Biology subjects

Jacob, J.

Publications and source records attributed to Jacob, J..

33 records · Page 2Linked to original sources

bioRxiv2023

Small in size but huge as reservoir -- insights into the virome of European white-toothed shrews

While the virome and immune system of bats and rodents have been extensively studied, comprehensive data are lacking for insectivores. Anthropogenic land use and outdoor recreational activities may lead to an expansion of the human-shrew interface with the risk of zoonotic infections, as reported for Borna disease virus 1. We investigated the virosphere of four white-toothed shrew species from Central Europe, addressing the One Health concept of spillover prevention. A high diversity of viruses was identified, including several co-infections. Whole genomes were generated for novel species of paramyxoviruses (n=3), nairoviruses (n=2) and hepevirus. Phylogenetically, they are closely related to WHO priority diseases, such as henipaviruses. High viral loads of orthoparamyxoviruses were detected in kidneys, in well-perfused organs for orthonairoviruses, and an association with liver and intestine was identified for orthohepevirus. Our study highlights the virus diversity present in shrews, not only in biodiversity hotspots but also in industrialised countries.

evolutionary biology↗

bioRxiv2023

Functional effects of TBC1 domain containing kinase deletion in immortalized B cells and plasma cells

TBC1 domain containing kinase (TBCK) is a ubiquitous protein pseudokinase highly expressed in neurons and glial cells. Mutations in TBCK disrupt nervous system function and cause a characteristic syndrome of intellectual disability and hypotonia with various other comorbidities occurring in non-nervous tissues. Previous studies have shown a vacuolation defect present in B cells from individuals with TBCK mutations, but it is unclear if this affects plasma cell differentiation or humoral immunity. Recent research has revealed that TBCK is part of the FERRY complex involved in mRNA transport and is implicated in regulating mTORC1 signaling, autophagy, and cellular processes like cell proliferation and migration. Yet, the exact role of TBCK in these processes is still not fully understood. In this study, TBCK knockout cell lines were generated to investigate its impact on B cells and plasma cells. TBCK knockout plasma cells showed reduced immunoglobulin secretion after one week in culture, suggesting a possible defect in recycling cell components or energy usage. However, more experiments are needed to confirm this observation.

immunology↗

bioRxiv2023

PTCH1 mutant human cerebellar organoids are associated with altered neural development and early pathways of medulloblastoma oncogenesis

Patched 1 (PTCH1) is the primary receptor for Sonic Hedgehog (SHH) ligand and negatively regulates SHH signalling, an essential pathway in human embryogenesis. Loss-of-function mutations in PTCH1 are associated with altered neuronal development and the malignant brain tumour medulloblastoma (MB). As a result of differences between murine and human development, molecular and cellular perturbations that arise from human PTCH1 mutations remain poorly understood. Here, we employ cerebellar organoids differentiated from human induced pluripotent stem cells (iPSC) combined with CRISPR/Cas9 gene editing to investigate the earliest molecular and cellular consequences of PTCH1 mutations on human cerebellar development. Our findings support the occurrence of developmental mechanisms in cerebellar organoids that mirror in vivo processes of regionalisation and SHH signalling, and offer new insight into early pathophysiological events of MB tumorigenesis. HiglightsO_LIDifferentiation of human iPSC into cerebellar organoids C_LIO_LIHomozygous LOF of PTCH1 prevents cerebellar organoid differentiation C_LIO_LIPTCH1+/- cerebellar organoids display tissue-specific effects of SHH signalling C_LIO_LIEarly altered gene expression relevant for MB in PTCH1+/- cerebellar organoids C_LI

neuroscience↗

bioRxiv2023

Micro to macro scale analysis of the intact human renal arterial tree with Synchrotron Tomography

The architecture of the kidney vasculature is essential for its function. Although structural profiling of the intact rodent kidney vasculature has been performed, it is challenging to map vascular architecture of larger human organs. We hypothesised that hierarchical phase-contrast tomography (HiP-CT) would enable quantitative analysis of the entire human kidney vasculature. Combining label-free HiP-CT imaging of an intact kidney from a 63-year-old male with topology network analysis, we quantitated vasculature architecture in the human kidney down to the scale of arterioles. Although human and rat kidney vascular topologies are comparable, vascular radius decreases at a significantly faster rate in humans as vessels branch from artery towards the cortex. At branching points of large vessels, radii are theoretically optimised to minimise flow resistance, an observation not found for smaller arterioles. Structural differences in the vasculature were found in different spatial zones of the kidney reflecting their unique functional roles. Overall, this represents the first time the entire arterial vasculature of a human kidney has been mapped providing essential inputs for computational models of kidney vascular flow and synthetic vascular architectures, with implications for understanding how the structure of individual blood vessels collectively scales to facilitate organ function.

bioengineering↗

bioRxiv2022

An antibody-drug conjugate targeting GPR56 demonstrates efficacy in preclinical models of colorectal cancer

Antibody-drug conjugates (ADCs) have become an increasingly successful class of anticancer therapy, particularly within the past few years. Though ADCs are in clinical trials for colorectal cancer (CRC), a candidate has yet to be approved. CRC continues to be a leading cause of cancer-related death, emphasizing the need to identify novel target antigens for ADC development. GPR56, a member of the 7TM receptor family, is upregulated in colorectal tumors compared to normal tissues and located on the surface of CRC cells, making it a promising ADC target. Furthermore, high GPR56 expression occurs in tumors that are microsatellite stable, negative for the CpG methylator phenotype, and show chromosomal instability. We previously reported the generation of a high affinity GPR56-specific monoclonal antibody, 10C7, and we have now mapped the epitope to the C-terminal end of the extracellular domain, proximal to the GPCR proteolysis site. Here, we describe the development of a duocarmycin-conjugated 10C7 ADC. 10C7 co-internalized with GPR56 and trafficked to the lysosomes of CRC cells, which is critical for efficient ADC payload release. Evaluation of the ADC in a panel of CRC cell lines and tumor organoids with different levels of GPR56 expression showed the ADC selectively induced cytotoxicity at low nanomolar concentrations in a GPR56-dependent manner. A nontargeting control ADC showed minimal to no activity. Furthermore, GPR56 ADC exhibited significant antitumor efficacy against GPR56-expressing patient-derived xenograft models of CRC. This study provides rationale for the development of a GPR56-targeted ADC approach to potentially treat a large fraction of CRC patients.

cancer biology↗

bioRxiv2022

Loss of LGR5 through plasticity or gene ablation is associated with therapy resistance and enhanced MET-STAT3 signaling in colorectal cancer cells

Plasticity plays a significant role in colorectal tumor initiation, progression, and drug resistance. LGR5 is highly expressed in colorectal cancer (CRC) and marks functional cancer stem cells (CSCs). While LGR5+ CSCs are tumor-initiating, the majority of CRC cells that disseminate to seed metastases are LGR5-; however, reemergence of LGR5+ CSCs is required to drive metastatic outgrowth. LGR5+ CSCs have been shown to convert to LGR5- CRC cells in response to chemotherapies and this loss of LGR5 promotes a more drug-resistant phenotype. However, the molecular mechanisms that mediate plasticity remain elusive. In this study, we demonstrate conversion of LGR5+ CRC cells to an LGR5- state in response to chemotherapy, LGR5-targeted antibody-drug conjugates (ADCs), or LGR5 gene ablation, led to activation of STAT3. Further investigation revealed increased STAT3 activation occurred a result of increased MET activity. LGR5 overexpression decreased MET-STAT3 activity and sensitized CRC cells to therapy. STAT3 inhibition suppressed MET phosphorylation, while constitutively active STAT3 reduced LGR5 levels and increased MET activity, suggesting a potential feedback mechanism. Combination treatment of MET-STAT3 inhibitors with irinotecan or ADCs substantiated synergistic effects in vitro. In CRC xenografts, STAT3 inhibition combined with irinotecan enhanced tumor growth suppression and prolonged survival. These findings suggest a mechanism by which drug-resistant LGR5- CRC cells acquire a survival advantage through activation of MET-STAT3 and provide rationale for new treatment strategies that target CRC cell plasticity. SignificanceThis study reveals that transition of highly plastic LGR5+ CRC cells to a more drug-resistant LGR5- state involves activation of MET-STAT3 signaling and provides new insight into therapeutic strategies to combat plasticity.

cancer biology↗

bioRxiv2021

A multiscale X-ray phase-contrast tomography dataset of whole human left lung

Technological advancements in X-ray imaging using bright and coherent synchrotron sources now allows to decouple sample size and resolution, while maintaining high sensitivity to the microstructure of soft, partially dehydrated tissues. The recently developed imaging technique, hierarchical phase-contrast tomography, is a comprehensive approach to address the challenge of organ-scale (up to tens of centimeters) soft tissue imaging with resolution and sensitivity down to the cellular level. Using this technique, we imaged ex vivo an entire human left lung at an isotropic voxel size of 25.08 m along with local zooms down to 6.05 - 6.5 m and 2.45 - 2.5 m in voxel size. The high tissue contrast offered by the fourth-generation synchrotron source at the European Synchrotron Radiation Facility reveals complex multiscale anatomical constitution of the human lung from the macroscopic (centimeter) down to the microscopic (micrometer) scale. The dataset provides complete organ-scale 3D information of the secondary pulmonary lobules and delineates the microstructure of lung nodules with unprecedented detail.

physiology↗

bioRxiv2021

Phumdis-A Promising Alternative Energy Resource

The inadequate sources of energy coupled with the increasing demands of power have necessitated the search for novel renewable energy resources. Phumdis are one such promising alternative. Phumdis are floating mats of heterogeneous mass of vegetation, soil and organic matter found in Loktak Lake in Manipur, North Eastern India. This paper delineates the use of Phumdis as an alternative energy source. Phumdis from Loktak Lake, Manipur were processed and analyzed for their biofuel capabilities. The Results indicate that the phumdis have high calorific content, cloudpoint and flash point indicating that they are at par with other fuels.

developmental biology↗

bioRxiv2021

Bacterioplankton diversity and pollution levels in the estuarine regions of Chaliyar and Anjarakkandi rivers, Kerala, India

Rivers and the estuarine regions play crucial roles in the environment by controlling the nutrient cycling, waste disposal, and also in supporting biodiversity. However, the estuarine regions of rivers are highly susceptible to disruptive changes by anthropogenic pollutants and eutrophication. Bacterioplankton diversity is the best indicators of the pollution level and eutrophication status of the rivers. The present study evaluated the eutrophication status and bacterioplankton diversity of the estuarine regions of Chaliyar and the Anjarakkandi originated from the Western Ghats. The present study observed the presence of increased incidence of bacterioplankton comprising of proteobacteria, actinobacteria and cyanobacteria in the estuarine regions of Anjarakkandi river compared to that of Chaliyar river. Further, the percentage incidence of faecal bacteria including Bacillus subtilis and E. coli, were also found to be in high in the Anjarakkandi river; in corroborating with this, the diversity of nitrogen metabolizing bacteria was also higher in the estuarine samples of Anjarakkandi river. Corroborating with this, the levels of water nutrients including nirates, nitrites and phosphates were high in the Anjarakkandi compared to the Chaliyar river. Together, it is possible that there might be increased faecal contamination and subsequent increased eutrophication in the estuarine regions of Anjarakkandi river. Altogether, the study concludes that the Anjarakkandi river may be more polluted and which is partly contributed by faecal contaminants and also responsible for the change in the bacterioplankton community of the estuaries.

microbiology↗

bioRxiv2021

Multiscale three-dimensional imaging of intact human organs down to the cellular scale using hierarchical phase-contrast tomography

Human organs are complex, three-dimensional and multiscale systems. Spatially mapping the human body down through its hierarchy, from entire organs to their individual functional units and specialised cells, is a major obstacle to fully understanding health and disease. To meet this challenge, we developed hierarchical phase-contrast tomography (HiP-CT), an X-ray phase propagation technique utilising the European Synchrotron Radiation Facilitys Extremely Brilliant Source: the worlds first high-energy 4th generation X-ray source. HiP-CT enabled three-dimensional and non-destructive imaging at near-micron resolution in soft tissues at one hundred thousand times the voxel size whilst maintaining the organs structure. We applied HiP-CT to image five intact human parenchymal organs: brain, lung, heart, kidney and spleen. These were hierarchically assessed with HiP-CT, providing a structural overview of the whole organ alongside detail of the organs individual functional units and cells. The potential applications of HiP-CT were demonstrated through quantification and morphometry of glomeruli in an intact human kidney, and identification of regional changes to the architecture of the air-tissue interface and alveolar morphology in the lung of a deceased COVID-19 patient. Overall, we show that HiP-CT is a powerful tool which can provide a comprehensive picture of structural information for whole intact human organs, encompassing precise details on functional units and their constituent cells to better understand human health and disease.

physiology↗

bioRxiv2020

96-Well Oxygen Control Using a 3D-Printed Device

Oxygen concentration varies tremendously within the body and has proven to be a critical variable in cell differentiation, proliferation, and drug metabolism among many other physiological processes. Currently, researchers study the gass role in biology using low-throughput gas-control incubators or hypoxia chambers in which all cells in a vessel are exposed to a single oxygen concentration. Here, we introduce a device which can simultaneously deliver 12 unique oxygen concentrations to cells in a 96-well plate and seamlessly integrate into biomedical research workflows. The device inserts into 96-well plates and delivers gas to the headspace thus avoiding undesirable contact with media. This simple approach isolates each well using gas-tight pressure resistant gaskets effectively creating 96 "mini-incubators". Each of the twelve columns of the plate is supplied by a distinct oxygen concentration from a gas-mixing gradient generator supplied by two feed gases. The wells within each column are then supplied by an equal flow-splitting distribution network. Using equal feed flow rates, concentrations ranging from 0.6% to 20.5% were generated within a single plate. A549 lung carcinoma cells were then used to show that O2 levels below 9% caused a stepwise increase in cell death for cells treated with the hypoxia-activated anti-cancer drug Tirapirizamine (TPZ). Additionally, the 96-well plate was further leveraged to simultaneously test multiple TPZ concentrations over an oxygen gradient and generate a 3D dose response landscape. The results presented here show how microfluidic technologies can be integrated into, rather than replace, ubiquitous biomedical labware allowing for increased throughput oxygen studies.

bioengineering↗

bioRxiv2019

RELA is Sufficient to Mediate Interleukin-1 (IL-1) Repression of Androgen Receptor Expression and Activity in LNCaP Disease Progression Model

BackgroundThe Androgen Receptor (AR) nuclear transcription factor is a therapeutic target for prostate cancer (PCa). Unfortunately, patients can develop resistance to AR-targeted therapies and progress to lethal disease, underscoring the importance of understanding the molecular mechanisms that underlie treatment resistance. Inflammation is implicated in PCa initiation and progression and we have previously reported that the inflammatory cytokine, interleukin-1 (IL-1), represses AR mRNA levels and activity in AR-positive (AR+) PCa cell lines concomitant with the upregulation of pro-survival biomolecules. Thus, we contend that IL-1 can select for AR-independent, treatment-resistant PCa cells.\n\nMethodsTo begin to explore how IL-1 signaling leads to the repression of AR mRNA levels, we performed comprehensive pathway analysis on our RNA sequencing data from IL-1-treated LNCaP PCa cells. Our pathway analysis predicted Nuclear Factor Kappa B (NF-{kappa}B) p65 subunit (RELA), a canonical IL-1 signal transducer, to be significantly active and potentially regulate many genes, including AR. We used siRNA to silence the NF-{kappa}B family of transcription factor subunits, RELA, RELB, c-REL, NFKB1, or NFKB2, in IL-1-treated LNCaP, C4-2, and C4-2B PCa cell lines. C4-2 and C4-2B cell lines are castration-resistant LNCaP sublines and represent progression towards metastatic PCa disease; and we have previously shown that IL-1 represses AR mRNA levels in C4-2 and C4-2B cells.\n\nResultssiRNA revealed that RELA alone is sufficient to mediate IL-1 repression of AR mRNA and AR activity. Intriguingly, while LNCaP cells are more sensitive to IL-1-mediated repression of AR than C4-2 and C4-2B cells, RELA siRNA led to a more striking de-repression of AR mRNA levels and AR activity in C4-2 and C4-2B cells than in LNCaP cells.\n\nConclusionsThese data indicate that there are RELA-independent mechanisms that regulate IL-1-mediated AR repression in LNCaP cells and suggest that the switch to RELA-dependent IL-1 repression of AR in C4-2 and C4-2B cells reflects changes in epigenetic and transcriptional programs that evolve during PCa disease progression.

cancer biology↗

bioRxiv2018

Longitudinal prediction of outcome in idiopathic pulmonary fibrosis using automated CT analysis

AIMSTo evaluate computer-derived (CALIPER) CT variables against FVC change as potential drug trials endpoints in IPF.\n\nMETHODS71 Royal Brompton Hospital (discovery cohort) and 23 Mayo Clinic Rochester and 24 St Antonius Hospital Nieuwegein IPF patients (validation cohort) were analysed. Patients had two CTs performed 5-30 months apart, concurrent FVC measurements and were not exposed to antifibrotics (to avoid confounding of mortality relationships from antifibrotic use). Cox regression analyses (adjusted for patient age and gender) evaluated outcome for annualized FVC and CALIPER vessel-related structures (VRS) change and examined the added prognostic value of thresholded VRS changes beyond standard FVC change thresholds.\n\nRESULTSChange in VRS was a stronger outcome predictor than FVC decline when examined as continuous variables, in discovery and validation cohorts. When FVC decline ([≥]10%) and VRS thresholds were examined together, the majority of VRS change thresholds independently predicted outcome, with no decrease in model fit. When analysed as co-endpoints, a VRS threshold of [≥]0{middle dot}40 identified 30% more patients reaching an endpoint than a [≥]10% FVC decline threshold alone.\n\nCONCLUSIONSChange in VRS is a strong predictor of outcome in IPF and can increase power in future drug trials when used as a co-endpoint alongside FVC change.\n\nEthics committee approvalApproval for this study of clinically indicated CT and pulmonary function data was obtained from Liverpool Research Ethics Committee (Reference: 14/NW/0028) and the Institutional Ethics Committee of the Royal Brompton Hospital, Mayo Clinic Rochester and St. Antonius Hospital, Nieuwegein. Informed patient consent was not required.\n\nTake home messageChange in the vessel-related structures, a computer-derived CT variable, is a strong predictor of outcome in idiopathic pulmonary fibrosis and can increase power in future drug trials when used as a co-endpoint alongside forced vital capacity change.

bioinformatics↗

bioRxiv2017

High-throughput single-cell DNA sequencing of AML tumors with droplet microfluidics

To enable the characterization of genetic heterogeneity in tumor cell populations, we developed a novel microfluidic approach that barcodes amplified genomic DNA from thousands of individual cancer cells confined to droplets. The barcodes are then used to reassemble the genetic profiles of cells from next generation sequencing data. Using this approach, we sequenced longitudinally collected AML tumor populations from two patients and genotyped up to 62 disease relevant loci across more than 16,000 individual cells. Targeted single-cell sequencing was able to sensitively identify tumor cells during complete remission and uncovered complex clonal evolution within AML tumors that was not observable with bulk sequencing. We anticipate that this approach will make feasible the routine analysis of heterogeneity in AML leading to improved stratification and therapy selection for the disease.

genomics↗

bioRxiv2017

A synthetic stroma-free germinal center niche using material-surface driven polyvalent signaling efficiently induces antigen-specific humoral immunity ex vivo

B cells play a major role in the adaptive immune response by producing antigen-specific antibodies against pathogens and imparting immunological memory. Following infection or vaccination, antibody-secreting B cells and memory B cells are generated in specialized regions of lymph nodes and spleens, called germinal centers. Here, we report a fully synthetic ex-vivo system that recapitulates the generation of antigen-specific germinal-center (GC) like B cells using material-surface driven polyvalent signaling. This synthetic germinal center (sGC) reaction was effectively induced using biomaterial-based artificial \"follicular T helper cells (TFH)\" that provided both natural CD40-CD40L ligation as well as crosslinking of CD40; and by mimicking artificial \"follicular dendritic cells (FDC)\" to provide efficient, polyvalent antigen presentation. The artificial sGC reaction resulted in efficient B cell expansion, immunoglobulin (Ig) class switching, and expression of germinal center phenotypes. Antigen presentation during sGC reaction selectively enhanced the antigen-specific B cell population and induced somatic hyper-mutations for potential affinity maturation. The resulting B cell population consisted primarily of GC-like B cells (centrocytes) as well as some plasma-like B cells expressing CD138. With concurrent cell sorting, we successfully created highly enriched populations of antigen-specific B cells. Adoptive transfer of these GC-like B cells into non-irradiated isogeneic or non-lethally irradiated congenic recipient mice showed successful engraftment and survival of the donor cells for the 4 week test period. We show that this material-surface driven sGC reaction can be successfully applied to not only splenic B cells but also B cells isolated from more therapeutically relevant sources such as peripheral blood mononuclear cells (PBMCs), thus making our current work an exciting prospect in the new era of personalized medicine and custom-immunotherapy.

bioengineering↗