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Effects of M. tuberculosis and HIV-1 infection on in vitro blood-brain barrier function.
No full textBACKGROUND: Tuberculous meningitis is the most severe form of tuberculosis and HIV-1 co-infection worsens the already poor prognosis. However, how Mycobacterium tuberculosis crosses the blood-brain barrier and how HIV-1 influences tuberculous meningitis pathogenesis remains unclear. METHODS: Using human pericytes, astrocytes, endothelial cells, and microglia alone and combined in an in vitro blood-brain barrier model, we investigated the effect of Mycobacterium tuberculosis +/- HIV-1 co-infection on central nervous system cell entry and function. Cells and the blood-brain barrier model were infected with Mycobacterium tuberculosis and/or HIV-1 and we evaluated the effects of both infection on (i) cells susceptibility to Mycobacterium tuberculosis and its growth in cells by flow cytometry; (ii) modulation of blood-brain barrier permeability and Mycobacterium tuberculosis passage through it; (iii) viral and bacterial cytopathogenicity using the xCELLigence system; (iv) cell metabolic activity and ROS release using colorimetric assays; (v) extracellular glutamate concentration by fluorometric assay; (vi) the inflammatory response by Luminex; and (vii) endoplasmic reticulum stress by quantitative PCR. RESULTS: We demonstrated that Mycobacterium tuberculosis infects and multiplies in all cell types with HIV-1 increasing entry to astrocytes and pericytes, and growth in HIV-1 positive pericytes and endothelial cells. Mycobacterium tuberculosis also induces an increase of the blood-brain barrier permeability resulting in translocation of bacilli across it. Cytopathic effects include (i) increased markers of cellular stress (mitochondrial metabolic activity, unfolded protein response); (ii) ROS release; (iii) the induction of neurotoxic astrocytes; (iv) and the secretion of the excitotoxic neurotransmitter glutamate. Lastly, we observed distinct cell-type specific production of inflammatory and effector mediators. CONCLUSION: These results indicate that Mycobacterium tuberculosis can translocate the blood-brain barrier directly to initiate meningitis.</p
Single-molecule localization microscopy as a tool to quantify di/oligomerization of receptor tyrosine kinases and G protein-coupled receptors
No full textDimerization and oligomerization of membrane receptors, including G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) are fundamental for regulating cell signaling and diversifying downstream responses to mediate range of physiological processes. Receptor di/oligomers play roles in diverse facets of receptor function. Changes in receptor di/oligomers have been implicated in a range of diseases, therefore better understanding of the specific composition and interactions between receptors in complexes is essential, especially for the development of di/oligomer specific drugs. Previously, different optical microscopy approaches, and proximity based biophysical assays have been used to demonstrate di/oligomerization of membrane receptors. However, in recent years, single-molecule super-resolution microscopy techniques have allowed researchers to quantify and uncover the precise dynamics and stoichiometry of specific receptor complexes. This allows the organization of membrane protein receptors to be mapped across the plasma membrane, to explore effects of factors such as ligands, effectors, membrane environment and therapeutic agents. Quantification of receptor complexes is required to better understand the intricate balance of distinct receptor complexes in cells. In this brief review, we provide an overview of single-molecule approaches for quantification of receptor di/oligomerization. We will discuss the techniques commonly employed to study membrane receptor di/oligomerization and their relative advantages and limitations.</p
Evidence of substrate control of Cdk phosphorylation during the budding yeast cell cycle.
No full textA series of sequential events orchestrates cell growth and division, set in motion by cyclin-dependent kinases (Cdks). In the "qualitative model" for Cdk control, order is achieved by cell cycle stage-specific cyclins. However, single-cyclin cells retain cell cycle order. In an alternative "quantitative model," increasing Cdk activity triggers substrate phosphorylation at sequential thresholds. Here, we test a key prediction from the quantitative model: the best Cdk substrates should be the first to be phosphorylated. Phosphoproteome analysis of synchronous budding yeast cultures, against expectations, reveals little correlation between known in vitro Cdk phosphorylation rates and observed in vivo phosphorylation timing. Incorporating Cdk-counteracting phosphatases that impose phosphorylation thresholds does not improve the correlation. Instead of kinase-phosphatase control (i.e., "regulator control"), our phosphoproteome patterns reveal signatures of "substrate control," including substrate-defined phosphorylation waves. The changing behavior of the substrates themselves therefore contributes to ordering their Cdk phosphorylation during the budding yeast cell cycle
Polygenic scores for autism are associated with reduced neurite density in adults and children from the general population
No full textGenetic variants linked to autism are thought to change cognition and behaviour by altering the structure and function of the brain. Although a substantial body of literature has identified structural brain differences in autism, it is unknown whether autism-associated common genetic variants are linked to changes in cortical macro- and micro-structure. We investigated this using neuroimaging and genetic data from adults (UK Biobank, N = 31,748) and children (ABCD, N = 4928). Using polygenic scores and genetic correlations we observe a robust negative association between common variants for autism and a magnetic resonance imaging derived phenotype for neurite density (intracellular volume fraction) in the general population. This result is consistent across both children and adults, in both the cortex and in white matter tracts, and confirmed using polygenic scores and genetic correlations. There were no sex differences in this association. Mendelian randomisation analyses provide no evidence for a causal relationship between autism and intracellular volume fraction, although this should be revisited using better powered instruments. Overall, this study provides evidence for shared common variant genetics between autism and cortical neurite density
The essential host genome for Cryptosporidium survival exposes metabolic dependencies that can be leveraged for treatment.
No full textCryptosporidium is a leading cause of diarrheal disease, yet little is known regarding the infection cell biology of this intracellular intestinal parasite. To this end, we implemented an arrayed genome-wide CRISPR-Cas9 knockout screen to microscopically analyze multiple phenotypic features of a Cryptosporidium infection following individual host gene ablation. We discovered parasite survival within the host epithelial cell hinges on squalene, an intermediate metabolite in the host cholesterol biosynthesis pathway. A buildup of squalene within intestinal epithelial cells creates a reducing environment, making more reduced glutathione available for parasite uptake. Remarkably, the Cryptosporidium parasite has lost the ability to synthesize glutathione and has become dependent on this host import. This dependency can be leveraged for treatment with the abandoned drug lapaquistat, an inhibitor of host squalene synthase that shifts the redox environment, blocking Cryptosporidium growth in vitro and in vivo.</p
Three-dimensional modelling of lymphangiogenesis in-vitro using bioorthogonal click-crosslinked gelatin hydrogels.
No full textLymphangiogenesis, the formation of new lymphatic vessels from pre-existing vessels, is crucial for maintaining tissue homeostasis and immune function. Despite recent advances in understanding the molecular mechanisms regulating lymphangiogenesis, most in vitro studies rely on traditional two-dimensional (2D) cell cultures, with limited replication of the complex microenvironment that governs lymphangiogenesis in vivo. Here, we present a three-dimensional (3D) lymphangiogenesis model using gelatin hydrogels modified with click-chemistry motifs (tetrazine and norbornene, GelTN), providing a biomimetic and mechanically tunable extracellular matrix (ECM) for lymphatic endothelial cells. By encapsulating human dermal lymphatic endothelial cells (HDLEC) spheroids in GelTN, we established a robust and reliable in vitro sprouting assay (<48 h duration) to investigate the effects of GelTN stiffness on lymphangiogenesis. HDLEC encapsulated in low GelTN concentrations exhibited enhanced sprouting in response to vascular endothelial growth factor (VEGF)-C stimulation, compared to HDLEC encapsulated in higher GelTN concentrations. We also provide evidence for the involvement of β3 integrin in lymphangiogenesis. The reduced sprout length upon β3 integrin inhibition further decreased with combined inhibition of α5β1, suggesting a synergistic interaction of the integrin subunits in controlling HDLEC-ECM mechanotransduction. GelTN hydrogels were also evaluated for their translational potential, demonstrating sustained release of VEGF-C in vitro and supporting cellular infiltration and neo-vessel formation following subcutaneous injection in an in vivo mouse model. Overall, these findings highlight the versatility of GelTN hydrogels as a platform for studying lymphangiogenesis and their potential use for therapeutic applications that require controlled growth factor delivery in tissue engineering and regenerative medicine
Slow virologic control but strong immune and metabolic recovery with dolutegravir-anchored therapy in an HIV cohort in Ghana
No full textIntroduction
The West African HIV/AIDS epidemic, historically driven by HIV-1 CRF02_AG, other recombinant forms and HIV-2, remains less researched for various preventive and therapeutic interventions. We established the WACCBIP long-term HIV Infection Cohort (WHICH Study) to investigate the dynamics of HIV epidemic in Ghana. This report evaluates viral load dynamics, immune responses, and organ-level metabolic changes following antiretroviral therapy (ART) initiation.
Method
We collected blood samples, medical, and demographic data from ART-naïve individuals at baseline and six months post-ART, and from ART-experienced individuals at a single time point. Participants, aged 10 years and above, were purposively enrolled from six health facilities. Laboratory analyses included viral load, CD4 and CD8 counts, co-infection screening (hepatitis B/C, syphilis), liver and kidney function tests, haemoglobin estimation, and HIV-1/2 typing. Chi-square and logistic regression analyses were used to assess associations between participant demographics and clinical data with uncontrolled viremia and immune recovery.
Results
A total of 426 participants were recruited, comprising 159 ART-naïve and 267 ART-experienced individuals, with a mean age of 41.5 years. Median ART duration for ART-experienced was greater than 5 years. Infections included HIV-1 (78.6%), HIV-2 (2.1%), and dual HIV-1&2 (19.2%). Common comorbidities were anaemia (54.9%), hepatitis B (9.5%), and hypertension (8.2%). Most participant (97.9%) were on dolutegravir-anchored regimen. Among ART-naïve individuals, median viral load decreased from log10 5.16 at baseline to log10 4.64 copies/mL after six months (p = 0.0156). Median viral load for the ART-experienced arm was log10 3.23 copies/mL. Median CD4 count increased from 290 cells/mm³ in ART-naïve participants to 504 cells/mm³ at six-months post-ART (p = 0.0003) and 581 cells/mm³ in ART-experienced participants (p < 0.0001). ART-naïve participants were 19 times more likely to have unsuppressed viral loads at baseline compared to ART-experienced participants. ARTnaïve- participants had significantly decreased odds of immune recovery (aOR = 0.35, 95% CI: 0.140–0.85, p = 0.021), as did those with low CD4/CD8 ratio (aOR = 0.06, 95% CI: 0.02–0.20; p < 0.001). Kidney function and haemoglobin levels were significantly improved six-month post-ART among the ART-naïve group.
Conclusion
This study highlights the significant reduction in viral load and improved immune recovery following ART initiation despite uncontrolled viremia in a subset of participants. This cohort presents an opportunity to study Ghana’s local HIV epidemic, including HIV-1 and HIV-2, and impact of ART on disease progression
Highly pathogenic avian influenza: pandemic preparedness for a scenario of high lethality with no vaccines
No full textHighly Pathogenic Avian Influenza (HPAI) viruses, particularly H5N1 and H7N9, have long been considered potential pandemic threats, despite the absence of sustained human-to-human transmission. However, recent outbreaks in previously unaffected regions, such as Antarctica, suggest we may be shifting from theoretical risk to a more imminent threat. These viruses are no longer limited to avian populations. Their increasing appearance in mammals, including dairy cattle and domestic animals, raises the likelihood of viral reassortment and mutations that could trigger a human pandemic. If such a scenario unfolds, the world may face a crisis marked by high transmissibility and lethality, without effective vaccines readily available. Unlike the COVID-19 pandemic, when vaccines were rapidly developed despite inequities in access, the current influenza vaccine production model, largely reliant on slow, egg-based technologies, is insufficient for a fast-moving outbreak. While newer platforms show promise, they remain in early stages and cannot yet meet global demand, which alerts to the urgent need for accelerating vaccine and drug development, especially universal vaccines, next-generation vaccine platforms designed to provide broad, long-lasting protection against a wide spectrum of HPAI virus subtypes and strains. Here we propose a paradigmatic shift toward a more integrated, digitalized One Health surveillance system that links human, animal, and environmental data, especially in high-risk spillover regions. We underscore that Artificial Intelligence can revolutionize pandemic preparedness strategies, from improving early detection to speeding up vaccine and drug development and access to medical care, but should not be considered a stand-alone solution
Systematic identification of Y-chromosome gene functions in mouse spermatogenesis.
No full textThe mammalian Y chromosome is essential for male fertility, but which Y genes regulate spermatogenesis is unresolved. We addressed this by generating 13 Y-deletant mouse models. In Eif2s3y, Uty, and Zfy2 deletants, spermatogenesis was impaired. We found that Uty regulates spermatogonial proliferation, revealed a role for Zfy2 in promoting meiotic sex chromosome pairing, and uncovered unexpected effects of Y genes on the somatic testis transcriptome. In the remaining single Y-gene deletants, spermatogenesis appeared unperturbed, but testis transcription was still altered. Multigene deletions, including a human-infertility AZFa model, exhibited phenotypes absent in single Y deletants. Thus, Y genes may regulate spermatogenesis even if they show no phenotypes when deleted individually. This study advances our knowledge of Y evolution and infertility and provides a resource to dissect Y-gene functions in other tissues
Clonal hematopoiesis landscape in frequent blood donors.
No full textDonor blood saves lives, yet the potential impact of recurrent large-volume phlebotomy on donor health and hematopoietic stem cells (HSCs) remains largely unexplored. In our study, we conducted a comprehensive screening of 217 older male volunteer donors with a history of extensive blood donation (>100 life-time donations) to investigate the phenomenon of clonal hematopoiesis (CH). No significant difference in the overall incidence of CH was found in frequent donors (FD) compared to sporadic donors (<10 life-time donations, 212 donors). However, upon deeper analysis of mutations in DNMT3A, the most commonly affected gene in CH, we observed distinct mutational patterns between the FD and age/sex matched control donor (CD) cohorts. Functional analysis of FD enriched DNMT3A variants examined in CRISPR-edited human HSCs demonstrated their competitive outgrowth potential upon stimulation with erythropoietin (EPO), a hormone which increases in response to blood loss. In contrast, clones harboring leukemogenic DNMT3A R882 mutations increase upon stimulation with IFNy. Through concurrent mutational and immunophenotypic profiling of primary samples at single cell resolution, a myeloid bias of premalignant R882 mutant HSCs was found, while no significant lineage bias was observed in HSCs harboring EPO responsive DNMT3A variants. The latter exhibited preferential erythroid differentiation when persistent erythropoietic stress was applied to CRISPR-edited human HSC xenografts. Our data demonstrate a nuanced ongoing Darwinian evolution at the somatic stem cell level, with EPO identified as a novel environmental factor that favors HSCs carrying certain DNMT3A mutations