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Biochemical reconstitution of sister chromatid cohesion establishment during DNA replication
No full textConcomitant with DNA replication, the ring-shaped cohesin complex encircles both newly synthesized sister chromatids, enabling their faithful segregation during cell divisions. Our molecular understanding of how cohesin co-entraps both replication products remains incomplete. Here, we reconstitute sister chromatid cohesion establishment using purified budding yeast proteins. Cohesin rings, initially loaded onto template DNA, remain DNA bound during complete DNA synthesis. Some of these cohesin rings encircle both sister chromatids, consistent with the idea that replisomes traverse through cohesin rings. Often, however, cohesin ends up embracing only one of the two replication products, suggesting that a two-step capture mechanism operates during cohesion establishment. Additionally, DNA replication initiates new cohesin recruitment as a further means to generate sister chromatid cohesion. Our results illustrate that more than one pathway leads to sister chromatid cohesion, and they make cohesion establishment amenable to direct biochemical exploration
Description of tenofovir levels in South African adults in the EVOLVE study: A pilot project to support therapeutic drug monitoring in HBV infection
No full textThis poster has been presented at the European Association for the Study of the Liver (EASL) 2025, to support the new Evaluation of Vukuzazi LiVEr disease - Hepatitis B 'EVOLVE-HBV' study based at the Africa Health Research Institute (AHRI) in KwaZulu-Natal, South Africa.Hepatitis B virus (HBV) treatment is based on nucleos/tide analogue (NA) drugs for viral suppression, which overlap with therapy used for HIV; the first line agent is tenofovir (TFV). Therapeutic drug monitoring (TDM) has not been explored to optimize therapy in HBV. However, TDM is established in the HIV field, providing a framework to fill evidence gaps for HBV. In this pilot project, we describe the distribution of free tenofovir (TFV) and its metabolite, tenofovir-diphosphate (TFV-DP), in a South African (SA) population offered treatment for HIV monoinfection or HIV/HBV coinfection.We set out to generate pilot data, aiming to:Describe the distribution of TFV and TFV-DP, in South African (SA) adults offered treatment for HIV monoinfection or HIV/HBV coinfection.Explore the relationship between levels of TFV and TFV-DP.Identify any association between drug levels and suppression of HBV.MethodsThis project is part of HBV-EVOLVE, a study of HBV infection conducted at the Africa Health Research Institute (AHRI) in KwaZulu Natal. Samples for analysis were collected by the Vukuzazi programme, which recruited residents in the AHRI Health and Demographic Surveillance Systems (HDSS) area, who were aged ≥15 years between May 2018 and March 2020. Dried Blood Spot (DBS) samples were selected to represent 40 adults living with HIV/HBV coinfection, and 40 with HIV mono-infection. All 80 are receiving antiretroviral therapy containing tenofovir. We also selected 20 HIV/HBV negative (control samples, presumed untreated). We used liquid chromatography tandem mass spectrometry (LC-MS) to measure drug levels of free tenofovir (TFV) and drug metabolite (TFV-DP). HBV viral suppression was defined as plasma HBV Viral load (VL) below the lower limit of quantitation, LLQ (Statistical analysis was performed using Pearsons’s or Kruskal-Wallis test, or linear regression in R.</p
Malaria parasites undergo a rapid and extensive metamorphosis after invasion of the host erythrocyte.
No full textWithin the human host, the symptoms of malaria are caused by the replication of malaria parasites within erythrocytes. Growth inside the erythrocyte exposes the parasites to the normal surveillance of erythrocytes by the host organism, in particular the clearance of erythrocytes in the spleen. Here we show that the malaria parasite Plasmodium falciparum undergoes a rapid, multi-step metamorphosis that transforms the invasive merozoite into an amoeboid-shaped cell within minutes after invading erythrocytes. This transformation involves an increase in the parasite surface area and is mediated by factors already present in the merozoite, including the parasite phospholipid transfer protein PV6. Parasites lacking PV6 do not assume an amoeboid form and instead are spherical and have a smaller surface area than amoeboid forms. Furthermore, erythrocytes infected with P. falciparum parasites lacking PV6 undergo a higher loss of surface area upon infection, which affects the traversal of infected erythrocytes through the spleen. This is the first evidence that after invasion, the parasite undergoes a rapid, complex metamorphosis within the host erythrocyte that promotes survival in the host
Neuronal oscillations in cognition: Down syndrome as a model of mouse to human translation.
No full textDown syndrome (DS), a prevalent cognitive disorder resulting from trisomy of human chromosome 21 (Hsa21), poses a significant global health concern. Affecting approximately 1 in 800 live births worldwide, DS is the leading genetic cause of intellectual disability and a major predisposing factor for early-onset Alzheimer's dementia. The estimated global population of individuals with DS is 6 million, with increasing prevalence due to advances in DS health care. Global efforts are dedicated to unraveling the mechanisms behind the varied clinical outcomes in DS. Recent studies on DS mouse models reveal disrupted neuronal circuits, providing insights into DS pathologies. Yet, translating these findings to humans faces challenges due to limited systematic electrophysiological analyses directly comparing human and mouse. Additionally, disparities in experimental procedures between the two species pose hurdles to successful translation. This review provides a concise overview of neuronal oscillations in human and rodent cognition. Focusing on recent DS mouse model studies, we highlight disruptions in associated brain function. We discuss various electrophysiological paradigms and suggest avenues for exploring molecular dysfunctions contributing to DS-related cognitive impairments. Deciphering neuronal oscillation intricacies holds promise for targeted therapies to alleviate cognitive disabilities in DS individuals
Cell volume regulates terminal differentiation of cultured human epidermal keratinocytes.
No full textTo gain insights into the human epidermal stem cell niche we have previously identified micron-scale topographical substrates that regulate differentiation of spread keratinocytes. On one substrate (S1), cells interact with circular topographies, and differentiation is stimulated; on the other (S2), cells interact with triangular topographies, and differentiation is inhibited. Cell stiffness on S1 and S2 was similar; and nuclear localisation of the mechano-sensitive transcriptional regulator YAP1 was decreased on S1 and S2 compared to flat substrates. However, cells on S2 exhibited reduced cell volume, leading us to explore potential volume-regulated differentiation. Treatment with polyethylene glycol decreased cell volume and inhibited differentiation under a range of conditions. Conversely, deionised water increased cell volume and stimulated differentiation. Bulk RNA sequencing identified several substrate-responsive genes, including aquaporins and ion channels. A membrane permeable Ca2+ chelator and an inhibitor of the water channel aquaporin 3 blocked volume-induced differentiation. These studies identify cell volume as a mechanism by which keratinocyte-niche interactions regulate terminal differentiation
Active thymus in adult with lung cancer: preliminary results from the Adult Thymic Project.
No full textThymus is considered a non-functional remnant in adults, but some evidence suggest that it may harbor residual activity. Lung cancer patients represent the ideal model to study thymic residual activity, as their thymus can be easily harvested during surgery. This study was designed to confirm the presence of residual thymic activity both in adult mice (step 1) and in humans (step 2). In step 1, lung cancer was induced by activating k-ras mutation in a cohort of 20 young and adult mice. After killing, thymus and lungs were analyzed. Thymus was considered active when medullary was evident, cortico-medullary ratio was 50:50 or higher and adipose involution was present. In step 2, a cohort of 20 patients, undergoing surgery for lung cancer, had biopsy of pericardial fat pad, site of ectopic thymus. Thymus was considered present if Hassall's bodies were detected. In mice, active thymus was detected in a high proportion of cases, without significant difference between adult and young (70% vs 44.4% respectively). Two cases without evidence of lung tumor had a fully functional thymus. In humans, ectopic thymus was detected in the pericardial fat pad in 2 cases (10.5%), confirmed by immunohistochemistry. Signs of previous thymic activity were detected in 8 additional patients. Results confirmed thymus activity in animal models and humans with lung cancer, providing the rationale for future systematic mediastinal thymic biopsy. The comprehension of interactions between thymus, lymphocytes and tumor may open a new potentially targetable perspective in lung cancer
Combinatorial BMP4 and activin direct the choice between alternate routes to endoderm in a stem cell model of human gastrulation.
No full textLineage specification requires accurate interpretation of multiple signaling cues. However, how combinatorial signaling histories influence fate outcomes remains unclear. We combined single-cell transcriptomics, live-cell imaging, and mathematical modeling to explore how activin and bone morphogenetic protein 4 (BMP4) guide fate specification during human gastrulation. We see that these signals interact both synergistically and antagonistically to drive fate decisions. We find that definitive endoderm arises from lineage convergence: a direct route from pluripotency and an indirect route via a mesoderm progenitor state. Cells pass through temporal windows of signaling competency, and the relative concentration of activin and BMP4 dictates the trajectory choice. The efficiency between routes is underpinned by a dual role of BMP4 in inducing mesoderm genes while promoting pluripotency exit. This work underscores that the combination of signals a cell is exposed to not only directs its final fate but also the developmental route taken, suggesting lineage convergence enhances robustness in fate specification.</p
Cell-autonomous timing drives the vertebrate segmentation clock’s wave pattern
No full textRhythmic and sequential segmentation of the growing vertebrate body relies on the segmentation clock, a multi-cellular oscillating genetic network. The clock is visible as tissue-level kinematic waves of gene expression that travel through the presomitic mesoderm (PSM) and arrest at the position of each forming segment. Here, we test how this hallmark wave pattern is driven by culturing single maturing PSM cells. We compare their cell-autonomous oscillatory and arrest dynamics to those we observe in the embryo at cellular resolution, finding similarity in the relative slowing of oscillations and arrest in concert with differentiation. This shows that cell-extrinsic signals are not required by the cells to instruct the developmental program underlying the wave pattern. We show that a cell-autonomous timing activity initiates during cell exit from the tailbud, then runs down in the anterior-ward cell flow in the PSM, thereby using elapsed time to provide positional information to the clock. Exogenous FGF lengthens the duration of the cell-intrinsic timer, indicating extrinsic factors in the embryo may regulate the segmentation clock via the timer. In sum, our work suggests that a noisy cell-autonomous, intrinsic timer drives the slowing and arrest of oscillations underlying the wave pattern, while extrinsic factors in the embryo tune this timer’s duration and precision. This is a new insight into the balance of cell-intrinsic and -extrinsic mechanisms driving tissue patterning in development
A scaleable inducible knockout system for studying essential gene function in the malaria parasite.
No full textThe malaria parasite needs nearly half of its genes to propagate normally within red blood cells. Inducible ways to interfere with gene expression like the DiCre-lox system are necessary to study the function of these essential genes. However, existing DiCre-lox strategies are not well-suited to be deployed at scale to study several genes simultaneously. To overcome this, we have developed SHIFTiKO (frameshift-based trackable inducible knockout), a novel scaleable strategy that uses short, easy-to-construct, barcoded repair templates to insert loxP sites around short regions in target genes. Induced DiCre-mediated excision of the flanked region causes a frameshift mutation resulting in genetic ablation of gene function. Dual DNA barcodes inserted into each mutant enables verification of successful modification and induced excision at each locus and collective phenotyping of the mutants, not only across multiple replication cycles to assess growth fitness but also within a single cycle to identify specific phenotypic impairments. As a proof of concept, we have applied SHIFTiKO to screen the functions of malarial rhomboid proteases, successfully identifying their blood stage-specific essentiality. SHIFTiKO thus offers a powerful platform to conduct inducible phenotypic screens to study essential gene function at scale in the malaria parasite
Expedited SARS-CoV-2 main protease inhibitor discovery through modular 'direct-to-biology' screening.
No full textReactive fragment (RF) screening has emerged as an efficient method for ligand discovery across the proteome, irrespective of a target's perceived tractability. To date, however, the efficiency of subsequent optimisation campaigns has largely been low-throughput, constrained by the need for synthesis and purification of target compounds. We report a high-throughput platform for 'direct-to-biology' (D2B) screening of cysteine-targeting chloroacetamide RFs, wherein synthesis is performed in 384-well plates allowing direct assessment in downstream biological assays without purification. Here, the developed platform was used to optimise inhibitors of SARS-CoV-2 main protease (MPro), an established drug target for the treatment of COVID-19. An initial RF hit was developed into a series of potent inhibitors, and further exploration using D2B screening enabled a 'switch' to a reversible inhibitor series. This example of ligand discovery for MPro illustrates the acceleration that D2B chemistry can offer for optimising RFs towards covalent inhibitor candidates, as well as providing future impetus to explore the evolution of RFs into non-covalent ligands