Biology subjects

Ho, M.

Publications and source records attributed to Ho, M..

32 records · Page 2Linked to original sources

bioRxiv2022

Prefusion-Stabilized Lassa Virus Trimer Identifies Neutralizing Nanobodies and Reveals an Apex-Situated Site of Vulnerability

Lassa virus (LASV) is responsible for 100,000-300,000 zoonotic infections annually and poses a threat to public health. Development of antibody-based therapeutics or vaccines has been challenging because neutralizing antibodies - even among Lassa hemorrhagic fever survivors - are generally of low titer, and the target of neutralizing antibodies, the trimeric glycoprotein complex (GPC), a type 1-fusion machine with GP1 and GP2 subunits, has been difficult to produce. Here, we use structure-based design to obtain a soluble LASV GPC by engineering an inter-protomer disulfide (R207GCGP1-L326CGP2) and appending the T4-fibritin trimerization domain. We verified the antigenicity of this prefusion-stabilized LASV GPC against a panel of human antibodies and used electron microscopy (EM) to confirm its trimeric association. We panned the prefusion-stabilized LASV GPC against single domain nanobody libraries and identified one of camel origin, which we named D5, which bound GPC with 27 nM affinity and neutralized the Josiah strain of LASV with an IC50 of 12 {micro}g/ml when formatted into a bivalent IgG2a context. The cryo-EM structure of a ternary complex of the D5 nanobody, the antigen-binding fragment of human antibody 8.11G, and LASV GPC revealed D5 to recognize a site-of-vulnerability at the trimer apex. The recognized site appeared to be specific to GPC lacking cleavage of between GP1 and GP2 subunits. Collectively, our findings suggest that GPC-cleavage intermediates may be targets for LASV neutralization and define an apex-situated site of vulnerability for vaccine development. SignificanceLassa virus (LASV) infection is expanding outside its traditionally endemic areas in West Africa, posing a biothreat to the world. LASV-neutralizing antibodies, moreover, have proven difficult to elicit. To gain insight into requirements for antibody-mediated neutralization of LASV, we developed a prefusion-stabilized LASV glycoprotein trimer (GPC), panned it against phage libraries comprised single-domain antibodies or nanobodies from shark and camel, and identified one, D5, which - when placed into bivalent IgG2a context - could neutralize LASV. Cryo-EM analysis revealed D5 to recognize a cleavage-dependent site-of-vulnerability at the trimer apex. We propose this apex-situated site to be an attractive target for LASV vaccine and therapeutic development.

immunology↗

bioRxiv2021

Camel nanobodies broadly neutralize SARS-CoV-2 variants

With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two VHH nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of [≥]5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants. One-Sentence SummaryDromedary camel (Camelus dromedarius) VHH phage libraries were built for isolation of the nanobodies that broadly neutralize SARS-CoV-2 variants.

immunology↗

bioRxiv2021

Cyclin-G-associated kinase GAK/Aux orchestrates glial autophagy via Atg9 phosphorylation in Parkinson's disease

Autophagy is a major means for the elimination of protein inclusions in neurons in neurodegenerative diseases such as Parkinsons disease (PD). Yet, the mechanism of autophagy in the other brain cell type, glia, is less well characterized and remains largely unknown. Here we present evidence that the PD risk factor Cyclin G-associated kinase (GAK)/dAuxilin (dAux) is a new component in glial autophagy. GAK/dAux directly interacts with ULK1/Atg1 via its uncoating domain. Lack of GAK/dAux increases the autophagosome number and size in adult fly glia and mouse microglia, and generally upregulates levels of components in the initiation and PI3K class III complexes including ULK1/Atg1, demonstrating that GAK/dAux regulates the onset of glial autophagy. Consistently, lack of GAK/dAux enhances Atg1 and Atg9 trafficking to autophagosomes, promoting autophagy initiation. On the other hand, lack of GAK/dAux impairs the autophagic flux and blocks substrate degradation, suggesting that GAK/dAux might play additional roles in glial autophagy. Our findings identify a new autophagy factor in glia; considering the pivotal role of glia under pathological conditions, targeting glial autophagy is potentially a therapeutic strategy for PD.

neuroscience↗

bioRxiv2021

SNARE-dependent mitochondria-lysosome contacts regulate glial mitochondrial dynamics and dopaminergic neuron survival

Disrupted mitochondrial dynamics in neurons are linked to neurodegenerative diseases, but their regulation in glia remains poorly understood. Here, we show that the R-SNARE protein VAMP7 regulates the untethering of mitochondria-lysosome contacts (MLCs) in adult fly glia. Glial-specific knockdown of VAMP7 leads to prolonged MLCs and mitochondrial elongation related to altered fission/fusion dynamics. These VAMP7-deficient mitochondria exhibit hyperpolarized membrane potential, resulting in increased ROS activity, lipid droplet accumulation, and dopaminergic (DA) neuron degeneration. Mechanistically, VAMP7 interacts with the GTPase-activating protein TBC1D15-17 to promote Rab7 GTP hydrolysis. Without VAMP7, TBC1D15-17 remains bound to Rab7 but fails to activate its hydrolysis, resulting in elevated GTP-bound Rab7 and impaired MLCs untethering. Consistently, expression of GTP-locked Rab7Q67L or GAP-dead TBC1D15-17{Delta}GAP phenocopies the mitochondrial defects, while GDP-bound Rab7^T22N or wild-type TBC1D15-17 restores the MLC dynamics. Considering that SNARE proteins mediate membrane fusion, our results demonstrate a new role for VAMP7 in glial mitochondrial dynamics via organelle contacts, impacting neuron survival in a non-cell-autonomous manner.

cell biology↗

bioRxiv2021

A novel PD-L1-targeted shark VNAR single domain-based CAR-T strategy for treating breast cancer and liver cancer

Chimeric antigen receptor (CAR)-T cell therapy shows great potency against hematological malignancies, whereas it remains difficult to treat solid tumors mainly due to lack of appropriate antigenic targets and immunosuppressive tumor microenvironment (TME). Checkpoint molecule PD-L1 is widely overexpressed on multiple tumor types, and the PD-1/PD-L1 interaction is a key mediator of immunosuppression in TME. Here, we isolated anti-PD-L1 single domain antibodies from a newly constructed semi-synthetic nurse shark VNAR phage library. We found that one VNAR, B2, showed cross-reactivity to human, mouse, and canine PD-L1 antigens, and it partially blocked the interaction of human PD-1 to PD-L1. Furthermore, CAR (B2) T cells specifically lysed human breast cancer and liver cancer cells by targeting constitutive and inducible expression of PD-L1, and also hindered tumor metastasis. Importantly, the combination of CAR (B2) T cells with CAR-T cells targeting liver cancer-specific antigen GPC3 regress liver tumors in mice. We concluded that PD-L1-targeted shark VNAR single domain-based CAR-T therapy is a novel strategy to treat breast cancer and liver cancer. This provides a rationale for potential use of CAR (B2) T cells as a monotherapy or combination with a tumor-specific therapy in clinical studies.

cancer biology↗

bioRxiv2021

Dependable Algorithm for Visualizing Snoring Duration through Acoustic Analysis

BackgroundSnoring is a nuisance for the bed partners of people who snore and is also associated with chronic diseases. Estimating the snoring duration from a whole-night-sleep period is challenging. The authors present a dependable algorithm for visualizing snoring durations through acoustic analysis. MethodBoth instruments (Sony digital recorder and smartphones SnoreClock app) were placed within 30 cm from the examinees head during the sleep period. Subsequently, spectrograms were plotted based on audio files recorded from Sony recorders. The authors developed an algorithm to validate snoring durations through visualization of typical snoring segments. ResultsIn total, 37 snoring recordings obtained from six individuals were analyzed. The mean age of the participants was 44.6 {+/-} 9.9 years. A 3-s segment demonstrated the typical dominant frequency bands and amplitude waves of two snores. Every recorded file was tailored to a regular 600-s segment and plotted. Visualization revealed that the typical features of the clustered snores in the amplitude domains were near-isometric spikes (most had an ascending-descending trend). The recorded snores exhibited one or more visibly fixed frequency bands. Intervals were noted between the snoring clusters and were incorporated into the whole-night snoring calculation. The correlative coefficients of snoring rates of digitally recorded files examined by Examiners A and B were higher (0.865, p < 0.001) than those with SnoreClock app (0.757, p < 0.001; 0.787, p < 0.001, respectively). ConclusionA dependable algorithm with high reproducibility was developed for visualizing snoring durations.

physiology↗

bioRxiv2021

Specialization and selective social attention establishes the balance between individual and social learning

A key question individuals face in any social learning environment is when to innovate alone and when to imitate others. Previous simulation results have found that the best performing groups exhibit an intermediate balance, yet it is still largely unknown how individuals collectively negotiate this balance. We use an immersive collective foraging experiment, implemented in the Minecraft game engine, facilitating unprecedented access to spatial trajectories and visual field data. The virtual environment imposes a limited field of view, creating a natural trade-off between allocating visual attention towards individual search or to look towards peers for social imitation. By analyzing foraging patterns, social interactions (visual and spatial), and social influence, we shine new light on how groups collectively adapt to the fluctuating demands of the environment through specialization and selective imitation, rather than homogeneity and indiscriminate copying of others.

animal behavior and cognition↗

bioRxiv2020

Complete and haplotype-specific sequence assembly of segmental duplication-mediated genome rearrangements using targeted CRISPR-targeted ultra-long read sequencing (CTLR-Seq)

We have developed a generally applicable method based on CRISPR/Cas9-targeted ultra-long read sequencing (CTLR-Seq) to completely and haplotype-specifically resolve, at base-pair resolution, large, complex, and highly repetitive genomic regions that had been previously impenetrable to next-generation sequencing analysis such as large segmental duplication (SegDup) regions and their associated genome rearrangements that stretch hundreds of kilobases. Our method combines in vitro Cas9-mediated cutting of the genome and pulse-field gel electrophoresis to haplotype-specifically isolate intact large (200-550 kb) target regions that encompass previously unresolvable genomic sequences. These target fragments are then sequenced (amplification-free) to produce ultra-long reads at up to 40x on-target coverage using Oxford nanopore technology, allowing for the complete assembly of the complex genomic regions of interest at single base-pair resolution. We applied CTLR-Seq to resolve the exact sequence of SegDup rearrangements that constitute the boundary regions of the 22q11.2 deletion CNV and of the 16p11.2 deletion and duplication CNVs. These CNVs are among the strongest known risk factors for schizophrenia and autism. We then perform de novo assembly to resolve, for the first time, at single base-pair resolution, the sequence rearrangements of the 22q11.2 and 16p11.2 CNVs, mapping out exactly the genes and non-coding regions that are affected by the CNV for different carriers.

genomics↗

bioRxiv2020

Vegetation and Microbes Interact to Preserve Organic Matter in Wooded Peatlands

Peatlands have persisted as massive carbon sinks over millennia, even during past periods of climate change. The commonly accepted theory of abiotic controls (mainly anoxia and low temperature) over carbon decomposition cannot explain how vast low-latitude wooded peatlands consistently accrete peat under warm and seasonally unsaturated conditions. Similarly, that theory cannot accurately project the decomposition rate in boreal peatlands where warming and drought have decreased Sphagnum and increased shrub expansion. Here, by comparing composition and ecological traits of microbes between Sphagnum- and shrub-dominated peatlands, we present a previously unrecognized natural course that curbs carbon loss against climate change. Slow-growing microbes decisively dominate the studied wooded peatlands, concomitant with plant-induced, high recalcitrant carbon and phenolics. The slow-growing microbes inherently metabolize organic matter slowly. However, the fast-growing microbes that dominate our Sphagnum site (most boreal peatlands as well) decomposed labile carbon >30 times faster than the slow-growing microbes. We show that the high-phenolic shrub/tree induced shifts in microbial composition may compensate for positive effects of temperature and/or drought on metabolism over time in peatlands. This biotic self-sustaining process that modulates abiotic controls on carbon cycling may help better project long-term climate-carbon feedbacks in peatlands.

ecology↗

bioRxiv2020

Genome-wide molecular effects of the neuropsychiatric 16p11 CNVs in an iPSC-to-iN neuronal model

Copy number variants (CNVs), either deletions or duplications, at the 16p11.2 locus in the human genome are known to increase the risk for autism spectrum disorders (ASD), schizophrenia, and for several other developmental conditions. Here, we investigate the global effects on gene expression and DNA methylation using a 16p11.2 CNV patient-derived induced pluripotent stem cell (iPSC) to induced neuron (iN) cell model system. This approach revealed genome-wide and cell-type specific alterations to both gene expression and DNA methylation patterns and also yielded specific leads on genes potentially contributing to some of the known 16p11.2 patient phenotypes. PCSK9 is identified as a possible contributing factor to the symptoms seen in carriers of the 16p11.2 CNVs. The protocadherin (PCDH) gene family is found to have altered DNA methylation patterns in the CNV patient samples. The iPSC lines used for this study are available through a repository as a resource for research into the molecular etiology of the clinical phenotypes of 16p11.2 CNVs and into that of neuropsychiatric and neurodevelopmental disorders in general.

genetics↗

bioRxiv2020

ImmunoPET as Stoichiometric Sensor for Glypican-3 in Models of Hepatocellular Carcinoma

BackgroundHepatocellular carcinoma (HCC) is the fifth most common cancer worldwide. While conventional imaging approaches like ultrasound, CT, and MRI play critical roles in the diagnosis and surveillance of HCC, improved methods for detection and assessment of treatment response are needed. One promising approach is the use of radiolabeled antibodies for positron emission tomography (immunoPET) imaging. Glypican-3 (GPC3) is a proteoglycan that is highly expressed in the majority of HCC tumors. GPC3-specific antibodies are used to diagnose HCC histopathologically, and have been proposed as a treatment of HCC. Here, we design, synthesize and demonstrate that our humanized immunoPET agent, [89Zr]Zr-DFO-TAB-H14, can stoichiometrically bind to models of human liver cancer with varied GPC3 expression. Methods: The GPC3-specific monoclonal humanized IgG1, TAB-H14, was used as a scaffold for engineering our immunoPET agent. Fluorescent and deferroxamine (DFO) chelate conjugates of TAB-H14 were characterized using mass spectrometry. Binding affinity of TAB-H14 and conjugates for GPC3 was determined in cell-free biolayer interferometry, and cell-based radioimmunoassays. GPC3-expression was assessed by flow cytometry and immunofluorescence using commercially available anti-GPC3 antibodies and TAB-H14 in GPC3-(A431) and GPC3+ cell lines including an engineered line (A431-GPC3+, G1) and liver cancer lines (HepG2, Hep3B, and Huh7). DFO-TAB-H14, was radiolabeled with Zr-89. Mice were subcutaneously engrafted with the aforementioned cell lines and in vivo target engagement of the immunoPET agent [89Zr]Zr-DFO-TAB-H14 was determined using PET/CT, quantitative biodistribution, and autoradiography. Results: TAB-H14 demonstrated subnanomolar to nanomolar affinity for human GPC3. Fluorescently tagged TAB-H14 was able to bind to GPC3 on cell membranes of GPC3-expressing lines by flow cytometry. These results were confirmed by immunofluorescence staining of A431, G1 HepG2, Hep3B, and Huh7 tumor sections. ImmunoPET imaging with [89Zr]Zr-DFO-TAB-H14 showed stoichiometric tumor uptake corresponding to the cell surface expression levels. Autoradiography and immunostaining confirmed in vivo findings. Conclusion: We systematically demonstrate that the humanized immnoPET agent [89Zr]Zr-DFO-TAB-H14 specifically and stoichiometrically binds to GPC3 in several models of human liver cancer, serving as a promising in vivo GPC3 sensor. This agent may provide utility in HCC diagnosis and surveillance, and the selection of candidates for GPC3-directed therapies.

cancer biology↗

bioRxiv2019

Transcriptomically-guided mesendoderm induction of human pluripotent stem cells using a systematically defined culture scheme

Human pluripotent stem cells (hPSCs) are an essential cell source in tissue engineering, studies of development, and disease modeling. Efficient, broadly amenable protocols for rapid lineage induction of hPSCs are of great interest in the stem cell biology field. We describe a simple, robust method for differentiation of hPSCs into mesendoderm in defined conditions utilizing single-cell seeding (SCS) and BMP4 and Activin A (BA) treatment. Gene sets and gene ontology terms related to mesoderm and endoderm differentiation were enriched after 48 hours of BA treatment. BA treatment was readily incorporated into existing protocols for chondrogenic and endothelial progenitor cell differentiation. After prolonged differentiation in vitro or in vivo, BA pre-treatment resulted in higher mesoderm and endoderm levels at the expense of ectoderm formation. These data demonstrate that SCS with BA treatment is a powerful method for induction of mesendoderm that can be integrated into protocols for mesoderm and endoderm differentiation.

bioengineering↗

bioRxiv2019

Integrating Quality of Life and Survival Outcomes Cardiovascular Clinical Trials: Results from the PARTNER Trial

BackgroundSurvival and health status (e.g., symptoms and quality of life) are key outcomes in clinical trials of heart failure treatment. However, health status can only be recorded on survivors, potentially biasing treatment effect estimates when there is differential survival across treatment groups. Joint modeling of survival and health status can address this bias.\n\nMethods and ResultsWe analyzed patient-level data from the PARTNER 1B trial of transcatheter aortic valve replacement (TAVR) versus standard care. Health status was quantified with the Kansas City Cardiomyopathy Questionnaire (KCCQ) at randomization, 1, 6, and 12 months. We compared hazard ratios for survival and mean differences in KCCQ scores at 12 months using several models: the original growth curve model for KCCQ scores (ignoring death), separate Bayesian models for survival and KCCQ scores, and a Bayesian joint longitudinal-survival model fit to either 12 or 30 months of survival follow-up. The benefit of TAVR on 12-month KCCQ scores was greatest in the joint model fit to all survival data (mean difference = 33.7 points; 95% CrI: 24.2, 42.4), followed by the joint model fit to 12 months of survival follow-up (32.3 points; 95% CrI: 22.5, 41.5), a Bayesian model without integrating death (30.4 points; 95% CrI: 21.4, 39.3), and the original growth curve model (26.0 points; 95% CI: 18.7, 33.3). At 12 months, the survival benefit of TAVR was also greater in the joint model (hazard ratio = 0.50; 95% CrI: 0.32, 0.73) than in the non-joint Bayesian model (0.54; 95% CrI: 0.37, 0.75) or the original Kaplan-Meier estimate (0.55; 95% CI: 0.40, 0.74).\n\nConclusionsIn patients with severe symptomatic aortic stenosis and prohibitive surgical risk, the estimated benefits of TAVR on survival and health status compared with standard care were greater in joint Bayesian models than other approaches.

clinical trials↗

bioRxiv2018

Haplotype-phased Callithrix jacchus embryonic stem cell line for genome editing using CRISPR/Cas9

Due to anatomical and physiological similarities to humans, the common marmoset (Callithrix jacchus) is an ideal organism for the study human diseases. Researchers are currently leveraging genome-editing technologies such as CRISPR/Cas9 to genetically engineer marmosets for the in vivo biomedical modeling of human neuropsychiatric and neurodegenerative diseases. The genome characterization of these cell lines greatly reinforces these transgenic efforts. It also provides the genomic contexts required for the accurate interpretation of functional genomics data. We performed haplotype-resolved whole-genome characterization for marmoset ESC line cj367 from the Wisconsin National Primate Research Center. This is the first haplotype-resolved analysis of a marmoset genome and the first whole-genome characterization of any marmoset ESC line. We identified and phased single-nucleotide variants (SNVs) and Indels across the genome. By leveraging this haplotype information, we then compiled a list of cj367 ESC allele-specific CRISPR targeting sites. Furthermore, we demonstrated successful Cas9 Endonuclease Dead (dCas9) expression and targeted localization in cj367 as well as sustained pluripotency after dCas9 transfection by teratoma assay. Lastly, we show that these ESCs can be directly induced into functional neurons in a rapid, single-step process. Our study provides a valuable set of genomic resources for primate transgenics in this post-genome era.

genetics↗