Biology subjects

Dhakal, S.

Publications and source records attributed to Dhakal, S..

31 records · Page 2Linked to original sources

bioRxiv2022

Ganglioside enriched phospholipid vesicles induce cooperative Aβ oligomerization and membrane disruption

A major hallmark of Alzheimer disease (AD) is the accumulation of extracellular aggregates of amyloid-{beta} (A{beta}). Structural polymorphism observed among A{beta} fibrils in AD brains seem to correlate with the clinical sub-types suggesting a link between fibril polymorphism and pathology. Since fibrils emerge from a templated growth of low-molecular weight oligomers, understanding the factors affecting oligomer generation is important. The membrane lipids are key factors that influence early stages of A{beta} aggregation and oligomer generation, and cause membrane disruption. We have previously demonstrated that conformationally discrete A{beta} oligomers can be generated by modulating the charge, composition, chain length of lipids and surfactants. Here, we extend our studies into liposomal models by investigating A{beta} oligomerization on large unilamellar vesicles (LUVs) of total brain extracts (TBE), reconstituted lipid rafts (LRs) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Specifically, we varied the vesicle composition by varying the amount of GM1 gangliosides added as a constituent. We found that liposomes enriched in GM1 induce the formation of toxic, low-molecular weight oligomers that are isolable in a lipid-complexed form. Importantly, the data indicate that oligomer formation and membrane disruption are highly cooperative processes. Numerical simulations on the experimental data confirm cooperativity and reveal that GM1-enriched liposomes form twice as many numbers of pores as those without GM1. Overall, this study uncovers mechanisms of cooperativity between oligomerization and membrane disruption under controlled lipid compositional bias, and refocuses the significance of the early stages of A{beta} aggregation in polymorphism, propagation, and toxicity in AD. TOC figure O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

biophysics↗

bioRxiv2021

αS oligomers generated from polyunsaturated fatty acid and dopamine metabolite differentially interact with Aβ to enhance neurotoxicity

It is increasingly becoming clear that neurodegenerative diseases are not as discrete as originally thought to be but rather display significant overlap in histopathological and clinical presentations. For example, nearly half of the patients with Alzheimer disease (AD) and synucleinopathies such as Parkinson disease (PD) show symptoms and pathological features of one another. Yet, the molecular events and features that underlie such comorbidities in neurodegenerative diseases remain poorly understood. Here, inspired to uncover the molecular underpinnings of the overlap between AD and PD, we investigated the interactions between amyloid-{beta} (A{beta}) and -synuclein (S), aggregates of which form the major components of amyloid plaques and Lewy bodies, respectively. Specifically, we focused on S oligomers generated from the dopamine metabolite called dihydroxyphenylacetaldehyde (DOPAL), and a polyunsaturated fatty acid docosahexaenoic acid (DHA). Both S oligomers showed structural and conformational differences confirmed by their disparity in size, secondary structure, susceptibility to proteinase K digestion and cytotoxicity. More importantly, the two oligomers differentially modulated A{beta} aggregation. While both oligomers inhibited A{beta} aggregation to varying extents, they induced structurally different A{beta} assemblies. Furthermore, A{beta} seeded with DHA-derived S oligomers showed greater toxicity than DOPAL-derived S oligomers in SH-SY5Y neuroblastoma cells. These results provide insights into the interactions between two amyloid proteins with empirically distinctive biophysical and cellular manifestations, enunciating a basis for potentially ubiquitous cross-amyloid interactions across many neurodegenerative diseases.

biophysics↗

bioRxiv2021

A bacterial extracellular vesicle-based intranasal vaccine against SARS-CoV-2

Several vaccines have been introduced to combat the coronavirus infectious disease-2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Current SARS-CoV-2 vaccines include mRNA-containing lipid nanoparticles or adenoviral vectors that encode the SARS-CoV-2 Spike (S) protein of SARS-CoV-2, inactivated virus, or protein subunits. Despite growing success in worldwide vaccination efforts, additional capabilities may be needed in the future to address issues such as stability and storage requirements, need for vaccine boosters, desirability of different routes of administration, and emergence of SARS-CoV-2 variants such as the Delta variant. Here, we present a novel, well-characterized SARS-CoV-2 vaccine candidate based on extracellular vesicles (EVs) of Salmonella typhimurium that are decorated with the mammalian cell culture-derived Spike receptor-binding domain (RBD). RBD-conjugated outer membrane vesicles (RBD-OMVs) were used to immunize the golden Syrian hamster (Mesocricetus auratus) model of COVID-19. Intranasal immunization resulted in high titers of blood anti-RBD IgG as well as detectable mucosal responses. Neutralizing antibody activity against wild-type and Delta variants was evident in all vaccinated subjects. Upon challenge with live virus, hamsters immunized with RBD-OMV, but not animals immunized with unconjugated OMVs or a vehicle control, avoided body mass loss, had lower virus titers in bronchoalveolar lavage fluid, and experienced less severe lung pathology. Our results emphasize the value and versatility of OMV-based vaccine approaches.

immunology↗

bioRxiv2021

Progression and Resolution of SARS-1 CoV-2 Infection in Golden Syrian Hamsters

To catalyze SARS-CoV-2 research including development of novel interventive and preventive strategies, we characterized progression of disease in depth in a robust COVID-19 animal model. In this model, male and female golden Syrian hamsters were inoculated intranasally with SARS-CoV-2 USA-WA1/2020. Groups of inoculated and mock-inoculated uninfected control animals were euthanized at day 2, 4, 7, 14, and 28 days post-inoculation to track multiple clinical, pathology, virology, and immunology outcomes. SARS-CoV-2-inoculated animals consistently lost body weight during the first week of infection, had higher lung weights at terminal timepoints, and developed lung consolidation per histopathology and quantitative image analysis measurements. High levels of infectious virus and viral RNA were reliably present in the respiratory tract at days 2 and 4 post-inoculation, corresponding with widespread necrosis and inflammation. At day 7, when infectious virus was rare, interstitial and alveolar macrophage infiltrates and marked reparative epithelial responses (type II hyperplasia) dominated in the lung. These lesions resolved over time, with only residual epithelial repair evident by day 28 post-inoculation. The use of quantitative approaches to measure cellular and morphologic alterations in the lung provides valuable outcome measures for developing therapeutic and preventive interventions for COVID-19 using the hamster COVID-19 model.

pathology↗

bioRxiv2021

Cytoplasmic Colocalization of Granulins and TDP-43 Prion-like Domain Involves Electrostatically Driven Complex Coacervation Tuned by the Redox State of Cysteines.

Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. We were interested in understanding if and how cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of a genetic risk factor in FTLD called progranulin, interact with TDP-43. We show that extracellular GRNs internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show no presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Identification of the sequence determinants within GRNs for the observed phase transitions reveal the negative charges to be the drivers of LLPS modulated by the positive charges and the redox state of cysteines. Furthermore, RNA and GRNs compete and expunge one another from PrLD condensates, providing a basis for GRNs absence in SGs. Together, the results illustrate the potential mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize, and modulate cytoplasmic TDP-43 inclusions in proteinopathies.

biophysics↗

bioRxiv2021

Protective efficacy of an orf virus-vector encoding the hemmagglutinin and the nucleoprotein of influenza A virus in swine

Swine influenza is a highly contagious respiratory disease of pigs caused by influenza A viruses (IAV-S). IAV-S causes significant economic losses to the swine industry and poses constant challenges to public health due to its zoonotic potential. Thus effective IAV-S vaccines are highly desirable and would benefit both animal and human health. Here, we developed two recombinant orf viruses, expressing the hemagglutinin (HA) gene (OV-HA) or both the HA and the nucleoprotein (NP) genes of IAV-S (OV-HA-NP). The immunogenicity and protective efficacy of these two recombinant viruses were evaluated in pigs. Both OV-HA and OV-HA-NP recombinants elicited robust virus neutralizing antibody response in pigs. Notably, although both recombinant viruses elicited IAV-S-specific T-cell responses, the frequency of IAV-S specific proliferating T cells secreting IFN-{gamma} upon re-stimulation was higher in OV-HA-NP-immunized animals than in the OV-HA group. Importantly, IgG1/IgG2 isotype ELISAs revealed that immunization with OV-HA induced Th2-biased immune responses, whereas immunization with OV-HA-NP virus resulted in a Th1-biased immune response. While pigs immunized with either OV-HA or OV-HA-NP were protected when compared to non-immunized controls, immunization with OV-HA-NP resulted in better protective efficacy as evidenced by reduced virus shedding in nasal secretions and reduced viral load in the lung. This study demonstrates the potential of ORFV-based vector for control of swine influenza virus in swine. ImportanceEffective influenza A virus (IAV-S) vaccines capable of providing robust protection to genetically diverse IAV-S in swine are lacking. Here, we explored the potential of orf virus based vectors expressing the hemagglutining (HA) or both the HA and the nucleoprotein (NP) genes of influena A virus (IAV-S) in eliciting protection against IAV-S in pigs. We observed that both recombinant viruses elicited IAV-S-specific humoral and cell-mediated immune responses in pigs. Addition of the NP and co-expression of this protein with HA, another major influenza protective antigen, resulted in higher T cell responses which presumably led to better protection in OV-HA-NP immunized animals, as evidenced by lower levels of virus shedding and viral load in lungs. This study highlights the the potential of ORFV as a vector platform for vaccine delivery against IAV-S. Results here provide the foundation for future development of broadly protective ORFV-based vectors for IAV-S for use in swine.

immunology↗

bioRxiv2021

Sex differences in lung imaging and SARS-CoV-2 antibody responses in a COVID-19 golden Syrian hamster model

In the ongoing coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), more severe outcomes are reported in males compared with females, including hospitalizations and deaths. Animal models can provide an opportunity to mechanistically interrogate causes of sex differences in the pathogenesis of SARS-CoV-2. Adult male and female golden Syrian hamsters (8-10 weeks of age) were inoculated intranasally with 105 TCID50 of SARS-CoV-2/USA-WA1/2020 and euthanized at several time points during the acute (i.e., virus actively replicating) and recovery (i.e., after the infectious virus has been cleared) phases of infection. There was no mortality, but infected male hamsters experienced greater morbidity, losing a greater percentage of body mass, developing more extensive pneumonia as noted on chest computed tomography, and recovering more slowly than females. Treatment of male hamsters with estradiol did not alter pulmonary damage. Virus titers in respiratory tissues, including nasal turbinates, trachea, and lungs, and pulmonary cytokine concentrations, including IFN{beta} and TNF, were comparable between the sexes. However, during the recovery phase of infection, females mounted two-fold greater IgM, IgG, and IgA responses against the receptor-binding domain of the spike protein (S-RBD) in both plasma and respiratory tissues. Female hamsters also had significantly greater IgG antibodies against whole inactivated SARS-CoV-2 and mutant S-RBDs, as well as virus neutralizing antibodies in plasma. The development of an animal model to study COVID-19 sex differences will allow for a greater mechanistic understanding of the SARS-CoV-2 associated sex differences seen in the human population. ImportanceMen experience more severe outcomes from COVID-19 than women. Golden Syrian hamsters were used to explore sex differences in the pathogenesis of a human clinical isolate of SARS-CoV-2. After inoculation, male hamsters experienced greater sickness, developed more severe lung pathology, and recovered more slowly than females. Sex differences in disease could not be reversed by estradiol treatment in males and were not explained by either virus replication kinetics or the concentrations of inflammatory cytokines in the lungs. During the recovery period, antiviral antibody responses in the respiratory tract and plasma, including to newly emerging SARS-CoV-2 variants, were greater in females than male hamsters. Greater lung pathology during the acute phase combined with reduced antiviral antibody responses during the recovery phase of infection in males than females illustrate the utility of golden Syrian hamsters as a model to explore sex differences in the pathogenesis of SARS-CoV-2 and vaccine-induced immunity and protection. One Sentence SummaryFollowing SARS-CoV-2 infection, male hamsters experience worse clinical disease and have lower antiviral antibody responses than females.

microbiology↗

bioRxiv2020

Prion-like C-terminal domain of TDP-43 and α-Synuclein interact synergistically to generate neurotoxic hybrid fibrils

Aberrant aggregation and amyloid formation of tar DNA binding protein (TDP-43) and -synuclein (S) underlie frontotemporal dementia (FTD) and Parkinsons disease (PD), respectively. Amyloid inclusions of TDP-43 and S are also commonly co-observed in amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies (DLB) and Alzheimer disease (AD). Emerging evidence from cellular and animal models show colocalization of the TDP-43 and S aggregates, raising the possibility of direct interactions and co-aggregation between the two proteins. In this report, we set out to answer this question by investigating the interactions between S and prion-like pathogenic C-terminal domain of TDP-43 (TDP-43 PrLD). PrLD is an aggregation-prone fragment generated both by alternative splicing as well as aberrant proteolytic cleavage of full length TDP-43. Our results indicate that two proteins interact in a synergistic manner to augment each others aggregation towards hybrid fibrils. While monomers, oligomers and sonicated fibrils of S seed TDP-43 PrLD monomer aggregation, TDP-43 PrLD fibrils failed to seed S monomers indicating selective interactions. Furthermore, S modulates liquid droplets formed by TDP-43 PrLD and RNA to promote insoluble amyloid aggregates. Importantly, the cross-seeded hybrid aggregates show greater cytotoxicity as compared to the individual homotypic aggregates suggesting that the interactions between the two proteins have a discernable impact on cellular functions. Together, these results bring forth insights into TDP-43 PrLD - S interactions that could help explain clinical and pathological presentations in patients with co-morbidities involving the two proteins.

biophysics↗

bioRxiv2020

Genome wide identification of QTL associated with yield and yield components in two popular wheat cultivars TAM 111 and TAM 112

Two drought-tolerant wheat cultivars, TAM 111 and TAM 112, have been widely grown in the Southern Great Plains of the U.S. and used as parents in many wheat breeding programs worldwide. This study aimed to reveal genetic control of yield and yield components in the two cultivars under both dryland and irrigated conditions. A mapping population containing 124 F5:7 recombinant inbred lines (RILs) was developed from the cross of TAM 112/TAM 111. A set of 5,948 SNPs from the wheat 90K iSelect array and double digest restriction-site associated DNA sequencing was used to construct high-density genetic maps. Data for yield and yield components were obtained from 11 environments. QTL analyses were performed based on 11 individual environments, across all environments, within and across mega-environments. Thirty-six unique consistent QTL regions were distributed on 13 chromosomes including 1A, 1B, 1D, 2A, 2D, 3D, 4B, 4D, 6A, 6B, 6D, 7B, and 7D. Ten unique QTL with pleiotropic effects were identified on four chromosomes and eight were in common with the consistent QTL. These QTL increased dry biomass grain yield by 16.3 g m-2, plot yield by 28.1 g m-2, kernels spike-1 by 0.7, spikes m-2 by 14.8, thousand kernel weight by 0.9 g with favorable alleles from either parent. TAM 112 alleles mainly increased spikes m-2 and thousand kernel weight while TMA 111 alleles increased kernels spike-1, harvest index and grain yield. The saturated genetic map and markers linked to significant QTL from this study will be very useful in developing high throughput genotyping markers for tracking the desirable haplotypes of these important yield-related traits in popular parental cultivars.

genetics↗

bioRxiv2020

Post-intervention Epidemiology of STH in Bangladesh: data to sustain the gains

IntroductionIn 2008, Bangladesh initiated Preventive Chemotherapy (PCT) for school-age children (SAC) through bi-annual school-based mass drug administration (MDA) to control Soil-Transmitted Helminth (STH) infections. In 2016, the Ministry of Health and Family Welfares Program on Lymphatic Filariasis Elimination and STH (ELFSTH) initiated district-level community impact assessments with Children Without Worms (CWW) using standardized, population-based sampling to measure the post-intervention STH burden across all ages ([≥] 1 yr) for the three STH species. Methods & Principal FindingsThe Integrated Community-based Survey for Program Monitoring (ICSPM) was developed by CWW and was used to survey 12 districts in Bangladesh from 2017 - 2020. We combined the individual demographic and parasite-specific characteristics from 10 districts and linked them with the laboratory data for collective analysis. Our analysis identified district-specific epidemiologic findings, important for program decisions. Of the 17,874 enrolled individuals, 10,824 (61%) provided stool samples. Overall, the prevalence of any STH species was substantially reduced to 14% from 79.8% in 2005. The impact was similar across all ages. STH prevalence was below 10% in 10 districts collectively, but remained high in 4 districts, despite their high reported PCT coverage in previous years. Among all, Bhola district was unique because it was the only district with high Trichuris prevalence. ConclusionBangladesh successfully lowered STH prevalence across all ages despite targeting SAC only. Data from the survey indicate significant number of adults and pre-school age children (PSAC) were self-deworming with purchased pills. This may account for the flat impact curve across all ages. Overall prevalence varied across surveyed districts, with persistent high transmission in the northeastern districts and a district in the central flood zone, indicating possible service and ecological factors. Discrepancies in the impact between districts highlight the need for district-level data to evaluate program implementation after consistent high PCT coverage. Authors SummaryBangladesh government conducted school-based mass drug administration (MDA) for over 10 years to control soil-transmitted helminth (STH) infections. School-based evaluations of MDA indicate a reduction in STH burden among school-aged children (SAC). To further assess the impact on the community, Children Without Worms and the Ministry of Health and Family Welfares Program on Lymphatic Filariasis Elimination and STH (ELFSTH) initiated district-level community impact surveys in 12 districts. We share the results from the latter 10 districts here. Our analysis of 10,824 interviews and stool samples from 10 districts showed an estimated 14% of community members infected with at least one species of STH. This finding is substantially lower than the baseline STH prevalence (79.8%) estimated in 2005. Bangladeshs successful impact was achieved across all ages despite only treating SAC. Deworming source data showed significant numbers of adults and pre-school age children (PSAC) self-dewormed with locally purchased pills. Prevalence varied across the surveyed districts, with persistent high transmission in the northeastern districts and a district in the central flood zone, indicating possible ecological and service factors contributing to persistent infections. Discrepancies in the impact across districts highlights the need for sub-national level data to evaluate program performance fllowing consistent high intervention.

scientific communication and education↗

bioRxiv2020

Structural basis of mechano-chemical coupling by the mitotic kinesin KIF14

KIF14 is a mitotic kinesin protein important for cytokinesis. Its overexpression is associated with a variety of cancers and mutations in KIF14 result in cerebral and renal development defects. Like other kinesins, KIF14 contains a highly conserved catalytic motor domain where the energy from ATP hydrolysis is converted to directed movement along microtubules. Although much is known regarding the molecular mechanism of kinesin motility, there is a lack of structural information of kinesin-microtubule interactions at sufficient resolution to unambiguously assess how conformational changes related to ATP hydrolysis, microtubule binding and translocation are coupled. Here we determined the near-atomic resolution cryo-electron microscopy structures of five different KIF14 constructs bound to microtubules in the presence of different nucleotide analogues mimicking distinct steps of the ATPase cycle. Eighteen independent structures together with supporting functional assays provide a comprehensive view of the kinesin conformational changes occurring with microtubule and nucleotide binding. Our data shows that: 1) microtubule binding induces opening of the KIF14 nucleotide binding pocket; 2) AMP-PNP and ADP-AlFx induce closure of the nucleotide binding pocket in microtubule bound KIF14 and this conformational change is allosterically controlled by the neck-linker domain; 3) the neck-linker domain when undocked prevents the nucleotide-binding-pocket to fully close and dampens ATP hydrolysis; 4) fifteen neck-linker residues are required to assume the docked conformation; 5) the nucleotide analogue ADP-AlFx adopts a distinct configuration in an open nucleotide-binding-pocket; 6) the neck-linker position controls the hydrolysis step rather than nucleotide binding in the KIF14 ATPase cycle; 7) the two motor domains of a KIF14 dimer adopt distinct conformations when simultaneously bound to the microtubule. These observations provide the structural basis for a coordinated chemo-mechanical kinesin plus end translocation model.

cell biology↗

bioRxiv2020

Changing and stable chromatin accessibility supports transcriptional overhaul during neural stem cell activation

Adult neural stem cells are largely quiescent, and require transcriptional reprogramming to reenter the cell cycle and undergo neurogenesis. However, the precise mechanisms that underlie the rapid transcriptional overhaul during NSC activation remain undefined. Here, we identify the genome-wide chromatin accessibility differences between primary neural stem and progenitor cells in quiescent and activated states. We show that these distinct cellular states exhibit both shared and unique chromatin profiles, which are both associated with gene regulation. Interestingly, we find that accessible chromatin states specific to quiescent or activated cells are active enhancers bound by pro-neurogenic and quiescence factors, ASCL1 and NFI. In contrast, shared sites are gene promoters harboring constitutively accessible chromatin enriched for particular core promoter elements that are functionally associated with translation and metabolic functions. Together, our findings reveal how accessible chromatin states regulate a transcriptional overhaul and drive the switch between quiescence and proliferation in NSC activation.

molecular biology↗

bioRxiv2017

Inferring propensity amongst lung and breast carcinomas via overlapped gene expression profiles

Reconstruction of biological networks for topological analyses helps in correlation identification between various types of biomarkers. These networks have been vital components of System Biology in present era. Genes are the basic physical and structural unit of heredity. Genes act as instructions to make molecules called proteins. Alterations in the normal sequence of these genes are the root cause of various diseases and cancer is the prominent example disease caused by gene alteration or mutation. These slight alterations can be detected by microarray analysis. The high throughput data obtained by microarray experiments aid scientists in reconstructing cancer specific gene regulatory networks. The purpose of experiment performed is to find out the overlapping of the gene expression profiles of breast and lung cancer data, so that the common hub genes can be sifted and utilized as drug targets which could be used for the treatment of diseased conditions. In this study, first the differentially expressed genes have been identified (lung cancer and breast cancer), followed by a filtration approach and most significant genes are chosen using paired t-test and gene regulatory network construction. The obtained result has been checked and validated with the available databases and literature.

systems biology↗