Bioinformatics Analysis Of Data Research Articles

C9orf16 represents the aberrant genetic programs and drives the progression of PDAC

BackgroundPancreatic ductal adenocarcinoma (PDAC), constituting 90% of pancreatic cancers, is the fourth leading cause of cancer-related deaths in the world. Lack of early detection of PDAC contributes to its poor prognosis as patients are often diagnosed at an advanced stage of disease. This is mostly due to the lack of promising diagnostic and therapeutic targets and corresponding drugs.Methods and resultsHere, by bioinformatic analysis of single cell RNA-sequencing data on normal pancreas tissues, primary and metastatic PDAC tumors, we identified a promising PDAC biomarker, C9orf16. The expression of C9orf16, rarely detectable in normal epithelial cells, was upregulated in primary PDAC cancer cells and was further elevated in metastatic PDAC cancer cells. Gain or loss of function of C9orf16 demonstrated its critical functions in regulating the cell proliferation, invasion and chemotherapy resistance of cancer cells. Pathway analysis and functional studies identified MYC signaling pathways as the most activated pathways in regulating C9orf16 expression and in mediating the development and progression of PDAC.ConclusionsThese data suggested a crucial gene regulation system, MYC-C9orf16, which is actively involved in PDAC development and progression, and targeting this system should be a novel diagnostic and therapeutic target for PDAC.

A hybrid feature selection approach for Microarray datasets using graph theoretic-based method

The feature selection process plays an important role in different fields, particularly in bioinformatics and microarray gene expression data analysis, for choosing discriminative genes from high-dimensional datasets and selecting a subset of highly relevant features with low redundancy that may lead to build improved prediction models. Consequently, this study proposes a new feature selection method that integrates Preordonnances theory in terms of new Relevance and Complementarity criteria introduced here and also connectivity in undirected Weighted Graphs (PCRWG). The method can handle high-dimensional data. PCRWG retains the relevant and complementary features to select effective features in large scale gene datasets. The proposed algorithm operates in two phases: filtering and wrapping. The strength of the first phase is that it is preceded by a step that further reduces the number of predictors by removing those in disagreement with the target based on the new proposed relevance criterion. Then, the proposed heuristic uses the relevance-complementarity ratio between preordonnances to automatically update the compromise rule between relevance and complementarity. In the wrapping phase, the suggested graph-based approach using maximal clique is based on a powerful relevance-complementarity matrix to consolidate edges, two connected interdependent features are complementary to each other, and it is possible to have high discriminative power when they serve as a group. We highlight the fact that existing graph-based feature selection algorithms do not consider relevance and complementarity simultaneously. The experiments were carried out on three simulated scenarios and the thirteen most popular cancer microarray gene datasets. Formally, they are eight binary and five multi-class microarray data. A 10-fold cross validation was used to evaluate the Support Vector Machine (SVM), Naive Bayes (NB) and artificial Neural Network (NN) classifiers. The empirical results demonstrate the high performance of the proposed hybrid approach when compared to the most recently published articles.

Dietary Selenomethionine Reduce Mercury Tissue Levels and Modulate Methylmercury Induced Proteomic and Transcriptomic Alterations in Hippocampi of Adolescent BALB/c Mice.

Methylmercury (MeHg) is a well-known environmental contaminant, particularly harmful to the developing brain. The main human dietary exposure to MeHg occurs through seafood consumption. However, seafood also contains several nutrients, including selenium, which has been shown to interact with MeHg and potentially ameliorate its toxicity. The aim of this study was to investigate the combined effects of selenium (as selenomethionine; SeMet) and MeHg on mercury accumulation in tissues and the effects concomitant dietary exposure of these compounds exert on the hippocampal proteome and transcriptome in mice. Adolescent male BALB/c mice were exposed to SeMet and two different doses of MeHg through their diet for 11 weeks. Organs, including the brain, were sampled for mercury analyses. Hippocampi were collected and analyzed using proteomics and transcriptomics followed by multi-omics bioinformatics data analysis. The dietary presence of SeMet reduced the amount of mercury in several organs, including the brain. Proteomic and RNA-seq analyses showed that both protein and RNA expression patterns were inversely regulated in mice receiving SeMet together with MeHg compared to MeHg alone. Several pathways, proteins and RNA transcripts involved in conditions such as immune responses and inflammation, oxidative stress, cell plasticity and Alzheimer’s disease were affected inversely by SeMet and MeHg, indicating that SeMet can ameliorate several toxic effects of MeHg in mice.

Endothelial RBPJ Is Essential for the Education of Tumor-Associated Macrophages.

Endothelial Notch signaling favors immunosuppression by increasing CXCL2 secretion to stimulate CD44 expression in macrophages, facilitating their education by tumor cells.

Ningmitai capsules have anti-inflammatory and pain-relieving effects in the chronic prostatitis/chronic pelvic pain syndrome mouse model through systemic immunity.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) seriously affects the physical and mental health of approximately 90% of males. Due to its complex and unclear etiology, the treatment methods that are currently available for chronic prostatitis/chronic pelvic pain syndrome are controversial, and their efficacy is unsatisfactory. At present, most researchers believe that this kind of prostatitis is caused by autoimmune inflammation. Chinese herbs, which are the essence of traditional Chinese medicine (TCM), are emerging treatment options for inflammation and immune diseases. In this experiment, we investigated the effect of Ningmitai capsules (a kind of traditional Chinese medicine widely used to treat lower urinary tract inflammation and pain in males) on chronic prostatitis/chronic pelvic pain syndrome in a non-obese diabetes-experimental autoimmune prostatitis (NOD-EAP) mouse model. First, by using bioinformatics analysis of data from the Encyclopedia of Traditional Chinese Medicine (ETCM) database, we found that quercetin, which is one of the main components of Ningmitai capsules, could reduce the secretion of CCL2 by inhibiting the MAPK pathway. In animal experiments, it was found that after Ningmitai treatment, the inflammation in mouse prostates was alleviated, the expression of CCL2, which is related to pain, and MAPK pathway components were downregulated, and the activation of the inflammatory NF–κB and STAT3 pathways was reduced. Pelvic pain and inflammation were relieved in mice with EAP. Due to the presence of the blood–prostate barrier, the drug may not completely reach the prostate directly and take effect locally. However, we found that after Ningmitai treatment, the proportions of proinflammatory CD11b+Ly6Chigh immune cells in the spleen, bloodstream (systemic immunity), and prostate (local immunity) were reduced. The infiltration of CD11b+ immune cells into the spleen and prostate was decreased. These findings suggested that Ningmitai can treat chronic prostatitis/chronic pelvic pain syndrome by affecting systemic and local immunities through the CCL2–MAPK pathway.

Experimental validation that human microbiome phages use alternative genetic coding

Previous bioinformatic analyses of metagenomic data have indicated that bacteriophages can use genetic codes different from those of their host bacteria. In particular, reassignment of stop codon TAG to glutamine (a variation known as ‘genetic code 15’) has been predicted. Here, we use LC-MS/MS-based metaproteomics of human fecal samples to provide experimental evidence of the use of genetic code 15 in two crAss-like phages. Furthermore, the proteomic data from several phage structural proteins supports the reassignment of the TAG stop codon to glutamine late in the phage infection cycle. Thus, our work experimentally validates the expression of genetic code 15 in human microbiome phages.

Screening of Sepsis Biomarkers Based on Bioinformatics Data Analysis.

Methods Gene expression profiles of GSE13904, GSE26378, GSE26440, GSE65682, and GSE69528 were obtained from the National Center for Biotechnology Information (NCBI). The differentially expressed genes (DEGs) were searched using limma software package. Gene Ontology (GO) functional analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and protein-protein interaction (PPI) network analysis were performed to elucidate molecular mechanisms of DEGs and screen hub genes. Results A total of 108 DEGs were identified in the study, of which 67 were upregulated and 41 were downregulated. 15 superlative diagnostic biomarkers (CCL5, CCR7, CD2, CD27, CD274, CD3D, GNLY, GZMA, GZMH, GZMK, IL2RB, IL7R, ITK, KLRB1, and PRF1) for sepsis were identified by bioinformatics analysis. Conclusion 15 hub genes (CCL5, CCR7, CD2, CD27, CD274, CD3D, GNLY, GZMA, GZMH, GZMK, IL2RB, IL7R, ITK, KLRB1, and PRF1) have been elucidated in this study, and these biomarkers may be helpful in the diagnosis and therapy of patients with sepsis.

MED16 Promotes Tumour Progression and Tamoxifen Sensitivity by Modulating Autophagy through the mTOR Signalling Pathway in ER-Positive Breast Cancer.

Recent studies have shown that the mediator complex (MED) plays a vital role in tumorigenesis and development, but the role of MED16 (mediator complex subunit 16) in breast cancer (BC) is not clear. Increasing evidence has shown that the mTOR pathway is important for tumour progression and therapy. In this study, we demonstrated that the mTOR signalling pathway is regulated by the expression level of MED16 in ER+ breast cancer. With the analysis of bioinformatics data and clinical specimens, we revealed an elevated expression of MED16 in luminal subtype tumours. We found that MED16 knockdown significantly inhibited cell proliferation and promoted G1 phase cell cycle arrest in ER+ BC cell lines. Downregulation of MED16 markedly reduced the sensitivity of ER+ BC cells to tamoxifen and increased the stemness and autophagy of ER+ BC cells. Bioinformatic analysis of similar genes to MED16 were mainly enriched in autophagy, endocrine therapy and mTOR signalling pathways, and the inhibition of mTOR-mediated autophagy restored sensitivity to tamoxifen by MED16 downregulation in ER+ BC cells. These results suggest an important role of MED16 in the regulation of tamoxifen sensitivity in ER+ BC cells, crosstalk between the mTOR signalling pathway-induced autophagy, and together, with the exploration of tamoxifen resistance, may indicate a new therapy option for endocrine therapy-resistant patients.

Thymosin β4 and prothymosin α promote cardiac regeneration post-ischaemic injury in mice.

The adult mammalian heart is a post-mitotic organ. Even in response to necrotic injuries, where regeneration would be essential to reinstate cardiac structure and function, only a minor percentage of cardiomyocytes undergo cytokinesis. The gene programme that promotes cell division within this population of cardiomyocytes is not fully understood. In this study, we aimed to determine the gene expression profile of proliferating adult cardiomyocytes in the mammalian heart after myocardial ischaemia, to identify factors to can promote cardiac regeneration. Here, we demonstrate increased 5-ethynyl-2'deoxyuridine incorporation in cardiomyocytes 3 days post-myocardial infarction in mice. By applying multi-colour lineage tracing, we show that this is paralleled by clonal expansion of cardiomyocytes in the borderzone of the infarcted tissue. Bioinformatic analysis of single-cell RNA sequencing data from cardiomyocytes at 3 days post ischaemic injury revealed a distinct transcriptional profile in cardiomyocytes expressing cell cycle markers. Combinatorial overexpression of the enriched genes within this population in neonatal rat cardiomyocytes and mice at postnatal day 12 (P12) unveiled key genes that promoted increased cardiomyocyte proliferation. Therapeutic delivery of these gene cocktails into the myocardial wall after ischaemic injury demonstrated that a combination of thymosin beta 4 (TMSB4) and prothymosin alpha (PTMA) provide a permissive environment for cardiomyocyte proliferation and thereby attenuated cardiac dysfunction. This study reveals the transcriptional profile of proliferating cardiomyocytes in the ischaemic heart and shows that overexpression of the two identified factors, TMSB4 and PTMA, can promote cardiac regeneration. This work indicates that in addition to activating cardiomyocyte proliferation, a supportive environment is a key for regeneration to occur.

The effect of Nrf2 deletion on the proteomic signature in a human colorectal cancer cell line

BackgroundColorectal cancer is one of the most common cancer and the third leading cause of death worldwide. Increased generation of reactive oxygen species (ROS) is observed in many types of cancer cells. Several studies have reported that an increase in ROS production could affect the expression of proteins involved in ROS-scavenging, detoxification and drug resistance. Nuclear factor erythroid 2 related factor 2 (Nrf2) is a known transcription factor for cellular response to oxidative stress. Several researches exhibited that Nrf2 could exert multiple functions and expected to be a promising therapeutic target in many cancers. Here, Nrf2 was knocked down in colorectal cancer cell line HT29 and changes that occurred in signaling pathways and survival mechanisms were evaluated.MethodsThe influence of chemotherapy drugs (doxorubicin and cisplatin), metastasis and cell viability were investigated. To explore the association between specific pathways and viability in HT29-Nrf2−, proteomic analysis, realtime PCR and western blotting were performed.ResultsIn the absence of Nrf2 (Nrf2−), ROS scavenging and detoxification potential were dramatically faded and the HT29-Nrf2− cells became more susceptible to drugs. However, a severe decrease in viability was not observed. Bioinformatic analysis of proteomic data revealed that in Nrf2− cells, proteins involved in detoxification processes, respiratory electron transport chain and mitochondrial-related compartment were down regulated. Furthermore, proteins related to MAPKs, JNK and FOXO pathways were up regulated that possibly helped to overcome the detrimental effect of excessive ROS production.ConclusionsOur results revealed MAPKs, JNK and FOXO pathways connections in reducing the deleterious effect of Nrf2 deficiency, which can be considered in cancer therapy.

Identification of Age-Associated Transcriptomic Changes Linked to Immunotherapy Response in Primary Melanoma.

Melanoma is a lethal form of skin cancer. Immunotherapeutic agents such as anti-PD-1 (pembrolizumab and nivolumab) and anti-CTLA-4 (ipilimumab) have revolutionized melanoma treatment; however, drug resistance is rapidly acquired. Several studies have reported an increase in melanoma rates in older patients. Thus, the impact of ageing on transcriptional profiles of melanoma and response to immunotherapy is essential to understand. In this study, the bioinformatic analysis of RNA seq data of old and young melanoma patients receiving immunotherapy identifies the significant upregulation of extra-cellular matrix and cellular adhesion genes in young cohorts, while genes involved in cell proliferation, inflammation, non-canonical Wnt signaling and tyrosine kinase receptor ROR2 are significantly upregulated in the old cohort. Several Treg signature genes as well as transcription factors that are associated with dysfunctional T cell tumor infiltration are differentially expressed. The differential expression of several genes involved in oxidative phosphorylation, glycolysis and glutamine metabolism is also observed. Taken together, this study provides novel findings on the impact of ageing on transcriptional changes in melanoma, and novel therapeutic targets for future studies.

Illuminating the Artefacts of NGS Seen in PGT-A & PGT-SR

Background: Advancements in next generation sequencing (NGS) based genomics over the past 10 years have allowed the routine adoption of preimplantation genetic testing for aneuploidy (PGT-A), and/or segmental rearrangements (PGT-SR) of [Formula: see text]10Mb 1 in the assisted reproductive technology (ART) clinic. However, one challenge remains within the assessment of aligned short read sequencing data, in the ability to discriminate between embryonic profiles with genuine pathological copy number variations (CNVs) from artefacts arising from erroneous DNA amplification or library preparation. Aim: To identify the common artefacts seen in next generation sequencing (NGS) based PGT to reduce inconclusive or false positive results. Method: NGS profiles generated from biopsied human embryonic trophectoderm cells were manually analysed for common genomic artefacts. Following trophectoderm biopsy within a clinical ART cycle, cell samples underwent DNA amplification (Illumina SureplexTM DNA amplification system), sequencing (VeriSeqTM PGS kit on the MiSeq[Formula: see text] System, and bioinformatic data analysis (Bluefuse Multi v4.4, Illumina). Results: Common artefacts were identified affecting chromosomes 7, 11, and 19. Artefacts imitated small CNVs typically displaying a [Formula: see text]50% increase in centromeric sequence counts on chromosomes 7 and 11, and a global increase of sequence counts of [Formula: see text]40% across the entire chromosome 19 (except at the centromere, presenting as diploid). Interestingly a technical repeat of the library preparation and sequencing of the original DNA template somewhat normalised these artefacts. Conclusion: The study found three commonly occurring artefacts involving chromosomes 7, 11, and 19 artificially introduced during suboptimal NGS library preparation. Importantly these artefacts may be normalised through technical repeat of library preparation and sequencing. The awareness of these artefacts may reduce false positive and/or inconclusive results, increasing clinical embryonic utilisation following PGT.

MAP kinase and mammalian target of rapamycin are main pathways of gallbladder carcinogenesis: results from bioinformatic analysis of next generation sequencing data from a hospital-based cohort (NCT05404347).

Gallbladder Cancer (GBC) is one of the most common cancers of the biliary tract and the third commonest gastrointestinal (GI) malignancy worldwide. The disease is characterized by the late presentation and poor outcome despite treatment, and hence, newer therapies and targets need to be identified. The current study investigated various functionally enriched pathways in GBC pathogenesis involving the genes identified through Next Generation Sequencing (NGS) in a hospital-based cohort. The Pathway enrichment analysis and Gene Ontology (GO) were carried out after NGS, followed by the construction of the protein-protein interaction (PPI) network to discover associations among the genes. Of the thirty-three patients with GBC who were screened through next-generation sequencing (NGS), 27somatic mutations were identified. These mutations involved a total of 14 genes. The p53 and KRAS were commonly found to be mutated, while mutations in other genes were seen in one case each, the mean number of mutations were 1.2, and maximum mutation in a single case (eight) was seen in one case. The bioinformatics analysis identified MAP kinase, PI3K-AKT, EGF/EGFR, and Focal Adhesion PI3K-AKT-mTOR signaling pathways and cross-talk between these. The results suggest that the complex crosstalk between the mTOR, MAPK, and multiple interacting cell signaling cascades can promote GBC progression, and hence, mTOR-MAPK targeted treatment will be an attractive option.

Vitamin D3 promotes longevity in Caenorhabditis elegans.

Vitamin D deficiency is associated with a variety of age-related diseases and is becoming increasingly more prevalent in the population over time. Some diseases associated with deficiency are cardiovascular disease, cancer, and neurodegeneration. This association, as well as the fact that vitamin D has been demonstrated to play an important role in a variety of extraskeletal processes, has led some to claim that vitamin D is an essential longevity vitamin. However, the role of vitamin D in healthy aging has been difficult to determine. In order to study vitamin D in the context of aging, the model organism, Caenorhabditis elegans (C. elegans), was employed. To study vitamin D's impact on aging and age-related disease, lifespan and health span were measured across three different genetic strains of C. elegans. Strains investigated were wildtype (N2), worms with a mutant vitamin D receptor ortholog (nhr-8), and worms engineered to represent Alzheimer disease (gnals2). Bioinformatic analysis of available public data was also performed in order to identify the transcriptional response produced in N2 worms treated with vitamin D3. Treatment with vitamin D3 significantly extended the lifespan of N2 worms and rescued nhr-8 worms, which typically have decreased lifespans compared to N2. Treatment with vitamin D3 minimally extended the lifespan of gnals2 worms. Similar results were obtained for measures of health span, quantified as motility through time. Differentially expressed genes upon treatment with vitamin D3 were largely associated with biological processes such as the innate immune response and metabolism of xenobiotic compounds in the worms, which may explain the observed increase in lifespan and health span.

Tumor-derived miR-130b-3p induces cancer-associated fibroblast activation by targeting SPIN90 in luminal A breast cancer

Cancer-associated fibroblasts (CAFs) in the tumor microenvironment (TME) interact closely with cancer cells to promote tumor development. Downregulation of SPIN90 in CAFs has been reported to facilitate breast cancer progression, but the underlying mechanism has not been elucidated. Here, we demonstrate that miR-130b-3p directly downregulates SPIN90 in stromal fibroblasts, leading to their differentiation into CAFs. As the decrease of SPIN90 in CAFs was shown to be more prominent in estrogen receptor (ER)-positive breast tumors in this study, miR-130b-3p was selected by bioinformatics analysis of data from patients with ER-positive breast cancer. Ectopic expression of miR-130b-3p in fibroblasts accelerated their differentiation to CAFs that promote cancer cell motility; this was associated with SPIN90 downregulation. We also found that miR-130b-3p was generated in luminal A-type cancer cells and activated fibroblasts after being secreted via exosomes from cancer cells. Finally, miR-130b-3p increased in SPIN90-downregulated tumor stroma of luminal A breast cancer patients and MCF7 cell-xenograft model mice. Our data demonstrate that miR-130b-3p is a key modulator that downregulates SPIN90 in breast CAFs. The inverse correlation between miR-130b-3p and SPIN90 in tumor stroma suggests that the miR-130b-3p/SPIN90 axis is clinically significant for CAF activation during breast cancer progression.

MYCN and HIF-1 directly regulate TET1 expression to control 5-hmC gains and enhance neuroblastoma cell migration in hypoxia

ABSTRACT Ten-Eleven-Translocation 5-methylcytosine dioxygenases 1–3 (TET1-3) convert 5-methylcytosine to 5-hydroxymethylcytosine (5-hmC), using oxygen as a co-substrate. Contrary to expectations, hypoxia induces 5-hmC gains in MYCN-amplified neuroblastoma (NB) cells via upregulation of TET1. Here, we show that MYCN directly controls TET1 expression in normoxia, and in hypoxia, HIF-1 augments TET1 expression and TET1 protein stability. Through gene-editing, we identify two MYCN and HIF-1 binding sites within TET1 that regulate gene expression. Bioinformatic analyses of 5-hmC distribution and RNA-sequencing data from hypoxic cells implicate hypoxia-regulated genes important for cell migration, including CXCR4. We show that hypoxic cells lacking the two MYCN/HIF-1 binding sites within TET1 migrate slower than controls. Treatment of MYCN-amplified NB cells with a CXCR4 antagonist results in slower migration under hypoxic conditions, suggesting that inclusion of a CXCR4 antagonist into NB treatment regimens could be beneficial for children with MYCN-amplified NBs.

Bioinformatic analysis of RNA-seq data from TCGA database reveals prognostic significance of immune-related genes in colon cancer.

The tumor immune microenvironment is of crucial importance in cancer progression and anticancer immune responses. Thus, systematic exploration of the expression landscape and prognostic significance of immune-related genes (IRGs) to assist in the prognosis of colon cancer is valuable and significant.The transcriptomic data of 470 colon cancer patients were obtained from The Cancer Genome Atlas database and the differentially expressed genes were analyzed. After an intersection analysis, the hub IRGs were identified and a prognostic index was further developed using multivariable Cox analysis. In addition, the discriminatory ability and prognostic significance of the constructed model were validated and the characteristics of IRGs associated overall survival were analyzed to elucidate the underlying molecular mechanisms.A total of 465 differentially expressed IRGs and 130 survival-associated IRGs were screened. Then, 46 hub IRGs were identified by an intersection analysis. A regulatory network displayed that most of these genes were unfavorable for the prognosis of colon cancer and were regulated by transcription factors. After a least absolute shrinkage and selection operator regression analysis, 14 hub IRGs were ultimately chose to construct a prognostic index. The validation results illustrated that this model could act as an independent indicator to moderately separate colon cancer patients into low- and high-risk groups.This study ascertained the prognostic significance of IRGs in colon cancer and successfully constructed an IRG-based prognostic signature for clinical prediction. Our results provide promising insight for the exploration of diagnostic markers and immunotherapeutic targets in colon cancer.

6-Bromoindirubin-3'-Oxime Regulates Colony Formation, Apoptosis, and Odonto/Osteogenic Differentiation in Human Dental Pulp Stem Cells.

6-bromoindirubin-3′-oxime (BIO) is a candidate small molecule that effectively modulates Wnt signalling owing to its stable property. The present study investigated the influence of BIO on the odonto/osteogenic differentiation of human dental pulp stem cells (hDPSCs). hDPSCs were treated with 200, 400, or 800 nM BIO, and the effects on hDPSC responses and osteogenic differentiation were assessed. BIO-mediated Wnt activation was confirmed by β-catenin nuclear translocation detected by immunofluorescence staining. BIO attenuated colony formation and cell migration determined by in vitro wound-healing assay. BIO increased early apoptotic cell population evaluated using flow cytometry. For osteogenic induction, BIO promoted alkaline phosphatase (ALP) activity and mineralisation in a dose-dependent manner. ALP, RUNX2, OCN, OSX, ANKH, DMP1, and DSPP mRNA expression were significantly upregulated. The OPG/RANKL expression ratio was also increased. Further, BIO attenuated adipogenic differentiation as demonstrated by decreased lipid accumulation and adipogenic-related gene expression. Bioinformatic analysis of RNA sequencing data from the BIO-treated hDPSCs revealed that BIO modulated pathways related to autophagy and actin cytoskeleton regulation. These findings demonstrated that BIO treatment promoted hDPSC osteogenic differentiation. Therefore, this small molecule is a strong candidate as a bioactive molecule to enhance dentin repair.

Cebpb Regulates Skeletal Stem Cell Osteogenic Differentiation and Fracture Healing via the WNT/β-Catenin Pathway.

Fracture is the most common traumatic organ injury, and fracture nonunion is a critical clinical challenge. The research on the mechanisms of skeletal stem cell (SSC) differentiation and fracture healing may help develop new treatment strategies and improve the prognosis of patients at high risk of nonunion. Bioinformatic analysis of scRNA-seq data of mouse SSCs and mouse osteoprogenitors was applied to discover major transcription factors for the regulation of SSC differentiation. FACS was used to isolate SSCs prospectively. The expression of Cebpb, osteogenesis-related genes (Runx2, Sp7, and Bglap2), and markers for Notch, Hedgehog, MAPK, BMP2/SMAD, and WNT/β-catenin signaling pathways (Hes1, Gli1, p-Erk1/2, p-Smad1/5/9, and β-catenin) were detected in SSCs with qPCR or western blot, respectively. Alkaline phosphatase assay and alizarin red S staining were used to illustrate the osteogenic differentiation ability of SSCs in vitro. A WNT inhibitor, IWR-1, was further used to explore the mechanism of WNT signaling in the differentiation of SSCs. Micro-CT, mechanical testing, and immunohistochemistry of osteogenic and chondrogenic proteins (Sp7 and Col2α1) were used to demonstrate the capacity of Cebpb knockdown in promoting fracture healing in a monocortical defect model. We found that Cebpb was the crucial transcription factor regulating SSC differentiation. Inhibiting Cebpb in SSCs enhanced the expression of active β-catenin to promote the expression of WNT target genes, thus facilitating the osteogenic differentiation of SSCs. Bone mass, mechanical properties, and osteogenic protein expression were also increased in the Cebpb inhibition group compared to the group without Cebpb inhibition. Collectively, our results proved that Cebpb knockdown promotes SSC osteogenic differentiation and fracture healing via the WNT/β-catenin signaling pathway.

Potential Opportunities and Challenges of Deploying Next Generation Sequencing and CRISPR-Cas Systems to Support Diagnostics and Surveillance Towards Malaria Control and Elimination in Africa.

Recent developments in molecular biology and genomics have revolutionized biology and medicine mainly in the developed world. The application of next generation sequencing (NGS) and CRISPR-Cas tools is now poised to support endemic countries in the detection, monitoring and control of endemic diseases and future epidemics, as well as with emerging and re-emerging pathogens. Most low and middle income countries (LMICs) with the highest burden of infectious diseases still largely lack the capacity to generate and perform bioinformatic analysis of genomic data. These countries have also not deployed tools based on CRISPR-Cas technologies. For LMICs including Tanzania, it is critical to focus not only on the process of generation and analysis of data generated using such tools, but also on the utilization of the findings for policy and decision making. Here we discuss the promise and challenges of NGS and CRISPR-Cas in the context of malaria as Africa moves towards malaria elimination. These innovative tools are urgently needed to strengthen the current diagnostic and surveillance systems. We discuss ongoing efforts to deploy these tools for malaria detection and molecular surveillance highlighting potential opportunities presented by these innovative technologies as well as challenges in adopting them. Their deployment will also offer an opportunity to broadly build in-country capacity in pathogen genomics and bioinformatics, and to effectively engage with multiple stakeholders as well as policy makers, overcoming current workforce and infrastructure challenges. Overall, these ongoing initiatives will build the malaria molecular surveillance capacity of African researchers and their institutions, and allow them to generate genomics data and perform bioinformatics analysis in-country in order to provide critical information that will be used for real-time policy and decision-making to support malaria elimination on the continent.