Establishment of decellularized extracellular matrix scaffold derived from caprine pancreas as a novel alternative template over porcine pancreatic scaffold for prospective biomedical application.

In this study, the caprine pancreas has been presented as an alternative to the porcine organ for pancreatic xenotransplantation with lesser risk factors. The obtained caprine pancreas underwent a systematic cycle of detergent perfusion for decellularization. It was perfused using anionic (0.5% w/v sodium dodecyl sulfate) as well as non-ionic (0.1% v/v triton X-100, t-octyl phenoxy polyethoxy ethanol) detergents and washed intermittently with 1XPBS supplemented with 0.1% v/v antibiotic and nucleases in a gravitation-driven set-up. After 48 h, a white decellularized pancreas was obtained, and its extracellular matrix (ECM) content was examined for scaffold-like properties. The ECM content was assessed for removal of cellular content, and nuclear material was evaluated with temporal H&E staining. Quantified DNA was found to be present in a negligible amount in the resultant decellularized pancreas tissue (DPT), thus prohibiting it from triggering any immunogenicity. Collagen and fibronectin were confirmed to be preserved upon trichrome and immunohistochemical staining, respectively. SEM and AFM images reveal interconnected collagen fibril networks in the DPT, confirming that collagen was unaffected. sGAG was visualized using Prussian blue staining and quantified with DMMB assay, where DPT has effectively retained this ECM component. Uniaxial tensile analysis revealed that DPT possesses better elasticity than NPT (native pancreatic tissue). Physical parameters like tensile strength, stiffness, biodegradation, and swelling index were retained in the DPT with negligible loss. The cytocompatibility analysis of DPT has shown no cytotoxic effect for up to 72 h on normal insulin-producing cells (MIN-6) and cancerous glioblastoma (LN229) cells in vitro. The scaffold was recellularized using isolated mouse islets, which have established in vitro cell proliferation for up to 9 days. The scaffold received at the end of the decellularization cycle was found to be non-toxic to the cells, retained biological and physical properties of the native ECM, suitable for recellularization, and can be used as a safer and better alternative as a transplantable organ from a xenogeneic source.

MIF confers survival advantage to pancreatic CAFs by suppressing interferon pathway-induced p53-dependent apoptosis.

The presence of activated pancreatic stellate cells (PSCs) in the pancreatic ductal adenocarcinoma (PDAC) microenvironment plays a significant role in cancer progression. Macrophage migration inhibitory factor (MIF) is overexpressed in PDAC tissues and expressed by both cancer and stromal cells. The pathophysiological role of MIF in PDAC-associated fibroblasts or PSCs is yet to be elucidated. Here we report that the PSCs of mouse or cancer-associated fibroblast cells (CAFs) of human expresses MIF and its receptors, whose expression gets upregulated upon LPS or TNF-α stimulation. In vitro functional experiments showed that MIF significantly conferred a survival advantage to CAFs/PSCs upon growth factor deprivation. Genetic or pharmacological inhibition of MIF also corroborated these findings. Further, co-injection of mouse pancreatic cancer cells with PSCs isolated from Mif-/- or Mif+/+ mice confirmed the pro-survival effect of MIF in PSCs and also demonstrated the pro-tumorigenic role of MIF expressed by CAFs in vivo. Differential gene expression analysis and in vitro mechanistic studies indicated that MIF expressed by activated CAFs/PSCs confers a survival advantage to these cells by suppression of interferon pathway induced p53 dependent apoptosis.

Olaparib enhances the Resveratrol-mediated apoptosis in breast cancer cells by inhibiting the homologous recombination repair pathway.

Although sensitization of BRCA-mutated, homologous recombination (HR)-deficient breast cancer cells through PARP inhibitor is widely studied, not much is known about the treatment of BRCA-wild-type, HR-proficient breast cancer. Here, we aim to investigate whether a bioactive compound, Resveratrol (RES), can induce DNA double-strand breaks in HR-proficient breast cancer cells and Olaparib (OLA), a PARP inhibitor, can enhance the RES-mediated apoptosis by deregulating the HR repair pathway. The detailed mechanism of anti-cancer action of RES + OLA combination in breast cancer has been evaluated using in vitro, ex vivo, and in vivo preclinical model systems. OLA increased RES-mediated DNA damage, downregulated the HR pathway proteins, caused a late S/G2 cell cycle arrest, enhanced apoptosis and cell death in RES pre-treated breast cancer cells at much lower concentrations than their individual treatments. Direct measurement of HR pathway activity using a GFP plasmid-based assay demonstrated reduced HR efficiency in I-SceI endonuclease-transfected cells treated with OLA. Moreover, RES + OLA treatment also caused significant reduction in PARP1-mediated PARylation and efficiently trapped PARP1 at the DNA damage site. Upon RES treatment, PARylated PARP1 was found to interact with BRCA1, which then activated other HR pathway proteins. But after addition of OLA in RES pre-treated cells, PARP1 could not interact with BRCA1 due to inhibition of PARylation. This resulted in deregulation of HR pathway. To further confirm the role of BRCA1 in PARP1-mediated HR pathway activation, BRCA1 was knocked down that caused complete inhibition of HR pathway activity, and further enhanced apoptosis after RES + OLA treatment in BRCA1-silenced cells. In agreement with in vitro data, similar experimental results were obtained in ex vivo patient-derived breast cancer cells and in vivo xenograft mice. Thus, RES + OLA combination treatment enhanced breast cancer cell death by causing excessive DNA damage and also simultaneously inhibiting the HR pathway.

Fluvastatin sensitizes pancreatic cancer cells toward radiation therapy and suppresses radiation- and/or TGF-ß-induced tumor-associated fibrosis.

Pancreatic cancer (PC) is highly resistant to chemo and radiotherapy. Radiation-induced fibrosis (RIF) is a major cause of clinical concern for various malignancies, including PC. In this study, we aimed to evaluate the radiosensitizing and anti-RIF potential of fluvastatin in PC. Short-term viability and clonogenic survival assays were used to evaluate the radiosensitizing potential of fluvastatin in multiple human and murine PC cell lines. The expression of different proteins was analyzed to understand the mechanisms of fluvastatin-mediated radiosensitization of PC cells and its anti-RIF effects in both mouse and human pancreatic stellate cells (PSCs). Finally, these effects of fluvastatin and/or radiation were assessed in an immune-competent syngeneic murine model of PC. Fluvastatin radiosensitized multiple PC cell lines, as well as radioresistant cell lines in vitro, by inhibiting radiation-induced DNA damage repair response. Nonmalignant cells, such as PSCs and NIH3T3 cells, were less sensitive to fluvastatin-mediated radiosensitization than PC cells. Interestingly, fluvastatin suppressed radiation and/or TGF-ß-induced activation of PSCs, as well as the fibrogenic properties of these cells in vitro. Fluvastatin considerably augmented the antitumor effect of external radiation therapy and also suppressed intra-tumor RIF in vivo. These findings suggested that along with radiation, fluvastatin co-treatment may be a potential therapeutic approach against PC.

TRIM16 controls assembly and degradation of protein aggregates by modulating the p62-NRF2 axis and autophagy.

Sequestration of protein aggregates in inclusion bodies and their subsequent degradation prevents proteostasis imbalance, cytotoxicity, and proteinopathies. The underlying molecular mechanisms controlling the turnover of protein aggregates are mostly uncharacterized. Herein, we show that a TRIM family protein, TRIM16, governs the process of stress-induced biogenesis and degradation of protein aggregates. TRIM16 facilitates protein aggregate formation by positively regulating the p62-NRF2 axis. We show that TRIM16 is an integral part of the p62-KEAP1-NRF2 complex and utilizes multiple mechanisms for stabilizing NRF2. Under oxidative and proteotoxic stress conditions, TRIM16 activates ubiquitin pathway genes and p62 via NRF2, leading to ubiquitination of misfolded proteins and formation of protein aggregates. We further show that TRIM16 acts as a scaffold protein and, by interacting with p62, ULK1, ATG16L1, and LC3B, facilitates autophagic degradation of protein aggregates. Thus, TRIM16 streamlines the process of stress-induced aggregate clearance and protects cells against oxidative/proteotoxic stress-induced toxicity in vitro and in vivo Taken together, this work identifies a new mechanism of protein aggregate turnover, which could be relevant in protein aggregation-associated diseases such as neurodegeneration.

Temperature-Controlled Chemoselective Synthesis of Multisubstituted 4-Alkynyl/trans 4-Alkenyl Coumarins.

A temperature-controlled facile synthesis of multisubstituted 4-alkynyl/trans 4-alkenyl coumarins with a metal salt cascade approach is reported. H2O serves both as a nucleophile and hydrogen source. The presence of metal salt facilitates the reduction of alkyne. The present protocol bypasses the structural shortcomings of the existing Sonogashira and Heck coupling reactions. In addition, the obtained 2,3-disubstituted coumarins are readily transformed into 2,3-disubstituted dihydrocoumarins, which serve as important building blocks in organic transformations.

NIR irradiation enhances the apoptotic potentiality of quinacrine-gold hybrid nanoparticles by modulation of HSP-70 in oral cancer stem cells.

Cancer stem cells (CSCs) are the tumor cell subpopulations that can self-renew, differentiate, initiate and maintain tumor growth. CSCs are frequently drug-resistant, resulting in tumor recurrence, metastasis, and angiogenesis. Herein, using in vitro oral squamous cell carcinoma (OSCC) CSCs and in vivo xenograft mice model, we have systematically studied the apoptotic potentiality of quinacrine-gold hybrid nanoparticle (QAuNP) and its underlying mechanism after NIR irradiation. QAuNP + NIR caused DNA damage and induced apoptosis in SCC-9-CSCs by deregulating mitochondrial membrane potential (ΔΨm) and activation of ROS. Upregulation of CASPASE-3 and DR-5/DR-4 and reduction of heat shock protein (HSP-70) up to 5-fold were also noticed upon the treatment. The increased expression of DR-5 and CASPASE-3 and decreased expression of HSP-70, CD-44 and Ki-67 were also noted in the xenograft mice treated with QAuNP + NIR + TRAIL. Thus, data suggest that the combined treatment enhances apoptosis in OSCC-CSCs by modulating HSP-70 in the DISC.

Threats of climate change and land use patterns enhance the susceptibility of future floods in India.

The main objective of this work is the future prediction of the floods in India due to climate and land change. Human activity and related carbon emissions are the primary cause of land use and climate change, which has a substantial impact on extreme weather conditions, such as floods. This study presents high-resolution flood susceptibility maps of different future periods (up to 2100) using a combination of remote sensing data and GIS modelling. To quantify the future flood susceptibility various flood causative factors, Global circulation model (GCM) rainfall and land use and land cover (LULC) data are envisaged. The present flood susceptibility model has been evaluated through receiver operating characteristic (ROC) curve, where area under curve (AUC) value shows the 91.57% accuracy of this flood susceptibility model and it can be used for future flood susceptibility modelling. Based on the projected LULC, rainfall and flood susceptibility, the results of the study indicating maximum monthly rainfall will increase by approximately 40-50 mm in 2100, while the conversion of natural vegetation to agricultural and built-up land is about 0.071 million sq. km. and the severe flood event area will increase by up to 122% (0.15 million sq. km) from now on.

Varicella-Zoster Virus (VZV) Small Noncoding RNAs Antisense to the VZV Latency-Encoded Transcript VLT Enhance Viral Replication.

Small noncoding RNAs (sncRNA), including microRNA (miR), are expressed by many viruses to provide an additional layer of gene expression regulation. Our work has shown that varicella-zoster virus (VZV; also called human herpesvirus 3 [HHV3]), the human alphaherpesvirus causing varicella and herpes zoster, expresses 24 virally encoded sncRNA (VZVsncRNA) in infected cells. Here, we demonstrate that several VZVsncRNA can modulate VZV growth, including four VZVsncRNA (VZVsncRNA10, -11, -12, and -13) that are antisense to VLT, a transcript made in lytic infections and associated with VZV latency. The influence on productive VZV growth and spread was assessed in epithelial cells transfected with locked nucleotide analog antagonists (LNAA). LNAA to the four VZVsncRNA antisense to VLT significantly reduced viral spread and progeny titers of infectious virus, suggesting that these sncRNA promoted lytic infection. The LNAA to VZVsncRNA12, encoded in the leader to ORF61, also significantly increased the levels of VLT transcripts. Conversely, overexpression of VZVsncRNA13 using adeno-associated virus consistently increased VZV spread and progeny titers. These results suggest that sncRNA antisense to VZV may regulate VZV growth, possibly by affecting VLT expression. Transfection of LNAA to VZVsncRNA14 and VZVsncRNA9 decreased and increased VZV growth, respectively, while LNAA to three other VZVsncRNA had no significant effects on replication. These data strongly support the conclusion that VZV replication is modulated by multiple virally encoded sncRNA, revealing an additional layer of complexity of VZV regulation of lytic infections. This may inform the development of novel anti-sncRNA-based therapies for treatment of VZV diseases.IMPORTANCE Varicella-zoster virus (VZV) causes herpes zoster, a major health issue in the aging and immunocompromised populations. Small noncoding RNAs (sncRNA) are recognized as important actors in modulating gene expression. This study extends our previous work and shows that four VZVsncRNA clustering in and near ORF61 and antisense to the latency-associated transcript of VZV can positively influence productive VZV infection. The ability of multiple exogenous small oligonucleotides targeting VZVsncRNA to inhibit VZV replication strengthens the possibility that they may inform development of novel treatments for painful herpes zoster.

TPM, FPKM, or Normalized Counts? A Comparative Study of Quantification Measures for the Analysis of RNA-seq Data from the NCI Patient-Derived Models Repository.

BACKGROUND: In order to correctly decode phenotypic information from RNA-sequencing (RNA-seq) data, careful selection of the RNA-seq quantification measure is critical for inter-sample comparisons and for downstream analyses, such as differential gene expression between two or more conditions. Several methods have been proposed and continue to be used. However, a consensus has not been reached regarding the best gene expression quantification method for RNA-seq data analysis. METHODS: In the present study, we used replicate samples from each of 20 patient-derived xenograft (PDX) models spanning 15 tumor types, for a total of 61 human tumor xenograft samples available through the NCI patient-derived model repository (PDMR). We compared the reproducibility across replicate samples based on TPM (transcripts per million), FPKM (fragments per kilobase of transcript per million fragments mapped), and normalized counts using coefficient of variation, intraclass correlation coefficient, and cluster analysis. RESULTS: Our results revealed that hierarchical clustering on normalized count data tended to group replicate samples from the same PDX model together more accurately than TPM and FPKM data. Furthermore, normalized count data were observed to have the lowest median coefficient of variation (CV), and highest intraclass correlation (ICC) values across all replicate samples from the same model and for the same gene across all PDX models compared to TPM and FPKM data. CONCLUSION: We provided compelling evidence for a preferred quantification measure to conduct downstream analyses of PDX RNA-seq data. To our knowledge, this is the first comparative study of RNA-seq data quantification measures conducted on PDX models, which are known to be inherently more variable than cell line models. Our findings are consistent with what others have shown for human tumors and cell lines and add further support to the thesis that normalized counts are the best choice for the analysis of RNA-seq data across samples.

Exploration of Benzo[b]carbazole-6,11-diones as anticancer agents: Synthesis and studies of hTopoIIα inhibition and apoptotic effects.

Two series of (hetero)arylamino-naphthoquinones and benzo-fused carbazolequinones were considered for study with the rationale that related structural motifs are present in numerous drugs, clinical trial agents, natural products and hTopoIIα inhibitors. Total 42 compounds were synthesized by reactions including dehydrogenative CN and Pd-catalyzed CC bond forming transformations. These compounds were screened against numerous cancer cells including highly metastatic one (MCF-7, MDA-MB-231, H-357 and HEK293T), and normal cells (MCF 10A). Some of the active compounds were evaluated for clonogenic cell survival and apoptotic effects in cancer cells (DAPI nuclear staining, Comet assay, Annexin-V-FITC/PI dual staining, flow cytometry, and western blot analysis with relevant proteins). All compounds were tested for hTopoIIα inhibitory activity. The investigated series compounds showed important properties like significant apoptotic antiproliferation in cancer cells with cell cycle arrest at S-phase and downregulation of NF- κß signaling cascade, relatively less cytotoxicity to normal cells, and hTopoIIα inhibition with more efficiency compared to an anticancer drug etoposide.

Estrogen receptor-1 is a key regulator of HIV-1 latency that imparts gender-specific restrictions on the latent reservoir.

Unbiased shRNA library screens revealed that the estrogen receptor-1 (ESR-1) is a key factor regulating HIV-1 latency. In both Jurkat T cells and a Th17 primary cell model for HIV-1 latency, selective estrogen receptor modulators (SERMs, i.e., fulvestrant, raloxifene, and tamoxifen) are weak proviral activators and sensitize cells to latency-reversing agents (LRAs) including low doses of TNF-α (an NF-κB inducer), the histone deacetylase inhibitor vorinostat (soruberoylanilide hydroxamic acid, SAHA), and IL-15. To probe the physiologic relevance of these observations, leukapheresis samples from a cohort of 12 well-matched reproductive-age women and men on fully suppressive antiretroviral therapy were evaluated by an assay measuring the production of spliced envelope (env) mRNA (the EDITS assay) by next-generation sequencing. The cells were activated by T cell receptor (TCR) stimulation, IL-15, or SAHA in the presence of either ß-estradiol or an SERM. ß-Estradiol potently inhibited TCR activation of HIV-1 transcription, while SERMs enhanced the activity of most LRAs. Although both sexes responded to SERMs and ß-estradiol, females showed much higher levels of inhibition in response to the hormone and higher reactivity in response to ESR-1 modulators than males. Importantly, the total inducible RNA reservoir, as measured by the EDITS assay, was significantly smaller in the women than in the men. We conclude that concurrent exposure to estrogen is likely to limit the efficacy of viral emergence from latency and that ESR-1 is a pharmacologically attractive target that can be exploited in the design of therapeutic strategies for latency reversal.

Escherichia coli, a common constituent of benign prostate hyperplasia-associated microbiota induces inflammation and DNA damage in prostate epithelial cells.

BACKGROUND: The role of microbiota in the pathophysiology of benign prostate hyperplasia (BPH), especially in creating an inflammatory milieu may not be avoided. The major objectives of this study were to investigate the microbial composition of BPH tissues, its association with inflammation and check the effect of clinically isolated bacteria on prostate epithelial cells. METHODS: The study includes 36 patients with a pathological diagnosis of BPH. Following strict aseptic measures, tissues were collected after transurethral resection of prostate, multiple pieces of the resected tissues were subjected to histopathological analysis, bacterial culture and genomic DNA extraction. Microbial composition was analyzed by culture and/or next-generation sequencing methods. Annotation of operational taxonomy unit has been done with an in-house algorithm. The extent of inflammation was scored through histological evaluation of tissue sections. The effect of clinical isolates on nuclear factor-κB (NF-κB) activity and induction of DNA-damage in the prostate epithelial cells were evaluated. RESULTS: Histopathological analysis of the BPH tissues showed the presence of inflammation in almost all the tissues with a varied level at different regions of the same tissue section and the level of overall inflammation was different from patients to patients. Microbial culture of tissue samples showed the presence of live bacteria in 55.5% (20 out of 36) of the patient tissues. Majority of the isolates were coagulase-positive Staphylococcus, E. coli and Micrococcus spp. Further, V3 16S rRNA sequencing of the DNA isolated from BPH tissues showed the presence of multiple bacteria and the most common phylum in the BPH tissues were found to be Proteobacteria, Actinobacteria, Firmicutes, and Bacteroidetes. The E. coli, isolated from one of the tissue was able to activate NF-κB and induce DNA damage in prostate epithelial cells. Phospho-histone γH2A.X staining confirmed the presence of cells with damaged DNA lesion in BPH tissues and also correlated with the severity of inflammation. CONCLUSION: Our study has shown that the BPH tissues do have a divergent microbial composition including the commonly found E. coli (phylum Proteobacteria), and these bacteria might contribute to the BPH-associated inflammation and/or tissue damage. The BPH-associated E. coli induced NF-κB signaling and DNA damage in prostate epithelial cells in vitro.

Quinacrine Based Gold Hybrid Nanoparticles Caused Apoptosis through Modulating Replication Fork in Oral Cancer Stem Cells.

The presence of cancer stem cells (CSCs) in the tumor microenvironment is responsible for the development of chemoresistance and recurrence of cancer. Our previous investigation revealed the anticancer mechanism of quinacrine-based silver and gold hybrid nanoparticles (QAgNP and QAuNP) in oral cancer cells, but to avoid cancer recurrence, it is important to study the effect of these nanoparticles (NPs) on CSCs. Here, we developed an in vitro CSCs model using SCC-9 oral cancer cells and validated via FACS analysis. Then, 40-60% of cells were found to be CD44+/CD133+ and CD24-. QAuNP showed excellent anti-CSC growth potential against SCC-9-cancer stem like cells (IC50 = 0.4 µg/mL) with the down-regulation of representative CSC markers. Prolonged exposure of QAuNP induced the S-phase arrest and caused re-replication shown by the extended G2/M population and apoptosis to SCC-9-CSC like cells. Up-regulation of BAX, PARP cleavage, and simultaneous down-regulation of Bcl-xL in prolonged treatment to CSCs suggested that the majority of the cells have undergone apoptosis. QAuNP treatment also caused a loss in DNA repair in CSCs. Mostly, the base excision repair (BER) components (Fen-1, DNA ligase-1, Pol-ß, RPA, etc.) were significantly down-regulated after QAuNP treatment, which suggested its action against DNA repair machinery. The replication fork maintenance-related proteins, RAD 51 and BRCA-2, were also deregulated. Very surprisingly, depletion of WRN (an interacting partner for Pre-RC and Fen-1) and a significant increase in expression of fork-degrading nuclease MRE-11 in 96 h treated NPs were observed. Results suggest QAuNP treatment caused excessive DNA damage and re-replication mediated replication stress (RS) and stalling of the replication fork. Inhibition of BER components hinders the flap clearance activity of Fen-1, and it further caused RS and stopped DNA synthesis. Overall, QAuNP treatment led to irreparable replication fork movement, and the stalled replication fork might have degraded by MRE-11, which ultimately results in apoptosis and the death of the CSCs.

The guiding role of dissipation in kinetic proofreading networks: Implications for protein synthesis.

Major biological polymerization processes achieve remarkable accuracy while operating out of thermodynamic equilibrium by utilizing the mechanism known as kinetic proofreading. Here, we study the interplay of the thermodynamic and kinetic aspects of proofreading by exploring the dissipation and catalytic rate, respectively, under the realistic constraint of fixed chemical potential difference. Theoretical analyses reveal no-monotonic variations of the catalytic rate and total entropy production rate (EPR), the latter quantifying the dissipation, at steady state. Applying this finding to a tRNA selection network in protein synthesis, we observe that the network tends to maximize both the EPR and catalytic rate, but not the accuracy. Simultaneously, the system tries to minimize the ratio of the EPRs due to the proofreading steps and the catalytic steps. Therefore, dissipation plays a guiding role in the optimization of the catalytic rate in the tRNA selection network of protein synthesis.

Potential of the fluoroketolide RBx 14255 against Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae in an experimental murine meningitis model.

BACKGROUND: RBx 14255 is a fluoroketolide in pre-clinical evaluation with potent activity against MDR Gram-positive pathogens. OBJECTIVES: To investigate the efficacy of RBx 14255 against bacterial meningitis caused by Streptococcus pneumoniae, Neisseria meningitidis or Haemophilus influenzae in an experimental murine meningitis model. METHODS: In vitro activity of RBx 14255 was evaluated against clinical isolates of S. pneumoniae, N. meningitidis and H. influenzae. The in vivo efficacy of RBx 14255 was evaluated against bacterial meningitis, induced with S. pneumoniae 3579 erm(B), S. pneumoniae MA 80 erm(B), N. meningitidis 1852 and H. influenzae B1414 in a murine meningitis model. RESULTS: RBx 14255 showed strong in vitro bactericidal potential against S. pneumoniae, N. meningitidis and H. influenzae with MIC ranges of 0.004-0.1, 0.03-0.5 and 1-4 mg/L, respectively. In a murine meningitis model, a 50 mg/kg dose of RBx 14255, q12h, resulted in significant reduction of bacterial counts in the brain compared with the pretreatment control. The concentration of RBx 14255 in brain tissue correlated well with the efficacy in this mouse model. CONCLUSIONS: RBx 14255 showed superior bactericidal activity in time-kill assays in vitro and in vivo in an experimental murine meningitis model. RBx 14255 could be a promising candidate for future drug development against bacterial meningitis.

Development and interlaboratory evaluation of a NIST Reference Material RM 8366 for EGFR and MET gene copy number measurements.

Background The National Institute of Standards and Technology (NIST) Reference Material RM 8366 was developed to improve the quality of gene copy measurements of EGFR (epidermal growth factor receptor) and MET (proto-oncogene, receptor tyrosine kinase), important targets for cancer diagnostics and treatment. The reference material is composed of genomic DNA prepared from six human cancer cell lines with different levels of amplification of the target genes. Methods The reference values for the ratios of the EGFR and MET gene copy numbers to the copy numbers of reference genes were measured using digital PCR. The digital PCR measurements were confirmed by two additional laboratories. The samples were also characterized using Next Generation Sequencing (NGS) methods including whole genome sequencing (WGS) at three levels of coverage (approximately 1 ×, 5 × and greater than 30 ×), whole exome sequencing (WES), and two different pan-cancer gene panels. The WES data were analyzed using three different bioinformatic algorithms. Results The certified values (digital PCR) for EGFR and MET were in good agreement (within 20%) with the values obtained from the different NGS methods and algorithms for five of the six components; one component had lower NGS values. Conclusions This study shows that NIST RM 8366 is a valuable reference material to evaluate the performance of assays that assess EGFR and MET gene copy number measurements.

Experimental models of pancreatic cancer desmoplasia.

Desmoplasia is a fibro-inflammatory process and a well-established feature of pancreatic cancer. A key contributor to pancreatic cancer desmoplasia is the pancreatic stellate cell. Various in vitro and in vivo methods have emerged for the isolation, characterization, and use of pancreatic stellate cells in models of cancer-associated fibrosis. In addition to cell culture models, genetically engineered animal models have been established that spontaneously develop pancreatic cancer with desmoplasia. These animal models are currently being used for the study of pancreatic cancer pathogenesis and for evaluating therapeutics against pancreatic cancer. Here, we review various in vitro and in vivo models that are being used or have the potential to be used to study desmoplasia in pancreatic cancer.

Large deviation theory for the kinetics and energetics of turnover of enzyme catalysis in a chemiostatic flow.

In the framework of large deviation theory, we have characterized nonequilibrium turnover statistics of enzyme catalysis in a chemiostatic flow with externally controllable parameters, like substrate injection rate and mechanical force. In the kinetics of the process, we have shown the fluctuation theorems in terms of the symmetry of the scaled cumulant generating function (SCGF) in the transient and steady state regime and a similar symmetry rule is reflected in a large deviation rate function (LDRF) as a property of the dissipation rate through boundaries. Large deviation theory also gives the thermodynamic force of a nonequilibrium steady state, as is usually recorded experimentally by a single molecule technique, which plays a key role responsible for the dynamical symmetry of the SCGF and LDRF. Using some special properties of the Legendre transformation, here, we have provided a relation between the fluctuations of fluxes and dissipation rates, and among them, the fluctuation of the turnover rate is routinely estimated but the fluctuation in the dissipation rate is yet to be characterized for small systems. Such an enzymatic reaction flow system can be a very good testing ground to systematically understand the rare events from the large deviation theory which is beyond fluctuation theorem and central limit theorem.