Establishing African genomics and bioinformatics programs through annual regional workshops.

The African BioGenome Project (AfricaBP) Open Institute for Genomics and Bioinformatics aims to overcome barriers to capacity building through its distributed African regional workshops and prioritizes the exchange of grassroots knowledge and innovation in biodiversity genomics and bioinformatics. In 2023, we implemented 28 workshops on biodiversity genomics and bioinformatics, covering 11 African countries across the 5 African geographical regions. These regional workshops trained 408 African scientists in hands-on molecular biology, genomics and bioinformatics techniques as well as the ethical, legal and social issues associated with acquiring genetic resources. Here, we discuss the implementation of transformative strategies, such as expanding the regional workshop model of AfricaBP to involve multiple countries, institutions and partners, including the proposed creation of an African digital database with sequence information relating to both biodiversity and agriculture. This will ultimately help create a critical mass of skilled genomics and bioinformatics scientists across Africa.

Scattered Crypt Intestinal Epithelial Cell Apoptosis Induces Necrotizing Enterocolitis Via Intricate Mechanisms.

BACKGROUND & AIMS: Necrotizing enterocolitis (NEC) is a life-threatening disease affecting mostly the ileum of preemies. Intestinal epithelial cell (IEC) apoptosis contributes to NEC pathogenesis. However, how scattered crypt IEC apoptosis leads to NEC with excessive villus epithelial necrosis remains unclear. METHODS: A novel triple-transgenic mouse model, namely, 3xTg-iAPcIEC (inducible apoptosis phenotype in crypt-IEC), was developed to induce IEC-specific overexpression of Fasl transgene using doxycycline (Dox)-inducible tetO-rtTA system and villin-cre technology. The 3-days-old neonatal 3xTg-iAPcIEC mice and their littermate controls were subcutaneously (s.c.) challenged with a single dose of Dox. Intestinal tissues were processed at different time points to examine scattered crypt IEC apoptosis-mediated NEC development. Gene knockout technology, antibody-mediated cell depletion, and antibiotic-facilitated Gram-positive bacteria depletion were used to study mechanisms. RESULTS: Treatment of 3xTg-iAPcIEC mouse pups with Dox induces scattered crypt IEC apoptosis followed by crypt inflammation and excessive villous necrosis resembling NEC. This progression correlated with elevated Ifng, Rip3, CD8+ T cells, and Gram-positive bacteria in the ileum. Mechanistically, IFN-γ and RIP3-activated signals mediate the effect of scattered crypt IEC apoptosis on the induction of intestinal crypt inflammation and villous necrosis. Meanwhile, pathophysiological events of CD8+ T cell infiltration and dysbiosis with Gram-positive bacteria primarily contribute to excessive villous inflammation and necrosis. Notably, blocking any of these events protects against NEC development in 3xTg-iAPcIEC mouse pups, underlining their central roles in NEC pathogenesis. CONCLUSIONS: Scattered crypt IEC apoptosis induces NEC in mouse pups via IFN-γ, RIP3, CD8+ T cells, and Gram-positive bacteria-mediated comprehensive pathophysiological events. Our findings may advance knowledge in the prevention and treatment of NEC.

Diversity of CFTR variants across ancestries characterized using 454,727 UK biobank whole exome sequences.

BACKGROUND: Limited understanding of the diversity of variants in the cystic fibrosis transmembrane conductance regulator (CFTR) gene across ancestries hampers efforts to advance molecular diagnosis of cystic fibrosis (CF). The consequences pose a risk of delayed diagnoses and subsequently worsened health outcomes for patients. Therefore, characterizing the spectrum of CFTR variants across ancestries is critical for revolutionizing molecular diagnoses of CF. METHODS: We analyzed 454,727 UK Biobank (UKBB) whole-exome sequences to characterize the diversity of CFTR variants across ancestries. Using the PanUKBB classification, the participants were assigned into six major groups: African (AFR), American/American Admixed (AMR), Central South Asia (CSA), East Asian (EAS), European (EUR), and Middle East (MID). We segregated ancestry-specific CFTR variants, including those that are CF-causing or clinically relevant. The ages of certain CF-causing variants were determined and analyzed for selective pressure effects, and curated phenotype analysis was performed for participants with clinically relevant CFTR genotypes. RESULTS: We detected over 4000 CFTR variants, including novel ancestry-specific variants, across six ancestries. Europeans had the most unique CFTR variants [n = 2212], while the American group had the least unique variants [n = 23]. F508del was the most prevalent CF-causing variant found in all ancestries, except in EAS, where V520F was the most prevalent. Common EAS variants such as 3600G > A, V456A, and V520, which appeared approximately 270, 215, and 338 generations ago, respectively, did not show evidence of selective pressure. Sixteen participants had two CF-causing variants, with two being diagnosed with CF. We found 154 participants harboring a CF-causing and varying clinical consequences (VCC) variant. Phenotype analysis performed for participants with multiple clinically relevant variants returned significant associations with CF and its pulmonary phenotypes [Bonferroni-adjusted p < 0.05]. CONCLUSIONS: We leveraged the UKBB database to comprehensively characterize the broad spectrum of CFTR variants across ancestries. The detection of over 4000 CFTR variants, including several ancestry-specific and uncharacterized CFTR variants, warrants the need for further characterization of their functional and clinical relevance. Overall, the presentation of classical CF phenotypes seen in non-CF diagnosed participants with more than one CF-causing variant indicates that they may benefit from current CFTR modulator therapies.

p32 regulates glycometabolism and TCA cycle to inhibit ccRCC progression via copper-induced DLAT lipoylation oligomerization.

A key player in mitochondrial respiration, p32, often referred to as C1QBP, is mostly found in the mitochondrial matrix. Previously, we showed that p32 interacts with DLAT in the mitochondria. Here, we found that p32 expression was reduced in ccRCC and suppressed progression and metastasis in ccRCC animal models. We observed that increasing p32 expression led to an increase in oxidative phosphorylation by interacting with DLAT, thus, regulating the activation of the pyruvate dehydrogenase complex (PDHc). Mechanistically, reduced p32 expression, in concert with DLAT, suppresses PDHc activity and the TCA cycle. Furthermore, our research discovered that p32 has a direct binding affinity for copper, facilitating the copper-induced oligomerization of lipo-DLAT specifically in ccRCC cells. This finding reveals an innovative function of the p32/DLAT/copper complex in regulating glycometabolism and the TCA cycle in ccRCC. Importantly, our research provides important new understandings of the underlying molecular processes causing the abnormal mitochondrial metabolism linked to this cancer.

G3BP1 regulates breast cancer cell proliferation and metastasis by modulating PKCζ.

Breast cancer is a leading cause of death and morbidity among female cancers. Several factors, including hormone levels, lifestyle, and dysregulated RNA-binding proteins, have been associated with the development of breast cancer. Ras-GTPase-activating protein SH3 domain-binding protein 1 (G3BP1) and protein kinase C, Zeta isoform (PKCζ) are oncogenes implicated in numerous cancers, including breast cancer. However, their interaction and role in promoting breast cancer proliferation and metastasis have not been well-characterized. In the present study, we demonstrated that G3BP1 expression was elevated in breast cancer and that knockdown of G3BP1 diminished the proliferation and metastasis of breast cancer cells. Mechanistically, we identified proliferation and a series of metastasis-related properties, including chemotaxis, migration, Golgi polarity localization, and actin polymerization, that were modulated by G3BP1 knockdown. We found that G3BP1 and PKCζ were co-localized and interacted intracellularly, and they co-underwent membrane translocation under EGF stimulation. Following the knockdown of G3BP1, we observed the membrane translocation and phosphorylation of PKCζ were significantly impaired, suggesting that G3BP1 regulates the activation of PKCζ. Our findings indicate that G3BP1 plays multiple roles in breast cancer cell proliferation and metastasis. The activation of PKCζ by G3BP1 may be the specific mechanism underlying the process.

Addressing underrepresentation in genomics research through community engagement.

The vision of the American Society of Human Genetics (ASHG) is that people everywhere will realize the benefits of human genetics and genomics. Implicit in that vision is the importance of ensuring that the benefits of human genetics and genomics research are realized in ways that minimize harms and maximize benefits, a goal that can only be achieved through focused efforts to address health inequities and increase the representation of underrepresented communities in genetics and genomics research. This guidance is intended to advance community engagement as an approach that can be used across the research lifecycle. Community engagement uniquely offers researchers in human genetics and genomics an opportunity to pursue that vision successfully, including by addressing underrepresentation in genomics research.

CFTR-mediated monocyte/macrophage dysfunction revealed by cystic fibrosis proband-parent comparisons.

Cystic fibrosis (CF) is an inherited disorder caused by biallelic mutations of the CF transmembrane conductance regulator (CFTR) gene. Converging evidence suggests that CF carriers with only 1 defective CFTR copy are at increased risk for CF-related conditions and pulmonary infections, but the molecular mechanisms underpinning this effect remain unknown. We performed transcriptomic profiling of peripheral blood mononuclear cells (PBMCs) of CF child-parent trios (proband, father, and mother) and healthy control (HC) PBMCs or THP-1 cells incubated with the plasma of these participants. Transcriptomic analyses revealed suppression of cytokine-enriched immune-related genes (IL-1ß, CXCL8, CREM), implicating lipopolysaccharide tolerance in innate immune cells (monocytes) of CF probands and their parents. These data suggest that a homozygous as well as a heterozygous CFTR mutation can modulate the immune/inflammatory system. This conclusion is further supported by the finding of lower numbers of circulating monocytes in CF probands and their parents, compared with HCs, and the abundance of mononuclear phagocyte subsets, which correlated with Pseudomonas aeruginosa infection, lung disease severity, and CF progression in the probands. This study provides insight into demonstrated CFTR-related innate immune dysfunction in individuals with CF and carriers of a CFTR mutation that may serve as a target for personalized therapy.

Microarray profiling identifies extracellular circulating miRNAs dysregulated in cystic fibrosis.

Extracellular circulating miRNAs (ECmiRNAs) play a crucial role in cell-to-cell communication and serve as non-invasive biomarkers in a wide range of diseases, but their abundance and functional relevance in cystic fibrosis (CF) remain poorly understood. In this study, we employed microarray technology to identify aberrantly expressed plasma ECmiRNAs in CF and elucidate the functional relevance of their targets. Overall, we captured several ECmiRNAs abundantly expressed in CF. Expression levels of 11 ECmiRNAs differed significantly between CF and healthy control (HC) samples (FDR < 0.05, log2 FC≥2). Among these, 10 were overexpressed while only hsa-miR-598-3p was underexpressed in CF. The overexpressed miRNAs included three let-7 family members (hsa-let-7b-5p, hsa-let-7c-5p and hsa-let-7d-5p), three 103/107 family members (hsa-mir-103a-3p; hsa-mir-103b; hsa-mir-107), hsa-miR-486-5p, and other miRNAs. Using in silico methods, we identified 2,505 validated targets of the 11 differentially expressed miRNAs. Hsa-let-7b-5p was the most important hub in the network analysis. The top-ranked validated targets were involved in miRNA biogenesis and gene expression, including AGO1, DICER1, HMGA1, and MYC. The top pathways influenced by all targets were primarily signal transduction pathways associated with CF, including PI3K/Akt-, Wnt/ß catenin-, glucocorticoid receptor-, and mTor signaling pathways. Our results suggest ECmiRNAs may be clinically relevant in CF and warrant further study.

The interaction of YBX1 with G3BP1 promotes renal cell carcinoma cell metastasis via YBX1/G3BP1-SPP1- NF-κB signaling axis.

BACKGROUND: Renal cell carcinoma (RCC) is a deadly urological tumor that remains largely incurable. Our limited understanding of key molecular mechanisms underlying RCC invasion and metastasis has hampered efforts to identify molecular drivers with therapeutic potential. With evidence from our previous study revealing that nuclear overexpression of YBX1 is associated with RCC T stage and metastasis, we investigated the effects of YBX1 in RCC migration, invasion, and adhesion, and then characterized its interaction with RCC-associated proteins G3BP1 and SPP1. METHODS: Renal cancer cell lines, human embryonic kidney cells, and clinical samples were analyzed to investigate the functional role of YBX1 in RCC metastasis. YBX1 knockdown cells were established via lentiviral infection and subjected to adhesion, transwell migration, and invasion assay. Microarray, immunoprecipitation, dual-luciferase reporter assay, and classical biochemical assays were applied to characterize the mechanism of YBX1 interaction with RCC-associated proteins G3BP1 and SPP1. RESULTS: Knockdown of YBX1 in RCC cells dramatically inhibited cell adhesion, migration, and invasion. Mechanistic investigations revealed that YBX1 interaction with G3BP1 upregulated their downstream target SPP1 in vitro and in vivo, which led to an activated NF-κB signaling pathway. Meanwhile, knockdown of SPP1 rescued the YBX1/G3BP1-mediated activation of NF-κB signaling pathway, and RCC cell migration and invasion. We further showed that YBX1 expression was positively correlated with G3BP1 and SPP1 expression levels in clinical RCC samples. CONCLUSIONS: YBX1 interacts with G3BP1 to promote metastasis of RCC by activating the YBX1/G3BP1-SPP1-NF-κB signaling axis.

Transcriptional consequences of impaired immune cell responses induced by cystic fibrosis plasma characterized via dual RNA sequencing.

BACKGROUND: In cystic fibrosis (CF), impaired immune cell responses, driven by the dysfunctional CF transmembrane conductance regulator (CFTR) gene, may determine the disease severity but clinical heterogeneity remains a major therapeutic challenge. The characterization of molecular mechanisms underlying impaired immune responses in CF may reveal novel targets with therapeutic potential. Therefore, we utilized simultaneous RNA sequencing targeted at identifying differentially expressed genes, transcripts, and miRNAs that characterize impaired immune responses triggered by CF and its phenotypes. METHODS: Peripheral blood mononuclear cells (PBMCs) extracted from a healthy donor were stimulated with plasma from CF patients (n = 9) and healthy controls (n = 3). The PBMCs were cultured (1 × 105 cells/well) for 9 h at 37 ° C in 5% CO2. After culture, total RNA was extracted from each sample and used for simultaneous total RNA and miRNA sequencing. RESULTS: Analysis of expression signatures from peripheral blood mononuclear cells induced by plasma of CF patients and healthy controls identified 151 genes, 154 individual transcripts, and 41 miRNAs differentially expressed in CF compared to HC while the expression signatures of 285 genes, 241 individual transcripts, and seven miRNAs differed due to CF phenotypes. Top immune pathways influenced by CF included agranulocyte adhesion, diapedesis signaling, and IL17 signaling, while those influenced by CF phenotypes included natural killer cell signaling and PI3K signaling in B lymphocytes. Upstream regulator analysis indicated dysregulation of CCL5, NF-κB and IL1A due to CF while dysregulation of TREM1 and TP53 regulators were associated with CF phenotype. Five miRNAs showed inverse expression patterns with three target genes relevant in CF-associated impaired immune pathways while two miRNAs showed inverse expression patterns with two target genes relevant to a dysregulated immune pathway associated with CF phenotypes. CONCLUSIONS: Our results indicate that miRNAs and individual transcript variants are relevant molecular targets contributing to impaired immune cell responses in CF.

Transcriptome Profiling and Molecular Therapeutic Advances in Cystic Fibrosis: Recent Insights.

In cystic fibrosis (CF), mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene disrupt the capacity of the encoded protein to function as a channel to transport chloride ions and water across cell membranes. The consequences are deleterious, system-wide, and immensely variable, even among patients with the same CFTR genotype. This underscores the need to characterize the mechanisms contributing to CF pathophysiology. Gene replacement and gene editing therapies have been pursued intensively and are expected to provide a one-time treatment for CF. However, gene replacement therapy is limited by the lack of efficient vectors to deliver functional copies of CFTR to cells without immunological complications, while gene editing technologies such as CRISPR/Cas9 are still in their infancy, mainly useful in somatic cells and limited by off-target insertions. Small molecule treatments targeted at potentiating or correcting CFTR have shown clinical benefits, but they are limited to a few CFTR mutations and insufficient to overcome challenges related to clinical heterogeneity. Transcriptome profiling approaches have emerged as robust tools capable of characterizing phenotypic variability and revealing novel molecular targets with therapeutic potential for CF. We summarize current insights gained through transcriptome profiling approaches in CF studies and recent advances in molecular therapeutics.

Cystic Fibrosis Plasma Blunts the Immune Response to Bacterial Infection.

Cystic fibrosis (CF) is caused by mutations of the gene encoding the CF transmembrane conductance regulator. It remains unclear whether the abnormal immune response in CF involves extrinsic signals released from the external or internal environment. We sought to characterize the peripheral immune signatures in CF and its association with clinical phenotypes. Healthy peripheral blood mononuclear cells (PBMCs) were cultured with plasma from CF probands (CFPs) or healthy control subjects (HCs) followed by nCounter gene and microRNA (miRNA) profiling. A discovery cohort of 12 CFPs and 12 HCs and a validation cohort of 103 CFPs and 31 HCs (our previous microarray data [GSE71799]) were analyzed to characterize the composition of cultured immune cells and establish a miRNA‒mRNA network. Cell compositions and miRNA profiles were associated with clinical characteristics of the cohorts. Significantly differentially expressed genes and abundance of myeloid cells were downregulated in PMBCs after culture with CF plasma (P < 0.05). Top-ranked miRNAs that increased in response to CF plasma (adjusted P < 0.05) included miR-155 and miR-146a, which target many immune-related genes, such as IL-8. Pseudomonas aeruginosa infection was negatively associated with abundance of monocytes and the presence of those regulatory miRNAs. Extrinsic signals in plasma from patients with CF led to monocyte inactivation and miRNA upregulation in PBMCs. An improved understanding of the immune effects of extrinsic factors in CF holds great promise for integrating immunomodulatory cell therapies into current treatment strategies in CF.

Identification of molecular signatures of cystic fibrosis disease status with plasma-based functional genomics.

Although cystic fibrosis (CF) is attributed to dysfunction of a single gene, the relationships between the abnormal gene product and the development of inflammation and progression of lung disease are not fully understood, which limits our ability to predict an individual patient's clinical course and treatment response. To better understand CF progression, we characterized the molecular signatures of CF disease status with plasma-based functional genomics. Peripheral blood mononuclear cells (PBMCs) from healthy donors were cultured with plasma samples from CF patients ( n = 103) and unrelated, healthy controls ( n = 31). Gene expression levels were measured with an Affymetrix microarray (GeneChip Human Genome U133 Plus 2.0). Peripheral blood samples from a subset of the CF patients ( n = 40) were immunophenotyped by flow cytometry, and the data were compared with historical data for age-matched healthy controls ( n = 351). Plasma samples from another subset of CF patients ( n = 56) and healthy controls ( n = 16) were analyzed by multiplex enzyme-linked immunosorbent assay (ELISA) for numerous cytokines and chemokines. Principal component analysis and hierarchical clustering of induced transcriptional data revealed disease-specific plasma-induced PBMC profiles. Among 1,094 differentially expressed probe sets, 51 genes were associated with pancreatic sufficient status, and 224 genes were associated with infection with Pseudomonas aeruginosa. The flow cytometry and ELISA data confirmed that various immune modulators are relevant contributors to the CF molecular signature. This study provides strong evidence for distinct molecular signatures among CF patients. An understanding of these molecular signatures may lead to unique molecular markers that will enable more personalized prognoses, individualized treatment plans, and rapid monitoring of treatment response.

Investigation into the genetic diversity in toll-like receptors 2 and 4 in the European badger Meles meles.

The Toll-like receptor (TLR) genes are a conserved family of genes central to the innate immune response to pathogen infection. They encode receptor proteins, recognise pathogen associated molecular patterns (PAMPs) and trigger initial immune responses. In some host-pathogen systems, it is reported that genetic differences, such as single nucleotide polymorphisms (SNPs), associate with disease resistance or susceptibility. Little is known about TLR gene diversity in the European badger (Meles meles). We collected DNA from UK badgers, carried out PCR amplification of the badger TLR2 gene and exon 3 of TLR4 and determined DNA sequences for individual badgers for TLR2 (n = 61) and TLR4 exon 3 (n = 59). No polymorphism was observed in TLR4. Three TLR2 amino acid haplotype variants were found. Ninety five percent of badgers were homozygous for one common haplotype (H1), the remaining three badgers had genotypes H1/H3, H1/H2 and H2/H2. By broad comparison with other species, diversity in TLR genes in badgers seems low. This could be due to a relatively localised sampling or inherent low genetic diversity. Further studies are required to assess the generality of the low observed diversity and the relevance to the immunological status of badgers.

Increased Expression of Plasma-Induced ABCC1 mRNA in Cystic Fibrosis.

The ABCC1 gene is structurally and functionally related to the cystic fibrosis transmembrane conductance regulator gene (CFTR). Upregulation of ABCC1 is thought to improve lung function in patients with cystic fibrosis (CF); the mechanism underlying this effect is unknown. We analyzed the ABCC1 promoter single nucleotide polymorphism (SNP rs504348), plasma-induced ABCC1 mRNA expression levels, and ABCC1 methylation status and their correlation with clinical variables among CF subjects with differing CFTR mutations. We assigned 93 CF subjects into disease severity groups and genotyped SNP rs504348. For 23 CF subjects and 7 healthy controls, donor peripheral blood mononuclear cells (PBMCs) stimulated with plasma underwent gene expression analysis via qRT-PCR. ABCC1 promoter methylation was analyzed in the same 23 CF subjects. No significant correlation was observed between rs504348 genotypes and CF disease severity, but pancreatic insufficient CF subjects showed increased colonization with any form of Pseudomonas aeruginosa (OR = 3.125, 95% CI: 1.192-8.190) and mucoid P. aeruginosa (OR = 5.075, 95% CI: 1.307-28.620) compared to the pancreatic sufficient group. A significantly higher expression of ABCC1 mRNA was induced by CF plasma compared to healthy control plasma (p < 0.001). CF subjects with rs504348 (CC/CG) also had higher mRNA expression compared to those with the ancestral GG genotype (p < 0.005). ABCC1 promoter was completely unmethylated; therefore, we did not detect any association between methylation and CF disease severity. In silico predictions suggested that histone modifications are crucial for regulating ABCC1 expression in PBMCs. Our results suggest that ABCC1 expression has a role in CFTR activity thereby increasing our understanding of the molecular underpinnings of the clinical heterogeneity in CF.