP, SEM7A, PTK7); immune system processes (CO1A2, CO1A1, CATB, TSP1, GAS6, PTX3, C1 S, SEM7A, G3P, PXDN, SRCRL, CD248, SPON2, ENPP2, CD109, CFAB, CATL1, MFAP5, MIF, CXCL5, ADAM9, CATK); and reproduction (MMP2, CATB, FBLN1, SAP, MFGM, GDN, CYTC) were identified in hMSC-AT-CMs. These results indicate that a comprehensive expression analysis of proteins by LC-MS/MS is useful for investigating new factors associated with cellular components, biological processes, and molecular functions.

ObjectivesDark-cutting beef is a meat quality defect in which meat does not display the marketable bright-red color. Although previous studies have indicated that the ultimate pH of dark-cutting beef is greater than normal, the mechanistic basis for the occurrence is not clear. Various mitochondrial and glycolytic enzymes/proteins are involved in muscle metabolism and lowering of pH. However, limited knowledge is currently available on the muscle protein profile differences between dark-cutting and normal-pH beef. The objective of the current study was to identify proteins related to the development of the dark-cutting condition by comparing the protein expression differences between dark-cutting and normal-pH beef.Materials and MethodsDark-cutting and normal-pH beef samples were collected from six (n = 6) different animals after slaughter. Tissue samples (0.5 g) were digested in 5 mL of lysis buffer. Tissue lysates were homogenized, boiled, sonicated using a bioruptor and centrifuged at 10,000 g for 10 min. Samples were digested with trypsin/Lys-C overnight at 37°C, after which additional 2 µg/mL of protease was added and digestion was continued for another 8h. The resulting trypsinolytic peptides were acidified to 1% trifluoroacetic acid and purified by solid phase extraction with C18 affinity media. Protein expression profiles of both dark-cutting and normal-pH beef samples were determined using LC-MS/MS mass spectrometry-based proteomics. Collected raw data instrument files were searched against a bovine proteome database of 23,968 bovine proteome sequences using MaxQuant (V.1.5.3.8). Differential protein expression analysis was done in Perseus (V.1.5.1.3). Ingenuity pathway analysis (IPA) was utilized to determine the significant pathways of the differentially expressed proteins in dark-cutting and normal-pH beef. Gene ontology enrichment pathway analysis was performed to determine the main functions of the differentially expressed proteins in dark-cutting and normal-pH beef identified in our samples.ResultsMass spectrometry analysis identified 1148 proteins, and 97 of these proteins were differentially expressed between normal-pH and dark-cutting beef (P < 0.05). Fold change of 1.5 was observed for 29 proteins. Dark-cutting beef had 19 abundant proteins, while normal-pH beef had 10 abundant proteins. The majority of the upregulated proteins in dark-cutting beef were involved in mitochondrial functioning and metabolism, while the majority of the downregulated proteins were important in glycogen degradation, calcium signaling, α-adrenergic signaling, n-NOS-signaling and the proteasome pathways.ConclusionThe results identify new protein biomarkers associated with dark-cutting and suggest new mechanistic explanations for the dark-cutting phenotype.

Motivation Multi-modal profiling of single cells represents one of the latest technological advancements in molecular biology. Among various single-cell multi-modal strategies, cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) allows simultaneous quantification of two distinct species: RNA and cell-surface proteins. Here, we introduce CiteFuse, a streamlined package consisting of a suite of tools for doublet detection, modality integration, clustering, differential RNA and protein expression analysis, antibody-derived tag evaluation, ligand–receptor interaction analysis and interactive web-based visualization of CITE-seq data. Results We demonstrate the capacity of CiteFuse to integrate the two data modalities and its relative advantage against data generated from single-modality profiling using both simulations and real-world CITE-seq data. Furthermore, we illustrate a novel doublet detection method based on a combined index of cell hashing and transcriptome data. Finally, we demonstrate CiteFuse for predicting ligand–receptor interactions by using multi-modal CITE-seq data. Collectively, we demonstrate the utility and effectiveness of CiteFuse for the integrative analysis of transcriptome and epitope profiles from CITE-seq data. Availability and implementation CiteFuse is freely available at http://shiny.maths.usyd.edu.au/CiteFuse/ as an online web service and at https://github.com/SydneyBioX/CiteFuse/ as an R package. Contact pengyi.yang@sydney.edu.au Supplementary information Supplementary data are available at Bioinformatics online.

Preeclampsia is one of the main causes of maternal and perinatal mortality and morbidity. The pathogenesis of preeclampsia remains unclear until now. It is believed that regulation of apoptosis in trophoblast cells plays an important role in the pathophysiology of preeclampsia. Failure of spiral arteries remodeling will eventually lead to placental hypoxia lead to excessive trophoblast apoptosis. The molecular mechanism of apoptosis is very complicated involving many signaling molecules included Bcl-2 proteins. The Bcl- 2 protein group consists of proapoptosis proteins (Bax) and apoptosis inhibitor proteins (Bcl-2 and Bcl-xL). The aimed of this stuty was to compare the expression of Bcl-xL protein in placental trophoblast cells of pregnancy complicated by severe preeclampsia with that normotensive pregnancy. This study was an observational study with cross sectional design involving 43 pregnancy patients with severe preeclampsia and 38 normotensive pregnancy who treated in Dr. Sardjito General Hospital, Yogyakarta from October 2011 until March 2012. Placenta samples were obtained from all subjects for Bcl-xL protein expression analysis using immunohistochemistry technique. Data were analyzed using independent t-test, chi-square test, and logistic regression. A p value