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The canonical role of the transcription factor E2F is to control the expression of cell cycle genes by binding to the E2F sites in their promoters. However, the list of putative E2F target genes is extensive and includes many metabolic genes, yet the significance of E2F in controlling the expression of these genes remains largely unknown. Here, we used the CRISPR/Cas9 technology to introduce point mutations in the E2F sites upstream of five endogenous metabolic genes in Drosophila melanogaster . We found that the impact of these mutations on both the recruitment of E2F and the expression of the target genes varied, with the glycolytic gene, Phosphoglycerate kinase ( Pgk) , being mostly affected. The loss of E2F regulation on the Pgk gene led to a decrease in glycolytic flux, tricarboxylic acid cycle intermediates levels, adenosine triphosphate (ATP) content, and an abnormal mitochondrial morphology. Remarkably, chromatin accessibility was significantly reduced at multiple genomic regions in Pgk ΔE2F mutants. These regions contained hundreds of genes, including metabolic genes that were downregulated in Pgk ΔE2F mutants. Moreover, Pgk ΔE2F animals had shortened life span and exhibited defects in high-energy consuming organs, such as ovaries and muscles. Collectively, our results illustrate how the pleiotropic effects on metabolism, gene expression, and development in the Pgk ΔE2F animals underscore the importance of E2F regulation on a single E2F target, Pgk .

Tyrosine kinase inhibitors were found to be clinically effective for treatment of patients with certain subsets of cancers carrying somatic mutations in receptor tyrosine kinases. However, the duration of clinical response is often limited, and patients ultimately develop drug resistance. Here, we use single-cell RNA sequencing to demonstrate the existence of multiple cancer cell subpopulations within cell lines, xenograft tumors and patient tumors. These subpopulations exhibit epigenetic changes and differential therapeutic sensitivity. Recurrently overrepresented ontologies in genes that are differentially expressed between drug tolerant cell populations and drug sensitive cells include epithelial-to-mesenchymal transition, epithelium development, vesicle mediated transport, drug metabolism and cholesterol homeostasis. We show analysis of identified markers using the LINCS database to predict and functionally validate small molecules that target selected drug tolerant cell populations. In combination with EGFR inhibitors, crizotinib inhibits the emergence of a defined subset of EGFR inhibitor-tolerant clones. In this study, we describe the spectrum of changes associated with drug tolerance and inhibition of specific tolerant cell subpopulations with combination agents., It has been proposed that resistance to targeted therapies in non-small cell lung carcinoma (NSCLC) is due to a nonhomogeneous cell population. Here the authors analyse preclinical NSCLC models using single-cell RNA-seq and identify drug tolerant cell states and subpopulations, as well as associated genes.

Background Due to its rapid lethal effect in the early development stage of shrimp, acute hepatopancreatic necrosis disease (AHPND) has been causing great economic losses, since its first outbreak in southeast China in 2009. Vibrio parahaemolyticus, carrying the pirA and pirB toxin genes is known to cause AHPND in shrimp. The overall objective of this study was to sequence the whole genome of AHPND positive V. parahaemolyticus strains isolated from shrimp (Peneaus monodon) of the south-west region of Bangladesh in 2016 and 2017 and characterize the genomic features and emergence pattern of this marine pathogen. Results Two targeted AHPND positive V. parahaemolyticus strains were confirmed using PCR with 16S rRNA, ldh, AP3 and AP4 primers. The assembled genomes of strain MSR16 and MSR17 were comprised of a total of 5,393,740 bp and 5,241,592 bp, respectively. From annotation, several virulence genes involved in chemotaxis and motility, EPS type II secretion system, Type III secretion system-1 (T3SS-1) and its secreted effectors, thermolabile hemolysin were found in both strains. Importantly, the ~ 69 kb plasmid was identified in both MSR16 and MSR17 strains containing the two toxin genes pirA and pirB. Antibiotic resistance genes were predicted against β-lactam, fluoroquinolone, tetracycline and macrolide groups in both MSR16 and MSR17 strains. Conclusions The findings of this research may facilitate the tracking of pathogenic and/or antibiotic-resistant V. parahaemolyticus isolates between production sites, and the identification of candidate strains for the production of vaccines as an aid to control of this devastating disease. Also, the emergence pattern of this pathogen can be highlighted to determine the characteristic differences of other strains found all over the world.

Osteoporosis is skeletal fragility caused by the excessive bone resorption due to osteoclastogenesis. But current drugs are less bioavailable and possess higher toxicity. Our study was conducted to identify safe oral bioavailable drugs from Fenugreek steroidal saponins and to delineate underlying mechanism of them to lower the osteoclastogenic bone resorption. We observed higher molecular docked binding affinities in finally selected eight hit compounds within the range of −11.0 to −10.1 kcal/mol which was greater than currently used drugs. Molecular Dynamics simulation with Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Solvent Accessible Surface Area (SASA) and Gyration trajectory projection reinforced the stability of the protein-ligand complexes. Pharmacokinetics analysis confirmed bioavailability of seven compounds out of eight, and drug likeliness and bioavailability profile evaluation indicated that they all are eligible to be developed as a potent oral inhibitor of CSF-1R. By literature mining knowledge-driven analysis, RNAseq data and Molecular Dynamics Simulation, we proposed that, the hit derivatives block the CSF-1/CSF-1R induced phosphorylation signaling pathway in both osteoclast and osteoblast resulting in hindrance of RANK expression and formation of Reactive oxygen species (ROS) in osteoclast and osteoblast respectively, thus declines the RANKL/OPG ratio, lowering the osteoclast survival, proliferation and differentiation.

Antiprotozoal drug nitazoxanide (NTZ) has shown diverse pharmacological properties and has appeared in several clinical trials. Herein we present the synthesis, characterization, in vitro biological investigation, and in silico study of four hetero aryl amide analogs of NTZ. Among the synthesized molecules, compound 2 and compound 4 exhibited promising antibacterial activity against Escherichia coli (E. coli), superior to that displayed by the parent drug nitazoxanide as revealed from the in vitro antibacterial assay. Compound 2 displayed zone of inhibition of 20 mm, twice as large as the parent drug NTZ (10 mm) in their least concentration (12.5 µg/ml). Compound 1 also showed antibacterial effect similar to that of nitazoxanide. The analogs were also tested for in vitro cytotoxic activity by employing cell counting kit‐8 (CCK‐8) assay technique in HeLa cell line, and compound 2 was identified as a potential anticancer agent having IC50 value of 172 µg which proves it to be more potent than nitazoxanide (IC50 = 428 µg). Furthermore, the compounds were subjected to molecular docking study against various bacterial and cancer signaling proteins. The in vitro test results corroborated with the in silico docking study as compound 2 and compound 4 had comparatively stronger binding affinity against the proteins and showed a higher docking score than nitazoxanide toward human mitogen‐activated protein kinase (MAPK9) and fatty acid biosynthesis enzyme (FabH) of E. coli. Moreover, the docking study demonstrated dihydrofolate reductase (DHFR) and thymidylate synthase (TS) as probable new targets for nitazoxanide and its synthetic analogs. Overall, the study suggests that nitazoxanide and its analogs can be a potential lead compound in the drug development., Four heteroaryl nitazoxanide analogues were synthesized efficiently. Among the synthesized analogues, two compounds showed prominent antibacterial and anticancer potentials through in vitro and in silico studies.

Background Although it is becoming evident that individual’s immune system has a decisive influence on SARS-CoV-2 disease progression, pathogenesis is largely unknown. In this study, we aimed to profile the host transcriptome of COVID-19 patients from nasopharyngeal samples along with virus genomic features isolated from respective host, and a comparative analyses of differential host responses in various SARS-CoV-2 infection systems. Results Unique and rare missense mutations in 3C-like protease observed in all of our reported isolates. Functional enrichment analyses exhibited that the host induced responses are mediated by innate immunity, interferon, and cytokine stimulation. Surprisingly, induction of apoptosis, phagosome, antigen presentation, hypoxia response was lacking within these patients. Upregulation of immune and cytokine signaling genes such as CCL4, TNFA, IL6, IL1A, CCL2, CXCL2, IFN, and CCR1 were observed in lungs. Lungs lacked the overexpression of ACE2 as suspected, however, high ACE2 but low DPP4 expression was observed in nasopharyngeal cells. Interestingly, directly or indirectly, viral proteins specially non-structural protein mediated overexpression of integrins such as ITGAV, ITGA6, ITGB7, ITGB3, ITGA2B, ITGA5, ITGA6, ITGA9, ITGA4, ITGAE, and ITGA8 in lungs compared to nasopharyngeal samples suggesting the possible way of enhanced invasion. Furthermore, we found comparatively highly expressed transcription factors such as CBP, CEBP, NFAT, ATF3, GATA6, HDAC2, TCF12 which have pivotal roles in lung injury. Conclusions Even though this study incorporates a limited number of cases, our data will provide valuable insights in developing potential studies to elucidate the differential host responses on the viral pathogenesis in COVID-19, and incorporation of further data will enrich the search of an effective therapeutics.

The constant rise of the death toll and cases of COVID-19 has made this pandemic a serious threat to human civilization. Understanding of host-SARS-CoV-2 interaction in viral pathogenesis is still in its infancy. In this study, we utilized a blend of computational and knowledgebase approaches to model the putative virus-host interplay in host signaling pathways by integrating the experimentally validated host interactome proteins and differentially expressed host genes in SARS-CoV-2 infection. While searching for the pathways in which viral proteins interact with host proteins, we discovered various antiviral immune response pathways such as hypoxia-inducible factor 1 (HIF-1) signaling, autophagy, retinoic acid-inducible gene I (RIG-I) signaling, Toll-like receptor signaling, fatty acid oxidation/degradation, and IL-17 signaling. All these pathways can be either hijacked or suppressed by the viral proteins, leading to improved viral survival and life cycle. Aberration in pathways such as HIF-1 signaling and relaxin signaling in the lungs suggests the pathogenic lung pathophysiology in COVID-19. From enrichment analysis, it was evident that the deregulated genes in SARS-CoV-2 infection might also be involved in heart development, kidney development, and AGE-RAGE signaling pathway in diabetic complications. Anomalies in these pathways might suggest the increased vulnerability of COVID-19 patients with comorbidities. Moreover, we noticed several presumed infection-induced differentially expressed transcription factors and epigenetic factors, such as miRNAs and several histone modifiers, which can modulate different immune signaling pathways, helping both host and virus. Our modeling suggests that SARS-CoV-2 integrates its proteins in different immune signaling pathways and other cellular signaling pathways for developing efficient immune evasion mechanisms while leading the host to a more complicated disease condition. Our findings would help in designing more targeted therapeutic interventions against SARS-CoV-2.

Background Cervical cancer is a gynecologic cancer type that develops in the cervix, accounting for 8% mortality of all female cancer patients. Infection with specific human papillomavirus (HPV) types is considered the most severe risk factor for cervical cancer. In the context of our socioeconomic conditions, an increasing burden of this disease and high mortality rate prevail in Bangladesh. Although several researches related to the epidemiology, HPV vaccination, and treatment modalities were conducted, researches on the mutation profiles of marker genes in cervical cancer in Bangladesh remain unexplored. Methods In this study, five different genomic regions within the top three most frequently mutated genes (EGFR, KRAS and PIK3CA) in COSMIC database with a key role in the development of cervical cancers were selected to study the mutation frequency in Bangladeshi patients. In silico analysis was done in two steps: nucleotide sequence analysis and its corresponding amino acid analysis. Results DNA from 46 cervical cancer tissue samples were extracted and amplified by PCR, using 1 set of primers designed for EGFR and 2 sets of primers designed for two different regions of both PIK3CA and KRAS gene. In total, 39 mutations were found in 26 patient samples. Eleven different mutations (23.91%), twenty-four different mutations (52.17%) and four mutations (8.7%) were found in amplified EGFR, PIK3CA and KRAS gene fragments, respectively; among which 1 (EGFR) was common in seven patient samples and 2 (PIKCA) were found in more than 1 patient. Our study shows that except for KRAS, the frequency of observed mutations in our patients is higher than those reported earlier in other parts of the world. Most of the exonic mutations were found only in the PIK3CA and EGFR genes. Conclusions The study can be used as a basis to build a mutation database for cervical cancer in Bangladesh with the possibility of targetable oncogenic mutations. Further explorations are needed to establish future diagnostics, personalized medicine decisions, and other pharmaceutical applications for specific cancer subtypes.

Background MicroRNAs are ~ 22-nucleotide-long biological modifiers that act as the post-transcriptional modulator of gene expression. Some of them are identified to be embedded within the introns of protein-coding genes, these miRNAs are called the intronic miRNAs. Previous findings state that these intronic miRNAs are co-expressed with their host genes. This co-expression is necessary to maintain the robustness of the biological system. Till to date, only a few experiments are performed discretely to elucidate the functional relationship between few co-expressed intronic miRNAs and their associated host genes. Results In this study, we have interpreted the underlying modulatory mechanisms of intronic miRNA hsa-miR-933 on its target host gene ATF2 and found that aberration can lead to several disease conditions. A protein-protein interaction network-based approach was adopted, and functional enrichment analysis was performed to elucidate the significantly over-represented biological functions and pathways of the common targets. Our approach delineated that hsa-miR-933 might control the hyperglycemic condition and hyperinsulinism by regulating ATF2 target genes MAP4K4, PRKCE, PEA15, BDNF, PRKACB, and GNAS which can otherwise lead to the development of type II diabetes mellitus. Moreover, we showed that hsa-miR-933 can regulate a target of ATF2, brain-derived neurotrophic factor (BDNF), to modulate the optimal expression of ATF2 in neuron cells to render neuroprotection for the inhibition of neurodegenerative diseases. Conclusions Our in silico model provides interesting resources for experimentations in a model organism or cell line for further validation. These findings may extend the common perception of gene expression analysis with new regulatory functionality.

Studies in three mouse models of breast cancer identified profound discrepancies between cell autonomous and systemic Akt1 or Akt2 deletion on breast cancer tumorigenesis and metastasis. First, unlike systemic Akt1 deletion, which inhibits metastasis, cell autonomous Akt1 deletion does not. Second, systemic Akt2 deletion does not inhibit mammary tumorigenesis and metastasis, but cell autonomous Akt2 deletion eliminates ErbB2 expressing cells in the mammary gland and prevents tumorigenesis. However, the elevation in insulin by Akt2 systemic deletion hyperactivates tumor Akt, enabling ErbB2 expression, and exacerbates mammary tumorigenesis. Decreasing insulin level inhibits accelerated tumorigenesis by systemic Akt2 deletion. Single cell mRNA sequencing revealed that systemic Akt1 deletion maintains the pro-metastatic cluster within primary tumors but ablates pro-metastatic neutrophils. Systemic Akt1 deletion inhibits metastasis by impairing the survival and mobilization of tumor-associated neutrophils. Importantly, neutrophil-specific deletion of Akt1 is sufficient to exert resistance to metastasis. The results underscore the importance of determining systemic effects rather than cell autonomous effects as a proof of concept for cancer therapy.

As the COVID-19 pandemic progresses, fatality and cases of new infections are also increasing at an alarming rate. SARS-CoV-2 follows a highly variable course and it is becoming more evident that individual’s immune system has a decisive influence on the progression of the disease. However, the detailed underlying molecular mechanisms of the SARS-CoV-2 mediate disease pathogenesis are largely unknown. Only a few host transcriptional responses in COVID-19 have been reported so far from the Western world, but no such data has been generated from the South-Asian region yet to correlate the conjectured lower fatality around this part of the globe. In this context, we aimed to perform the transcriptomic profiling of the COVID-19 patients from Bangladesh along with the reporting of the SARS-CoV-2 isolates from these patients. Moreover, we performed a comparative analysis to demonstrate how differently the various SARS-CoV-2 infection systems are responding to the viral pathogen. We detected a unique missense mutation at 10329 position of ORF1ab gene, annotated to 3C like proteinase, which is found in 75% of our analyzed isolates; but is very rare globally. Upon the functional enrichment analyses of differentially modulated genes, we detected a similar host induced response reported earlier; this response was mainly mediated by the innate immune system, interferon stimulation, and upregulated cytokine expression etc. in the Bangladeshi patients. Surprisingly, we did not perceive the induction of apoptotic signaling, phagosome formation, antigen presentation and production, hypoxia response within these nasopharyngeal samples. Furthermore, while comparing with the other SARS-CoV-2 infection systems, we spotted that lung cells trigger the more versatile immune and cytokine signaling which was several folds higher compared to our reported nasopharyngeal samples. We also observed that lung cells did not expressACE2in a very high amount as suspected, however, the nasopharyngeal cells are found overexpressingACE2. But the amount ofDPP4expression within the nasal samples was significantly lower compared to the other cell types. Surprisingly, we observed that lung cells express a very high amount of integrins compared to the nasopharyngeal samples, which might suggest the putative reasons for an increased amount of viral infections in the lungs. From the network analysis, we got clues on the probable viral modulation for the overexpression of these integrins. Our data will provide valuable insights in developing potential studies to elucidate the roles of ethnicity effect on the viral pathogenesis, and incorporation of further data will enrich the search of an effective therapeutics.

A detailed understanding of the molecular mechanism of SARS-CoV-2 pathogenesis is still elusive, and there is a need to address its deadly nature and to design effective therapeutics. Here, we present a study that elucidates the interplay between the SARS-CoV and SARS-CoV-2 viruses' and host's miRNAs, an epigenetic regulator, as a mode of pathogenesis; and we explored how the SARS-CoV and SARS-CoV-2 infections differ in terms of their miRNA-mediated interactions with the host and the implications this has in terms of disease complexity. We have utilized computational approaches to predict potential host and viral miRNAs and their possible roles in different important functional pathways. We have identified several putative host antiviral miRNAs that can target the SARS viruses and also predicted SARS viruses-encoded miRNAs targeting host genes. In silico predicted targets were also integrated with SARS-infected human cell microarray and RNA-seq gene expression data. A comparison between the host miRNA binding profiles on 67 different SARS-CoV-2 genomes from 24 different countries with respective country's normalized death count surprisingly uncovered some miRNA clusters, which are associated with increased death rates. We have found that induced cellular miRNAs can be both a boon and a bane to the host immunity, as they have possible roles in neutralizing the viral threat; conversely, they can also function as proviral factors. On the other hand, from over representation analysis, our study revealed that although both SARS-CoV and SARS-CoV-2 viral miRNAs could target broad immune-signaling pathways; only some of the SARS-CoV-2 miRNAs are found to uniquely target some immune-signaling pathways, such as autophagy, IFN-I signaling, etc., which might suggest their immune-escape mechanisms for prolonged latency inside some hosts without any symptoms of COVID-19. Furthermore, SARS-CoV-2 can modulate several important cellular pathways that might lead to the increased anomalies in patients with comorbidities like cardiovascular diseases, diabetes, breathing complications, etc. This might suggest that miRNAs can be a key epigenetic modulator behind the overcomplications amongst the COVID-19 patients. Our results support that miRNAs of host and SARS-CoV-2 can indeed play a role in the pathogenesis which can be further concluded with more experiments. These results will also be useful in designing RNA therapeutics to alleviate the complications from COVID-19.

BackgroundSince December 2019, the world is experiencing an unprecedented crisis due to a novel coronavirus, SARS-CoV-2. Owing to poor understanding of pathogenicity, the virus is eluding treatment and complicating recovery. Regulatory roles of long non-coding RNAs (lncRNAs) during viral infection and associated antagonism of host antiviral immune responses has become more evident in last decade. To elucidate possible functions of lncRNAs in the COVID-19 pathobiology, we have utilized RNA-seq dataset of SARS-CoV-2 infected lung epithelial cells.ResultsOur analyses uncover 21 differentially expressed lncRNAs whose functions are broadly involved in cell survival and regulation of gene expression. By network enrichment analysis we find that these lncRNAs can directly interact with differentially expressed protein-coding genes ADAR, EDN1, KYNU, MALL, TLR2 and YWHAG; and also AKAP8L, EXOSC5, GDF15, HECTD1, LARP4B, LARP7, MIPOL1, UPF1, MOV10 and PRKAR2A, host genes that interact with SARS-CoV-2 proteins. These genes are involved in cellular signaling, metabolism, immune response and RNA homeostasis. Since lncRNAs have been known to sponge microRNAs and protect expression of upregulated genes, we also identified 9 microRNAs that are induced in viral infections; however, some lncRNAs are able to block their usual suppressive effect on overexpressed genes and consequently contribute to host defense and cell survival.ConclusionsOur investigation determines that deregulated lncRNAs in SARS-CoV-2 infection are involved in viral proliferation, cellular survival, and immune response, ultimately determining disease outcome and this information could drive the search for novel RNA therapeutics as a treatment option.

Since the first recorded case of the SARS-CoV-2, it has acquired several mutations in its genome while spreading throughout the globe. However, apart from some changes in protein coding, functional importance of these mutations in disease pathophysiology are still largely unknown. In this study, we investigated the significance of these mutations both from the host’s and virus’s perspective by analyzing the host miRNA binding and virus’s internal ribosome entry site (IRES), respectively. Strikingly, we observed that due to the acquired mutations, host miRNAs bind differently compared to the reference; where few of the miRNAs lost and few gained the binding affinity for targeting the viral genome. Moreover, functional enrichment analysis suggests that targets of both of these gained and lost miRNAs might be involved in various host immune signaling pathways. Also, we sought to shed some insights on the impacts of mutations on the IRES structure of SARS-CoV-2. Remarkably, we detected that three particular mutations in the IRES can disrupt its secondary structure which can further make the virus less functional. These results could be valuable in exploring the functional importance of the mutations of SARS-CoV-2 and could provide novel insights into the differences observed different parts of the world.

BackgroundmiRNAs are small non-coding RNAs that regulate the expression of genes by RNA silencing method. Like eukaryotic organisms, some viruses also produce miRNAs. While contribution of host miRNA in the prevention of viral pathogenesis has been studied, it is not known very well how viral miRNA can confer its survival in the host. Here we hypothesized that viral miRNAs can bind to the host target genes to confer their pathogenicity by down-regulating specific pathways and related genes that otherwise pose threat to cell survival.Methods and ResultsUsing targets of 168 viral miRNAs from 13 different viruses overrepresentation analysis was done. Functional enrichment analysis of the genes targeted by the miRNAs indicates that viruses target specific immune system and host defense related pathways via miRNA mediated gene silencing. Integration and analysis of the publicly available experimental host gene expression data by RNA-seq provided insight that viruses target host apoptosis process by switching off related genes through miRNA induced mechanisms and thus probably ensure their survival.ConclusionsAs switching off the apoptosis of host cells would provide the viruses with selective advantages in surviving inside host, our findings therefore envisage an important function of viral miRNA which demands further in vivo experiments for better understanding in this regard.

An incomplete understanding of the molecular mechanisms behind impairment of lung pathobiology by COVID-19 complicates its clinical management. In this study, we analyzed the gene expression pattern of cells obtained from biopsies of COVID-19-affected patient and compared to the effects observed in typical SARS-CoV-2 and SARS-CoV-infected cell-lines. We then compared gene expression patterns of COVID-19-affected lung tissues and SARS-CoV-2-infected cell-lines and mapped those to known lung-related molecular networks, including hypoxia induced responses, lung development, respiratory processes, cholesterol biosynthesis and surfactant metabolism; all of which are suspected to be downregulated following SARS-CoV-2 infection based on the observed symptomatic impairments. Network analyses suggest that SARS-CoV-2 infection might lead to acute lung injury in COVID-19 by affecting surfactant proteins and their regulators SPD, SPC, and TTF1 through NSP5 and NSP12; thrombosis regulators PLAT, and EGR1 by ORF8 and NSP12; and mitochondrial NDUFA10, NDUFAF5, and SAMM50 through NSP12. Furthermore, hypoxia response through HIF-1 signaling might also be targeted by SARS-CoV-2 proteins. Drug enrichment analysis of dysregulated genes has allowed us to propose novel therapies, including lung surfactants, respiratory stimulants, sargramostim, and oseltamivir. Our study presents a distinct mechanism of probable virus induced lung damage apart from cytokine storm.

Amongst the anti-TNF-α therapy for rheumatoid arthritis and other autoimmune diseases, Adalimumab mAb is one of the best candidates. However, several risk factors are found to be associated with higher doses. Improvement of the binding properties will therefore significantly increase its therapeutic efficacy, reduce the dosage requirements, and ultimately the associated toxicity and treatment cost. Here, we proposed a systematic in silico approach of finding newer mAb variants with improved binding properties. Using various bioinformatics tools, we have identified the significant amino acid residues on Adalimumab mAb. Next, we searched for the suitability of the other residues for mutating the significant residues and from the combinations of suitable mutations, variants were designed. To find the most significant ones, binding properties of the variants were compared with the wild type Adalimumab mAb using molecular docking scrutiny and molecular dynamics simulation. Finally, structural properties between the variant and wild type were analyzed. We have identified the six most significant residues on Adalimumab mAb involved in the antigen-antibody interactions. Using the suitable mutations replacing each of these residues, we have modeled 143 variants. From several docking analyses, we have found five significant variants and after molecular dynamics simulation, one most significant variant with improved binding affinity was identified whose structural properties are similar to the wild type Adalimumab mAb. Designed variant from this study, may provide newer insights on the structure-based affinity improvements of monoclonal antibodies and likewise modifications of the Fc region will also improve the therapeutic effector functions of antibodies too.

Detailed molecular mechanism of SARS-CoV-2 pathogenesis is still elusive to address its deadlier nature and to design effective theraputics. Here, we present our study elucidating the interplay between the SARS-CoV and SARS-CoV-2 viruses’; and host’s miRNAs, an epigenetic regulator, as a mode of pathogenesis, and enlightened how the SARS-CoV and SARS-CoV-2 infections differ in terms of their miRNA mediated interactions with host and its implications in the disease complexity. We have utilized computational approaches to predict potential host and viral miRNAs, and their possible roles in different important functional pathways. We have identified several putative host antiviral miRNAs that can target the SARS viruses, and also SARS viruses’ encoded miRNAs targeting host genes. In silico predicted targets were also integrated with SARS infected human cells microarray and RNA-seq gene expression data. Comparison of the host miRNA binding profiles on 67 different SARS-CoV-2 genomes from 24 different countries with respective country’s normalized death count surprisingly uncovered some miRNA clusters which are associated with increased death rates. We have found that induced cellular miRNAs can be both a boon and a bane to the host immunity, as they have possible roles in neutralizing the viral threat, parallelly, they can also function as proviral factors. On the other hand, from over representation analysis, interestingly our study revealed that although both SARS-CoV and SARS-CoV-2 viral miRNAs could target broad immune signaling pathways; only some of the SARS-CoV-2 miRNAs are found to uniquely target some immune signaling pathways like-autophagy, IFN-I signaling etc, which might suggest their immune-escape mechanisms for prolonged latency inside some hosts without any symptoms of COVID-19. Further, SARS-CoV-2 can modulate several important cellular pathways which might lead to the increased anomalies in patients with comorbidities like-cardiovascular diseases, diabetes, breathing complications, etc. This might suggest that miRNAs can be a key epigenetic modulator behind the overcomplications amongst the COVID-19 patients. Our results support that miRNAs of host and SARS-CoV-2 can indeed play a role in the pathogenesis which can be further concluded with more experiments. These results will also be useful in designing RNA therapeutics to alleviate the complications from COVID-19.

Vitamin D (Vit.D) has tantalizing effects as a neurosteroidal micronutrient in the brain...

SummaryMammalian IκB proteins (IκBs) exert their main function as negative regulators of NF-κB, a central signaling pathway controlling immunity and inflammation. An alternative chromatin role for IκBs has been shown to affect stemness and cell differentiation. However, the involvement of NF-κB in this function has not been excluded. NFKI-1 and IKB-1 are IκB homologs inCaenorhabditis elegans, which lacks NF-κB nuclear effectors. We found thatnfki-1andikb-1mutants display developmental defects that phenocopy mutations in Polycomb and UTX-1 histone demethylase, suggesting a role forC. elegansIκBs in chromatin regulation. Further supporting this possibility(i)we detected NFKI-1 in the nucleus of cells;(ii)NFKI-1 and IKB-1 bind to histones and Polycomb proteins,(iii)and associate with chromatinin vivo, and(iv)mutations innfki-1andikb-1alter chromatin marks. Based on these results, we propose that ancestral IκB inhibitors modulate Polycomb activity at specific gene subsets with an impact on development.

The receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. Here, we profile the Drosophila larval brain by single cell RNA-sequencing and identify Amalgam (Ama), encoding a cell adhesion protein of the immunoglobulin IgLON family, that regulates the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood-brain barrier (BBB) that leads to hemocyte infiltration and neuronal death. We show that Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPOUTY2 in glioblastoma cell lines suggesting that the relationship between Ama and Sty is conserved.

Since the first recorded case of the SARS-CoV-2, it has acquired several mutations in its genome while spreading throughout the globe. In this study, we investigated the significance of these mutations by analyzing the host miRNA binding and virus's internal ribosome entry site (IRES). Strikingly, we observed that due to the acquired mutations, five host miRNAs which lost their affinity for targeting the viral genome, and another five can target the mutated viral genome. Moreover, functional enrichment analysis suggests that targets of both of these miRNAs might be involved in various host immune signaling pathways. Remarkably, we detected that three particular mutations in the IRES can disrupt its secondary structure which can further make the virus less functional. These results could be valuable in exploring the functional importance of the mutations of SARS-CoV-2 and could provide novel insights into the differences observed different parts of the world.

The differential expression of two closelyassociated cyclooxygenase isozymes, COX-1 and COX-2, exhibited functions beyond eicosanoid metabolism. We hypothesized that COX-1 or COX-2 knockout lung fibroblasts may display altered protein profiles which may allow us to further differentiate the functional roles of these isozymes at the molecular level. Proteomic analysis shows constitutive production of macrophage migration inhibitory factor (MIF) in lung fibroblasts derived from COX-2-/- but not wild-type (WT) or COX-1-/- mice. MIF was spontaneously released in high levels into the extracellular milieu of COX2-/- fibroblasts seemingly from the preformed intracellular stores, with no change in the basal gene expression of MIF. The secretion and regulation of MIF in COX-2-/- was "prostaglandin-independent." GO analysis showed that concurrent with upregulation of MIF, there is a significant surge in expression of genes related to fibroblast growth, FK506 binding proteins, and isomerase activity in COX-2-/- cells. Furthermore, COX-2-/- fibroblasts also exhibit a significant increase in transcriptional activity of various regulators, antagonists, and co-modulators of p53, as well as in the expression of oncogenes and related transcripts. Integrative Oncogenomics Cancer Browser (IntroGen) analysis shows downregulation of COX-2 and amplification of MIF and/or p53 activity during development of glioblastomas, ependymoma, and colon adenomas. These data indicate the functional role of the MIF-COX-p53 axis in inflammation and cancer at the genomic and proteomic levels in COX-2-ablated cells. This systematic analysis not only shows the proinflammatory state but also unveils a molecular signature of a pro-oncogenic state of COX-1 in COX-2 ablated cells.

In Drosophila, the wing disc‐associated muscle precursor cells give rise to the fibrillar indirect flight muscles (IFM) and the tubular direct flight muscles (DFM). To understand early transcriptional events underlying this muscle diversification, we performed single‐cell RNA‐sequencing experiments and built a cell atlas of myoblasts associated with third instar larval wing disc. Our analysis identified distinct transcriptional signatures for IFM and DFM myoblasts that underlie the molecular basis of their divergence. The atlas further revealed various states of differentiation of myoblasts, thus illustrating previously unappreciated spatial and temporal heterogeneity among them. We identified and validated novel markers for both IFM and DFM myoblasts at various states of differentiation by immunofluorescence and genetic cell‐tracing experiments. Finally, we performed a systematic genetic screen using a panel of markers from the reference cell atlas as an entry point and found a novel gene, Amalgam which is functionally important in muscle development. Our work provides a framework for leveraging scRNA‐seq for gene discovery and details a strategy that can be applied to other scRNA‐seq datasets., A single‐cell atlas of the Drosophila wing disc identifies the transcriptional profiles and markers for diverse cell types. The myoblasts exhibit distinct Notch‐dependent states of differentiation, and diversification into fiber fate specification.

SummaryInDrosophila, the wing disc-associated adult muscle precursors (AMPs) give rise to the fibrillar indirect flight muscles (IFM) and the tubular direct flight muscles (DFM). To understand early transcriptional events underlying this muscle diversification, we performed single cell RNA-sequencing experiments and built a cell atlas of AMPs associated with third instar larval wing disc. Our analysis identified distinct transcriptional signatures for IFM and DFM precursors that underlie the molecular basis of their divergence. The atlas further revealed various states of differentiation of AMPs, thus illustrating previously unappreciated spatial and temporal heterogeneity among them. We identified and validated novel markers for both IFM and DFM precursors at various states of differentiation by immunofluorescence and genetic tracing experiments. Finally, we performed a systematic genetic screen using a panel of markers from the reference cell atlas as an entry point and found a novel gene,Ama, which is functionally important in muscle development. Thus, our work provides a framework of leveraging scRNA-seq for gene discovery and therefore, this strategy can be applied to other scRNA-seq datasets.

Long intergenic noncoding RNAs (lincRNAs) are more than 200 bases long, transcribed from intergenic genomic regions and do not undergo translation. They have regulatory roles in differentiation and development. However, how their transcription is activated and how their expression is differentially modulated in differentiation is quite unclear. In this study, we explored and analyzed data at the transcriptomic and epigenetic level to address these questions. Here, we identified novel lincRNAs that are differentially expressed in neuronal and hematopoietic differentiation and showed that such differential modulations are achieved under epigenetic regulations. lincRNAs that are upregulated in mature cells than in progenitor are activated from a bivalent poised state where activating H3K4me3/H3K9ac/H3K27ac and suppressive H3K9me3/H3K27me3 marks are colocalized. And, lincRNAs that are downregulated in mature cells after differentiation are suppressed by the addition of H3K9me3/H3K27me3 marks. Moreover, here we show a tissue-specific expression pattern of lincRNAs in various cell lines and normal tissues. The study reveals bidirectional histone marks as an epigenetic means of directing the differential expression of lincRNAs which are found to be involved in the process of cellular differentiation.

Liquid Chromatography tandem mass spectrometry (LC-MS/MS) is used for the diagnosis of more than 30 inborn errors of metabolisms (IEMs). Accurate and reliable diagnosis of IEMs by quantifying amino acids (AAs) and acylcarnitines (ACs) using LC-MS/MS systems depend on the establishment of age-specific cut-offs of the analytes. This study aimed to (1) determine the age-specific cut-off values of AAs and ACs in Bangladesh and (2) validate the LC-MS/MS method for diagnosis of the patients with IEMs. A total of 570 enrolled healthy participants were divided into 3 age groups, namely, (1) newborns (1-7 days), (2) 8 days–7 years, and (3) 8–17 years, to establish the age-specific cut-offs for AAs and ACs. Also, 273 suspected patients with IEMs were enrolled to evaluate the reliability of the established cut-off values. Quantitation of AAs and ACs was performed on an automated LC-MS/MS system using dried blood spot (DBS) cards. Then the specimens of the enrolled clinically suspected patients were analyzed by the established method. Nine patients came out as screening positive for different IEMs, including two borderline positive cases of medium-chain acyl-CoA dehydrogenase deficiency (MCAD). A second-tier test for confirmation of the screening positive cases was conducted by urinary metabolic profiling using gas chromatography- mass spectrometry (GC-MS). Out of 9 cases that came out as screening positive by LC-MS/MS, seven cases were confirmed by urinary GC-MS analysis including 3 cases with phenylketonuria, 1 with citrullinemia type II, 1 with methylmalonic acidemia, 1 with isovaleric acidemia and 1 with carnitine uptake defect. Two borderline positive cases with MCAD were found negative by urinary GC-MS analysis. In conclusion, along with establishment of a validated LC-MS/MS method for quantitation of AAs and ACs from the DBS cards, the study also demonstrates the presence of predominantly available IEMs in Bangladesh.

Aim: In silico approach is used to identify most potent epitope and drug against pathogenic Hantavirus against which no approved therapeutics exist. Methods: Nucleocapsid protein sequences were retrieved, aligned and conserved regions were analyzed for the presence of B- and T-cell epitopes, and pockets for potential drugs. Results: T-cell epitope SYLRRTQSM and B-cell epitopes SYLRRTQ and YLRRTQSM appeared to be highly conserved, antigenic, nonallergenic. The T-cell epitope bound to maximum alleles. Thus, SYLRRTQSM would likely elicit both T- and B-cell immunity. High-throughput screening of Traditional Chinese Medicine database by docking technique revealed a potential drug, compound 46547 (1R,11S,15S,18S,20S,21R,22S)-12-oxa-8,17-diazaheptacyclo[15.5.2.0^{1,18}.0^{2,7}.0^{8,22}.0^{11,21}.0^{15,20}]tetracosa-2,4,6-trien-9-one. Conclusion: Our results predict potential therapeutics against multiple strains of pathogenic Hantavirus, but requires validation by in vivo experimentation.

Heart muscle maintains blood circulation, while skeletal muscle powers skeletal movement. Despite having similar myofibrilar sarcomeric structures, these striated muscles differentially express specific sarcomere components to meet their distinct contractile requirements. The mechanism responsible is still unclear. We show here that preservation of the identity of the two striated muscle types depends on epigenetic repression of the alternate lineage gene program by the chromatin remodeling complex Chd4/NuRD. Loss of Chd4 in the heart triggers aberrant expression of the skeletal muscle program, causing severe cardiomyopathy and sudden death. Conversely, genetic depletion of Chd4 in skeletal muscle causes inappropriate expression of cardiac genes and myopathy. In both striated tissues, mitochondrial function was also dependent on the Chd4/NuRD complex. We conclude that an epigenetic mechanism controls cardiac and skeletal muscle structural and metabolic identities and that loss of this regulation leads to hybrid striated muscle tissues incompatible with life. We thank Drs. Richard Harvey (University of Sydney, Australia) and Michael Schneider (Imperial College, London, UK) for providing Nkx2.5cre/+ and α-MHCcre/+ transgenic lines, respectively, Dr. S. Bartlett for English editing, and Gemma Benito and Ana Guío for technical assistance. J.M.R. is supported by the Spanish Ministry of Economy and Competitiveness (Ministerio de Economía y Competitividad; SAF 2012 34296 and SAF 2015 63633-R), the Spanish Ministry of Health (Ministerio de Sanidad y Consumo) Red de Investigación Cardiovascular (RIC; grant RD06/0042/0022), and the Fundación La Marató TV3 (264/C/2012). The RIC also supports J.L.P. (RD12/0042/0005) and L.J.J.-B. (RD12/0042/0056). RIC grants are partially funded by FEDER funds. P.M.-C., J.A.E., and J.L.P. are supported by SAF2012-38547, SAF2015-67369-R, SAF2012-1207, and SAF2013-45543-R, respectively. E.P. and P.M.-C. were also supported by SAF2015-67369-R. NGS experiments were performed at the CNIC Genomics Unit. IF of adult hearts was performed at the Advanced Fluorescence Microscopy Unit of (IBMB-CSIC) Barcelona. The CNIC is supported by the Spanish Ministry of Economy and Competitiveness and the Pro-CNIC Foundation and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505). R.A.-P. was supported by a Ramón y Cajal grant and is a holder of a Marie Curie grant (RA-P:UEO/MCA1108). M.Z. was a holder of a Marie Curie Outgoing International Fellowship (MOIF-CT-2006-039327), and P.G.A. was supported by a Ramón y Cajal grant from the Spanish Ministry of Education, Science and Sport.

The constitutively-expressed cyclooxygenase 1 (COX-1) and the inducible COX-2 are both involved in the conversion of arachidonic acid (AA) to prostaglandins (PGs). However, the functional roles of COX-1 at the cellular level remain unclear. We hypothesized that by comparing differential gene expression and eicosanoid metabolism in lung fibroblasts from wild-type (WT) mice and COX-2(-/-) or COX-1(-/-) mice may help address the functional roles of COX-1 in inflammation and other cellular functions. Compared to WT, the number of specifically-induced transcripts were altered descendingly as follows: COX-2(-/-)COX-1(-/-)WT+IL-1β. COX-1(-/-) or COX-2(-/-) cells shared about 50% of the induced transcripts with WT cells treated with IL-1β, respectively. An interactive "anti-inflammatory, proinflammatory, and redox-activated" signature in the protein-protein interactome map was observed in COX-2(-/-) cells. The augmented COX-1 mRNA (in COX-2(-/-) cells) was associated with the upregulation of mRNAs for glutathione S-transferase (GST), superoxide dismutase (SOD), NAD(P)H dehydrogenase quinone 1 (NQO1), aryl hydrocarbon receptor (AhR), peroxiredoxin, phospholipase, prostacyclin synthase, and prostaglandin E synthase, resulting in a significant increase in the levels of PGE2, PGD2, leukotriene B4 (LTB4), PGF1α, thromboxane B2 (TXB2), and PGF2α. The COX-1 plays a dominant role in shifting AA toward the LTB4 pathway and anti-inflammatory activities. Compared to WT, the upregulated COX-1 mRNA in COX-2(-/-) cells generated an "eicosanoid storm". The genomic characteristics of COX-2(-/-) is similar to that of proinflammatory cells as observed in IL-1β induced WT cells. COX-1(-/-) and COX-2(-/-) cells exhibited compensation of various eicosanoids at the genomic and metabolic levels.

The histone deacetylase HDAC7 interacts with and represses myeloid and T cell genes in pro–B cells. HDAC7 deletion blocks early B cell development and results in severe lymphopenia., Class IIa histone deacetylase (HDAC) subfamily members are tissue-specific gene repressors with crucial roles in development and differentiation processes. A prominent example is HDAC7, a class IIa HDAC that shows a lymphoid-specific expression pattern within the hematopoietic system. In this study, we explored its potential role in B cell development by generating a conditional knockout mouse model. Our study demonstrates for the first time that HDAC7 deletion dramatically blocks early B cell development and gives rise to a severe lymphopenia in peripheral organs, while also leading to pro–B cell lineage promiscuity. We find that HDAC7 represses myeloid and T lymphocyte genes in B cell progenitors through interaction with myocyte enhancer factor 2C (MEFC2). In B cell progenitors, HDAC7 is recruited to promoters and enhancers of target genes, and its absence leads to increased enrichment of histone active marks. Our results prove that HDAC7 is a bona fide transcriptional repressor essential for B cell development.

Although thyroid dyshormonogenesis (TDH) accounts for 10-20% of congenital hypothyroidism (CH), the molecular etiology of TDH is unknown in Bangladesh. Thyroid peroxidase (TPO) is most frequently associated with TDH and the present study investigated the spectrum of TPO mutations in Bangladeshi patients and analyzed the effects of mutations on TPO protein structure through in silico approach. Sequencing-based analysis of TPO gene revealed four mutations in 36 diagnosed patients with TDH including three nonsynonymous mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, and one synonymous mutation p.Pro715Pro. Homology modelling-based analysis of predicted structures of MPO-like domain (TPO142-738) and the full-length TPO protein (TPO1-933) revealed differences between mutant and wild type structures. Molecular docking studies were performed between predicted structures and heme. TPO1-933 predicted structure showed more reliable results in terms of interactions with the heme prosthetic group as the binding energies were -11.5 kcal/mol, -3.2 kcal/mol, -11.5 kcal/mol, and -7.9 kcal/mol for WT, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, respectively, implying that p.Ala373Ser and p.Thr725Pro mutations were more damaging than p.Ser398Thr. However, for the TPO142-738 predicted structures, the binding energies were -11.9 kcal/mol, -10.8 kcal/mol, -2.5 kcal/mol, and -5.3 kcal/mol for the wild type protein, mutant proteins with p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro substitutions, respectively. However, when the interactions between the crucial residues including residues His239, Arg396, Glu399, and His494 of TPO protein and heme were taken into consideration using both TPO1-933 and TPO142-738 predicted structures, it appeared that p.Ala373Ser and p.Thr725Pro could affect the interactions more severely than the p.Ser398Thr. Validation of the molecular docking results was performed by computer simulation in terms of quantum mechanics/molecular mechanics (QM/MM) and molecular dynamics (MD) simulation. In conclusion, the substitutions mutations, namely, p.Ala373Ser, p.Ser398Thr, and p.Thr725Pro, had been involved in Bangladeshi patients with TDH and molecular docking-based study revealed that these mutations had damaging effect on the TPO protein activity.

Treatment for acute myeloid leukemia (AML) remains suboptimal and many patients remain refractory or relapse upon standard chemotherapy based on nucleoside analogs plus anthracyclines. The crosstalk between AML cells and the BM stroma is a major mechanism underlying therapy resistance in AML. Lenalidomide and pomalidomide, a new generation immunomodulatory drugs (IMiDs), possess pleiotropic anti-leukemic properties including potent immune-modulating effects and are commonly used in hematological malignances associated with intrinsic dysfunctional BM such as myelodysplastic syndromes and multiple myeloma. Whether IMiDs may improve the efficacy of current standard treatment in AML remains understudied. Here, we have exploited in vitro and in vivo preclinical AML models to analyze whether IMiDs potentiate the efficacy of AraC/Idarubicin-based standard AML chemotherapy by interfering with the BM stroma-mediated chemoresistance. We report that IMiDs do not exert cytotoxic effects on either non-del5q/5q- AML cells nor BM-MSCs, but they enhance the immunomodulatory properties of BM-MSCs. When combined with AraC/Idarubicin, IMiDs fail to circumvent BM stroma-mediated resistance of non-del5q/5q- AML cells in vitro and in vivo but induce robust extramedullary mobilization of AML cells. When administered as a single agent, lenalidomide specifically mobilizes non-del5q/5q- AML cells, but not healthy CD34+ cells, to peripheral blood (PB) through specific downregulation of CXCR4 in AML blasts. Global gene expression profiling supports a migratory/mobilization gene signature in lenalidomide-treated non-del5q/5q- AML blasts but not in CD34+ cells. Collectively, IMiDs mobilize non-del5q/5q- AML blasts to PB through CXCR4 downregulation, but fail to potentiate AraC/Idarubicin activity in preclinical models of non-del5q/5q- AML. This work was supported by the European Research Council (CoG-2014-646903 to P.M), the Spanish Ministry of Economy and Competitiveness (SAF-SAF2013-43065, RTC-2016-4603-1 to P.M), the Asociación Española Contra el Cáncer (AECC-CI-2015), FERO Foundation, and the ISCIII/FEDER (PI14-01191) to C.B and the ‘‘Fundación Hay Esperanza’’ to E.A. P.M also acknowledges financial support from The Obra Social La Caixa-Fundaciò Josep Carreras, The Inocente Inocente Foundation and The Generalitat de Catalunya (SGR330). P.M an investigator of the Spanish Cell Therapy cooperative network (TERCEL). We thank Celgene Corporation (San Diego, CA) for providing IMiDs. We thank Judit Sopena and Mariano Graupera (IDIBELL, Barcelona) for their technical assistance with immunohistochamical analysis

DYRK1A is a dosage-sensitive protein kinase that fulfills key roles during development and in tissue homeostasis, and its dysregulation results in human pathologies. DYRK1A is present in both the nucleus and cytoplasm of mammalian cells, although its nuclear function remains unclear. Genome-wide analysis of DYRK1A-associated loci reveals that the kinase is recruited preferentially to promoters of genes actively transcribed by RNA polymerase II (RNAPII), which are functionally associated with translation, RNA processing, and cell cycle. DYRK1A-bound promoter sequences are highly enriched in a conserved palindromic motif, which is necessary to drive DYRK1A-dependent transcriptional activation. DYRK1A phosphorylates the C-terminal domain (CTD) of RNAPII at Ser2 and Ser5. Depletion of DYRK1A results in reduced association of RNAPII at the target promoters as well as hypophosphorylation of the RNAPII CTD along the target gene bodies. These results are consistent with DYRK1A being a transcriptional regulator by acting as a CTD kinase. This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO: BFU2010-15347, BFU2013-44513 to S.d.l.L., SAF2012-36199 to N.L.-B., and ‘Centro de Excelencia Severo Ochoa 2013-2017’-SEV-2012-0208) and the Secretariat of Universities and Research-Government of Catalonia (2014SGR674 to S.d.l.L.). C.D.V. and E.S. were FPI predoctoral fellows financed by MINECO (BES2008-002751 and BES2005-10136)

The protein p27Kip1 (p27), a member of the Cip-Kip family of cyclin-dependent kinase inhibitors, is involved in tumorigenesis and a correlation between reduced levels of this protein in human tumours and a worse prognosis has been established. Recent reports revealed that p27 also behaves as a transcriptional regulator. Thus, it has been postulated that the development of tumours with low amounts of p27 could be propitiated by deregulation of transcriptional programs under the control of p27. However, these programs still remain mostly unknown. The aim of this study has been to define the transcriptional programs regulated by p27 by first identifying the p27-binding sites (p27-BSs) on the whole chromatin of quiescent mouse embryonic fibroblasts. The chromatin regions associated to p27 have been annotated to the most proximal genes and it has been considered that the expression of these genes could by regulated by p27. The identification of the chromatin p27-BSs has been performed by Chromatin Immunoprecipitation Sequencing (ChIP-seq). Results revealed that p27 associated with 1839 sites that were annotated to 1417 different genes being 852 of them protein coding genes. Interestingly, most of the p27-BSs were in distal intergenic regions and introns whereas, in contrast, its association with promoter regions was very low. Gene ontology analysis of the protein coding genes revealed a number of relevant transcriptional programs regulated by p27 as cell adhesion, intracellular signalling and neuron differentiation among others. We validated the interaction of p27 with different chromatin regions by ChIP followed by qPCR and demonstrated that the expressions of several genes belonging to these programs are actually regulated by p27. Finally, cell adhesion assays revealed that the adhesion of p27-/- cells to the plates was much higher that controls, revealing a role of p27 in the regulation of a transcriptional program involved in cell adhesion.

The Scientific Foundation ‘Asociación Española Contra el Cáncer’ (AECC, Stable Coordinated Group, Hereditary Cancer); the BBVA Foundation; the Eugenio Rodríguez Pascual Foundation grant 2012; Generalitat de Catalunya AGAUR SGR 2012 grants 283, 290 and 312, and SGR 2014 grants 364, 530, and 535; Spanish Ministry of Health ISCIII FIS grants PI10/00057, PI10/00222, PI10/01422, PI12/01528, PI13/00132, and PI14/00336. ISCIII RTICC grants RD06/0020/1051, RD12/0036/0007, RD12/0036/0008 and RD12/0036/0063; Spanish Ministry of Science and Innovation, ‘Fondo Europeo de Desarrollo Regional (FEDER), una manera de hacer Europa’, MINECO grants SAF2010-20203 and SAF2013-46196; and the Telemaraton 2014 ‘Todos Somos Raros, Todos Somos Únicos’ grant P35 (...), Mateo, F.; Arenas, E. J.; Aguilar, H.; Serra-Musach, J.; de Garibay, G. Ruiz; Boni, J.; Maicas, M.; Du, S.; Iorio, F.; Herranz-Ors, C.; Islam, A.; Prado, X.; Llorente, A.; Petit, A.; Vidal, A.; Catala, I.; Soler, T.; Venturas, G.; Rojo-Sebastian, A.; Serra, H.; Cuadras, D.; Blanco, I.; Lozano, J.; Canals, F.; Sieuwerts, A. M.; de Weerd, V.; Look, M. P.; Puertas, S.; Garcia, N.; Perkins, A. S.; Bonifaci, N.; Skowron, M.; Gomez-Baldo, L.; Hernandez, V.; Martinez-Aranda, A.; Martinez-Iniesta, M.; Serrat, X.; Ceron, J.; Brunet, J.; Barretina, M. P.; Gil, M.; Falo, C.; Fernandez, A.; Morilla, I.; Pernas, S.; Pla, M. J.; Andreu, X.; Segui, M. A.; Ballester, R.; Castella, E.; Nellist, M.; Morales, S.; Valls, J.; Velasco, A.; Matias-Guiu, X.; Figueras, A.; Sanchez-Mut, J. V.; Sanchez-Cespedes, M.; Cordero, A.; Gomez-Miragaya, J.; Palomero, L.; Gomez, A.; Gajewski, T. F.; Cohen, E. E. W.; Jesiotr, M.; Bodnar, L.; Quintela-Fandino, M.; Lopez-Bigas, N.; Valdes-Mas, R.; Puente, X. S.; Vinals, F.; Casanovas, O.; Graupera, M.; Hernandez-Losa, J.; Ramon y Cajal, S.; Garcia-Alonso, L.; Saez-Rodriguez, J.; Esteller, M.; Sierra, A.; Martin-Martin, N.; Matheu, A.; Carracedo, A.; Gonzalez-Suarez, E.; Nanjundan, M.; Cortes, J.; Lazaro, C.; Odero, M. D.; Martens, J. W. M.; Moreno-Bueno, G.; Barcellos-Hoff, M. H.; Villanueva, A.; Gomis, R. R.; Pujana, M. A.

The cyclin-dependent kinase inhibitor p27Kip1 (p27) also behaves as a transcriptional repressor. Data showing that the p300/CBP-associated factor (PCAF) acetylates p27 inducing its degradation suggested that PCAF and p27 could collaborate in the regulation of transcription. However, this possibility remained to be explored. We analyzed here the transcriptional programs regulated by PCAF and p27 in the colon cancer cell line HCT116 by chromatin immunoprecipitation sequencing (ChIP-seq). We identified 269 protein-encoding genes that contain both p27 and PCAF binding sites being the majority of these sites different for PCAF and p27. PCAF or p27 knock down revealed that both regulate the expression of these genes, PCAF as an activator and p27 as a repressor. The double knock down of PCAF and p27 strongly reduced their expression indicating that the activating role of PCAF overrides the repressive effect of p27. We also observed that the transcription factor Pax5 interacts with both p27 and PCAF and that the knock down of Pax5 induces the expression of p27/PCAF target genes indicating that it also participates in the transcriptional regulation mediated by p27/PCAF. In summary, we report here a previously unknown mechanism of transcriptional regulation mediated by p27, Pax5 and PCAF., Ministerio de Economía y Competitividad (MINECO) [SAF2012-38078 to O.B. and BFU2015-65311-R to R.A.]; Instituto de Salud Carlos III [RD12/0036/0054 to O.B. and CB16/12/00244 (CIBERONC) to A.B.]; ‘La Caixa’ Foundation 2016-052407 (to R.A.). Funding for open access charge: MInisterio the Economia y Competitividad, Spain La Caixa Foundation. Spain.

We applied global gene expression arrays, quantitative real-time PCR, immunostaining, and functional assays to untangle the role of High Mobility Groups proteins (HMGs) in human osteoarthritis (OA)-affected cartilage. Bioinformatics analysis showed increased mRNA expression of Damage-Associated Molecular Patterns (DAMPs): HMGA, HMGB, HMGN, SRY, LEF1, HMGB1, MMPs, and HMG/RAGE-interacting molecules (spondins and S100A4, S100A10, and S100A11) in human OA-affected cartilage as compared with normal cartilage. HMGB2 was down-regulated in human OA-affected cartilage. Immunohistological staining identified HMGB1 in chondrocytes in the superficial cartilage. Cells of the deep cartilage and subchondral bone showed increased expression of HMGB1 in OA-affected cartilage. HMGB1 was expressed in the nucleus, cytosol, and extracellular milieu of chondrocytes in cartilage. Furthermore, HMGB1 was spontaneously released from human OA-affected cartilage in ex vivo conditions. The effects of recombinant HMGB1 was tested on human cartilage and chondrocytes in vitro. HMGB1 stimulated mRNA of 2 NFκB gene enhancers (NFκB1 and NFκB2), 16 CC and CXC chemokines (IL-8, CCL2, CCL20, CCL3, CCL3L1, CCL3L3, CCL4, CCL4L1, CCL4L2, CCL5, CCL8, CXCL1, CXCL10, CXCL2, CXCL3, and CXCL6) by ≥10-fold. Furthermore, HMGB1 and IL-1β and/or tumor necrosis factor α (but not HMGI/Y) also significantly induced inducible nitric oxide synthase, NO, and interleukin (IL)-8 production in human cartilage and chondrocytes. The recombinant HMGB1 utilized in this study shows properties that are similar to disulfide-HMGB1. The differential, stage and/or tissue-specific expression of HMGB1, HMGB2, and S100A in cartilage was associated with regions of pathology and/or cartilage homeostasis in human OA-affected cartilage. Noteworthy similarities in the expression of mouse and human HMGB1 and HMGB2 were conserved in normal and arthritis-affected cartilage. The multifunctional forms of HMGB1 and S100A could perpetuate damage-induced cartilage inflammation in late-stage OA-affected joints similar to sterile inflammation. The paracrine effects of HMGB1 can induce chemokines and NO that are perceived to change cartilage homeostasis in human OA-affected cartilage.

Guiu et al. use ChIP-on-chip analysis for the Notch partner RBPj, using embryonic tissue from the aorta-gonad-mesonephros region to identify potential novel Notch target genes involved in HSC emergence. They show that c-MYC–responsive gene Cdca7 is expressed in different HSC and progenitor subpopulations and that CDCA7 is important for maintaining the undifferentiated phenotype. Cdca7 acts downstream of Notch in HSCs in zebrafish, mouse, and human, indicating a highly conserved Notch/RBPj/Cdca7 axis in hematopoietic development., Hematopoietic stem cell (HSC) specification occurs in the embryonic aorta and requires Notch activation; however, most of the Notch-regulated elements controlling de novo HSC generation are still unknown. Here, we identify putative direct Notch targets in the aorta-gonad-mesonephros (AGM) embryonic tissue by chromatin precipitation using antibodies against the Notch partner RBPj. By ChIP-on-chip analysis of the precipitated DNA, we identified 701 promoter regions that were candidates to be regulated by Notch in the AGM. One of the most enriched regions corresponded to the Cdca7 gene, which was subsequently confirmed to recruit the RBPj factor but also Notch1 in AGM cells. We found that during embryonic hematopoietic development, expression of Cdca7 is restricted to the hematopoietic clusters of the aorta, and it is strongly up-regulated in the hemogenic population during human embryonic stem cell hematopoietic differentiation in a Notch-dependent manner. Down-regulation of Cdca7 mRNA in cultured AGM cells significantly induces hematopoietic differentiation and loss of the progenitor population. Finally, using loss-of-function experiments in zebrafish, we demonstrate that CDCA7 contributes to HSC emergence in vivo during embryonic development. Thus, our study identifies Cdca7 as an evolutionary conserved Notch target involved in HSC emergence.

SUMMARY The importance of the retinoblastoma tumor suppressor protein pRB in cell cycle control is well established. However, less is known about its role in differentiation during animal development. Here, we investigated the role of Rbf, the Drosophila pRB homolog, in adult skeletal muscles. We found that the depletion of Rbf severely reduced muscle growth and altered myofibrillogenesis but only minimally affected myoblast proliferation. We identified an Rbf-dependent transcriptional program in late muscle development that is distinct from the canonical role of Rbf in cell cycle control. Unexpectedly, Rbf acts as a transcriptional activator of the myogenic and metabolic genes in the growing muscles. The genomic regions bound by Rbf contained the binding sites of several factors that genetically interacted with Rbf by modulating Rbf-dependent phenotype. Thus, our results reveal a distinctive role for Rbf as a direct activator of the myogenic transcriptional program that drives late muscle differentiation., Graphical Abstract, In Brief Inactivation of the tumor suppressor RB, an obligatory step in most cancers, results in unrestrained cell cycle progression. Zappia et al. show that Rbf, the RB Drosophila ortholog, directly activates the metabolic program that accompanies muscle development. This work expands the understanding of the plethora of Rbf functions.