Biology subjects

Yu, J.

Publications and source records attributed to Yu, J..

At least 379 records · Page 21Linked to original sources

bioRxiv2021

A monoclonal antibody that neutralizes SARS-CoV-2 variants, SARS-CoV, and other sarbecoviruses

The repeated emergence of highly pathogenic human coronaviruses as well as their evolving variants highlight the need to develop potent and broad-spectrum antiviral therapeutics and vaccines. By screening monoclonal antibodies (mAbs) isolated from COVID-19-convalescent patients, we found one mAb, 2-36, with cross-neutralizing activity against SARS-CoV. We solved the cryo-EM structure of 2-36 in complex with SARS-CoV-2 or SARS-CoV spike, revealing a highly conserved epitope in the receptor-binding domain (RBD). Antibody 2-36 neutralized not only all current circulating SARS-CoV-2 variants and SARS-COV, but also a panel of bat and pangolin sarbecoviruses that can use human angiotensin-converting enzyme 2 (ACE2) as a receptor. We selected 2-36-escape viruses in vitro and confirmed that K378T in SARS-CoV-2 RBD led to viral resistance. Taken together, 2-36 represents a strategic reserve drug candidate for the prevention and treatment of possible diseases caused by pre-emergent SARS-related coronaviruses. Its epitope defines a promising target for the development of a pan-sarbecovirus vaccine.

microbiology↗

bioRxiv2021

A sense-antisense RNA interaction promotes breast cancer metastasis via regulation of NQO1 expression

Antisense RNAs are ubiquitous in human cells, yet the role that they play in healthy and diseased states remains largely unexplored. Here, we developed a computational framework to catalog and profile antisense RNAs and applied it to poorly and highly metastatic breast cancer cell lines. We identified one antisense RNA that plays a functional role in driving breast cancer progression by upregulating the redox enzyme NQO1, and hence named NQO1-antisense RNA or NQO1-AS. This upregulation occurs via a stabilizing interaction between NQO1-AS and its complementary region in the 3UTR of NQO1 mRNA. By increasing expression of NQO1 protein, breast cancer cells are able to tolerate higher levels of oxidative stress, enabling them to colonize the lung. During this process the cancer cells become dependent on NQO1 to protect them from ferroptosis. We have shown that this dependence can be exploited therapeutically in xenograft models of metastasis. Together, our findings establish a previously unknown role for NQO1-AS in the progression of breast cancer by serving as a post-transcriptional regulator of RNA processing and decay for its sense mRNA.

cancer biology↗

bioRxiv2021

HOXB13 suppresses de novo lipogenesis through HDAC3-mediated epigenetic reprogramming

HOXB13, a homeodomain transcription factor, critically regulates androgen receptor (AR) function and promotes androgen-dependent prostate cancer (PCa) growth. However, the functions of HOXB13 in an AR-independent context remain elusive. Here we report an essential role of HOXB13 in directly suppressing lipogenic transcriptional programs in both AR-positive and -negative PCa cells. The MEIS domain (aa70-150) of HOXB13 interacts with the histone deacetylase HDAC3, which is disrupted by HOXB13 G84E mutation that has been associated with early-onset PCa. Thus, HOXB13 wildtype (WT), but not G84E mutant, recruits HDAC3 to lipogenic enhancers to catalyze histone de-acetylation and suppress lipogenic programs. HOXB13 knockdown unleashes the expression of key lipogenic regulators such as fatty acid synthase (FASN), requiring HDAC3. Analysis of human tissues revealed that HOXB13 is lost in about 30% of metastatic castration-resistant PCa, at least in part, through DNA hypermethylation. Functionally, loss of HOXB13 leads to massive lipid accumulation in PCa cells, thereby promoting cell motility in vitro and fueling xenograft tumor metastasis in vivo, which is mitigated by pharmaceutical inhibitors of FASN. In summary, our study discovers an essential AR-independent function of HOXB13 in repressing de novo lipogenesis and inhibiting tumor metastasis and defines a subclass of PCa that may benefit from lipogenic pathway inhibitors.

cancer biology↗

bioRxiv2021

Metformin modulates microbiota-derived inosine and ameliorates methamphetamine-induced anxiety and depression-like withdrawal symptoms in mice

ObjectiveMetformin exhibits therapeutic potential in behavioural deficits induced by methamphetamine (METH) in rats. Emerging studies suggest gut microbiota may impact psychiatric symptoms, but there is no direct evidence supporting metformins participation in the pathophysiology of withdrawal symptoms via modulation of gut microbiota. MehodsIn order to define the functional contributions by gut microbiota and metformin to the behavioural deficits during METH withdrawal, we utilized a combination of fecal microbiota transplantation (FMT), high-throughput sequencing, and untargeted metabolomics technologies. ResultsFirst, METH addicts exhibited higher diversity and distinct microbial structures compared to heathy controls. In particular, the relative abundance of Rikenellaceae was positively correlated with the severity of anxiety and depression. Second, both human-to-mouse and mouse-to-mouse FMTs confirmed that METH-altered-microbiota transplantation is sufficient to promote anxiety and depression-like behaviours in recipient germ-free mice, and these behavioural disturbances could be ameliorated by metformin. In-depth analysis revealed that METH significantly altered the bacterial composition and structure as well as relative abundance of several bacterial taxa and metabolites, including Rikenellaceae and inosine, respectively, whereas add-on metformin could remodel these alterations. Finally, the inosine complementation successfully restored METH-induced anxiety and depression-like behaviours in mice. DiscussionThis study demonstrates that METH withdrawal-induced anxiety and depression-like behaviours are convertible and transmissible via gut microbiota in a mouse model. The therapeutic effects of metformin on psychiatric manifestations are associated with microbiota-derived metabolites, highlighting the role of the gut microbiota in substance use disorders and the pathophysiology of withdrawal symptoms. Study HighlightsO_ST_ABSWhat is known?C_ST_ABSO_LIThere are no targeted therapies for substance withdrawal syndrome, but there is considerable evidence that withdrawal-associated psychiatric manifestations contribute to the poor adherence to rehabilitation treatment as well as the relapse rates. C_LIO_LIMetformin has shown its therapeutic potential against METH-induced neurobehavioural changes and neurodegeneration in rats through CREB/BDNF and Akt/GSK3 signaling pathways in the anxiety-related brain nuclei. C_LI What is new here?O_LIMETH withdrawal-induced anxiety and depression-like behaviours are convertible and transmissible via gut microbiota in a mouse model. C_LIO_LIThe therapeutic effects of metformin on psychiatric manifestations are associated with microbiota derived metabolites. C_LIO_LIInosine complementation could restore METH withdrawal-induced anxiety and depression-like behaviours. C_LI

animal behavior and cognition↗

bioRxiv2021

Genome sequencing of white-blotched river stingray (Potamotrygon leopoldi) provides novel clues for niche-adaptation and skeleton formation

The white-blotched river stingray (Potamotrygon leopoldi) is a cartilaginous fish native to the Xingu River, a tributary of the Amazon River system. It possesses a lot of unique biological features such as disc-like body shape, bizarre color pattern and living in freshwater habitat while most stingrays and their close relatives are sea dwellers. As a member of the Potamotrygonidae family, P. leopoldi bears evolutionary signification in fish phylogeny, niche adaptation and skeleton formation. In this study, we present its draft genome of 4.11 Gb comprised of 16,227 contigs and 13,238 scaffolds, which has contig N50 of 3,937 kilobases and scaffold N50 of 5,675 kilobases in size. Our analysis shows that P. leopoldi is a slow-evolving fish, diverged from elephant shark about 96 million years ago. We find that two gene families related to immune system, immunoglobulin heavy constant delta genes, and T-cell receptor alpha/delta variable genes, stand out expanded in P. leopoldi only, suggesting robustness in response to freshwater pathogens in adapting novel environments. We also identified the Hox gene clusters in P. leopoldi and discovered that seven Hox genes shared by five representative fishes are missing in P. leopoldi. The RNA-seq data from P. leopoldi and other three fish species demonstrate that fishes have a more diversified tissue expression spectrum as compared to the corresponding mammalian data. Our functional studies suggest that the lack of genes encoding vitamin D-binding protein in cartilaginous (both P. leopoldi and Callorhinchus milii) fishes could partly explain the absence of hard bone in their endoskeleton. Overall, this genome resource provides new insights into the niche-adaptation, body plan and skeleton formation of P. leopoldi as well as the genome evolution in cartilaginous fish.

genomics↗

bioRxiv2021

De novo variants in Chinese ASD trios reveal genetic basis underlying autism without developmental delay and intellectual disabilities

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder that causes a range of social communication and behavioral impairments. ASD typically manifests in young children, often with developmental delay or intellectual disabilities (DD/ID) as comorbidities. Accruing evidence indicates that ASD is highly heritable and genomewide studies on ASD cohorts have defined numerous genetic contributors. Notably, most of these studies have been performed with individuals of European and Hispanic ancestry and thus there is a paucity of genetic analyses of ASD in the East Asian population. Here, we performed whole-exome sequencing on 772 ASD trios from China, combining with a previous study of 369 Chinese ASD trios, to identify de novo variants in a total of 1141 ASD trios. We found that ASD probands without DD/ID carried less disruptive de novo variants, including protein-truncating and missense variants, than ASD with DD/ID. Surprisingly, we showed that expression of genes with de novo variants found in ASD probands without DD/ID were enriched in a specific group of neural progenitor cells, suggesting a potential mechanism underlying high-functioning autism. Importantly, some ASD risk genes from this study are not present in the current ASD gene database, suggesting that there are novel genetic contributors to ASD in East Asian populations. We validated one such novel ASD risk gene - SLC35G1 by showing that mice harboring heterozygous deletion of Slc35g1 exhibited defects in social interaction behaviors. Together, this work nominates novel ASD risk genes and indicates that ASD genetic studies in different geographic populations are essential to reveal the comprehensive genetic architecture of ASD.

neuroscience↗

bioRxiv2021

Protective Efficacy of Gastrointestinal SARS-CoV-2 Delivery Against Intranasal and Intratracheal SARS-CoV-2 Challenge in Rhesus Macaques

Live oral vaccines have been explored for their protective efficacy against respiratory viruses, particularly for adenovirus serotypes 4 and 7. The potential of a live oral vaccine against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), however, remains unclear. In this study, we assessed the immunogenicity of live SARS-CoV-2 delivered to the gastrointestinal tract in rhesus macaques and its protective efficacy against intranasal and intratracheal SARS-CoV-2 challenge. Post-pyloric administration of SARS-CoV-2 by esophagogastroduodenoscopy resulted in limited virus replication in the gastrointestinal tract and minimal to no induction of mucosal antibody titers in rectal swabs, nasal swabs, and bronchoalveolar lavage. Low levels of serum neutralizing antibodies were induced and correlated with modestly diminished viral loads in nasal swabs and bronchoalveolar lavage following intranasal and intratracheal SARS-CoV-2 challenge. Overall, our data show that post-pyloric inoculation of live SARS-CoV-2 is weakly immunogenic and confers partial protection against respiratory SARS-CoV-2 challenge in rhesus macaques. ImportanceSARS-CoV-2 remains a global threat, despite the rapid deployment but limited coverage of multiple vaccines. Alternative vaccine strategies that have favorable manufacturing timelines, greater ease of distribution and improved coverage may offer significant public health benefits, especially in resource-limited settings. Live oral vaccines have the potential to address some of these limitations; however no studies have yet been conducted to assess the immunogenicity and protective efficacy of a live oral vaccine against SARS-CoV-2. Here we report that oral administration of live SARS-CoV-2 in non-human primates may offer prophylactic benefits, but that formulation and route of administration will require further optimization.

microbiology↗

bioRxiv2021

Multiplex knockout of trichome-regulating MYBs in poplar affects light sensitivity and triterpene accumulation

As the focus for CRISPR edited plants moves from proof-of-concept to real world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one gRNA per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in Populus tremula x P. alba INRA 717-1B4. Unexpected duplications of MYB186 and MYB138 resulted in a total of eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and PCR analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the non-glandular trichomes of poplar. ONE SENTENCE SUMMARYTargeting conserved sequences with a single gRNA allowed efficient mutagenesis of a multigene family and the recovery of trichomeless and triterpene-free poplar mutants.

plant biology↗

bioRxiv2021

An adjuvanted SARS-CoV-2 RBD nanoparticle elicits neutralizing antibodies and fully protective immunity in aged mice

Development of affordable and effective vaccines that can also protect vulnerable populations such as the elderly from COVID-19-related morbidity and mortality is a public health priority. Here we took a systematic and iterative approach by testing several SARS-CoV-2 protein antigens and adjuvants to identify a combination that elicits neutralizing antibodies and protection in young and aged mice. In particular, SARS-CoV-2 receptorbinding domain (RBD) displayed as a protein nanoparticle (RBD-NP) was a highly effective antigen, and when formulated with an oil-in-water emulsion containing Carbohydrate fatty acid MonoSulphate derivative (CMS) induced the highest levels of cross-neutralizing antibodies compared to other oil-in-water emulsions or AS01B. Mechanistically, CMS induced antigen retention in the draining lymph node (dLN) and expression of cytokines, chemokines and type I interferon-stimulated genes at both injection site and dLN. Overall, CMS:RBD-NP is effective across multiple age groups and is an exemplar of a SARS-CoV-2 subunit vaccine tailored to the elderly.

immunology↗

bioRxiv2021

Drug targeting to sites of oxidative stress using the Baeyer-Villiger reaction

The antioxidant nuclear factor erythroid 2-related factor 2 (Nrf2) is a desirable therapeutic target for a broad range of pathologies, including chronic diseases of the lung and liver, and autoimmune, neurodegenerative, and cardiovascular disorders. However, current Nrf2 activators are limited by unwanted effects due to non-specificity, and systemic distribution and action. Here we report that a 1,2-dicarbonyl moiety masks the electrophilic reactivity of the Nrf2 activator monomethyl fumarate (MMF), otherwise responsible for its non-specific effects. The 1,2-dicarbonyl compound is highly susceptible to Baeyer-Villiger oxidation, with generation of MMF specifically on exposure to pathological levels of hydrogen peroxide or peroxynitrite. Oral treatment with the MMF generating 1,2-dicarbonyl compound reversed chronic neuropathic and osteoarthritis pain in mice, and selectively activated Nrf2 at sites of oxidative stress. This 1,2-dicarbonyl platform may be used to treat additional disorders of oxidative stress, and to selectively target other therapeutics to sites of redox imbalance.

pharmacology and toxicology↗

bioRxiv2021

Clinically-relevant T cell expansion protocols activate distinct cellular metabolic programs and phenotypes

Ex vivo expansion conditions used to generate T cells for immunotherapy are thought to adopt metabolic phenotypes that impede therapeutic efficacy in vivo. The comparison of five different culture media used for clinical T cell expansion revealed unique optima based on different output variables including proliferation, differentiation, function, activation and mitochondrial phenotypes. T cells adapted their metabolism to match their media expansion condition as shown by glucose and glutamine uptake, and patterns of glucose isotope labeling. However, adoption of these metabolic phenotypes was uncoupled to T cell function. Furthermore, T cell products cultured in ascites from ovarian cancer patients displayed suppressed mitochondrial activity and function irrespective of the ex vivo expansion media. In one case, culturing in ascites resulted in increased glucose uptake which was insufficient to rescue T cell function. Thus, ex vivo T cell expansion conditions have profound impacts on metabolism and function.

immunology↗

bioRxiv2021

Optimization of Non-Coding Regions Improves Protective Efficacy of an mRNA SARS-CoV-2 Vaccine in Nonhuman Primates

The CVnCoV (CureVac) mRNA vaccine for SARS-CoV-2 has recently been evaluated in a phase 2b/3 efficacy trial in humans. CV2CoV is a second-generation mRNA vaccine with optimized non-coding regions and enhanced antigen expression. Here we report a head-to-head study of the immunogenicity and protective efficacy of CVnCoV and CV2CoV in nonhuman primates. We immunized 18 cynomolgus macaques with two doses of 12 ug of lipid nanoparticle formulated CVnCoV, CV2CoV, or sham (N=6/group). CV2CoV induced substantially higher binding and neutralizing antibodies, memory B cell responses, and T cell responses as compared with CVnCoV. CV2CoV also induced more potent neutralizing antibody responses against SARS-CoV-2 variants, including B.1.351 (beta), B.1.617.2 (delta), and C.37 (lambda). While CVnCoV provided partial protection against SARS-CoV-2 challenge, CV2CoV afforded robust protection with markedly lower viral loads in the upper and lower respiratory tract. Antibody responses correlated with protective efficacy. These data demonstrate that optimization of non-coding regions can greatly improve the immunogenicity and protective efficacy of an mRNA SARS-CoV-2 vaccine in nonhuman primates.

immunology↗

bioRxiv2021

An ultrapotent neutralizing bispecific antibody with broad spectrum against SARS-CoV-2 variants

In spite of the successful development of effective countermeasures against Covid-19, variants have and will continue to emerge that could compromise the efficacy of currently approved neutralizing antibodies and vaccines. Consequently, novel and more efficacious agents are urgently needed. We have developed a bispecific antibody, 2022, consisting of two antibodies, 2F8 and VHH18. 2F8 was isolated from our proprietary fully synthetic human IDEAL (Intelligently Designed and Engineered Antibody Library)-VH/VL library and VHH18 is a single domain antibody isolated from IDEAL-nanobody library. 2022 was constructed by attaching VHH18 to the C-terminal of Fc of 2F8. 2022 binds two non-overlapping epitopes simultaneously on the RBD of the SARS-CoV-2 spike protein and blocks the binding of RBD to human angiotensin-converting enzyme 2 (ACE2). 2022 potently neutralizes SARS-CoV-2 and all of the variants tested in both pseudovirus and live virus assays, including variants carrying mutations known to resist neutralizing antibodies approved under EUA and that reduce the protection efficiency of current effective vaccines. The half-maximum inhibitory concentration (IC50) of 2022 is 270 pM, 30 pM, 20 pM, and 1 pM, for wild-type, alpha, beta, and delta pseudovirus, respectively. In the live virus assay, 2022 has an IC50 of 26.4 pM, 13.3 pM, and 88.6 pM, for wild-type, beta, and delta live virus, respectively. In a mouse model of SARS-CoV-2, 2022 showed strong prophylactic and therapeutic effects. A single administration of 2022 intranasal (i.n.) or intraperitoneal (i.p.) 24 hours before virus challenge completely protected all mice from bodyweight loss, as compared with up to 20% loss of bodyweight in placebo treated mice. In addition, the lung viral titers were undetectable (FRNT assay) in all mice treated with 2022 either prophylactically or therapeutically, as compared with around 1x105 pfu/g lung tissue in placebo treated mice. In summary, bispecific antibody 2022 showed potent binding and neutralizing activity across a variety of SARS-CoV-2 variants and could be an attractive weapon to combat the ongoing waves of the COVID-19 pandemic propagated mainly by variants, especially, the much more contagious delta variant.

microbiology↗

bioRxiv2021

Low-level overexpression of wild type TDP-43 causes late-onset, progressive neurodegeneration and paralysis in mice

Modestly increased expression of transactive response DNA binding protein (TDP-43) gene have been reported in amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neuromuscular diseases. However, whether this modest elevation triggers neurodegeneration is not known. Although high levels of TDP-43 overexpression have been modeled in mice and shown to cause early death, models with low-level overexpression that mimic the human condition have not been established. In this study, transgenic mice overexpressing wild type TDP-43 at less than 60% above the endogenous CNS levels were constructed, and their phenotypes analyzed by a variety of techniques, including biochemical, molecular, histological, behavioral techniques and electromyography. The TDP-43 transgene was expressed in neurons, astrocytes, and oligodendrocytes in the cortex and predominantly in astrocytes and oligodendrocytes in the spinal cord. The mice developed a reproducible progressive weakness ending in paralysis in mid-life. Detailed analysis showed [~]30% loss of large pyramidal neurons in the layer V motor cortex; in the spinal cord, severe demyelination was accompanied by oligodendrocyte injury, protein aggregation, astrogliosis and microgliosis, and elevation of neuroinflammation. Surprisingly, there was no loss of lower motor neurons in the lumbar spinal cord despite the complete paralysis of the hindlimbs. However, denervation was detected at the neuromuscular junction. These results demonstrate that low-level TDP-43 overexpression can cause diverse aspects of ALS, including late-onset and progressive motor dysfunction, neuroinflammation, and neurodegeneration. Our findings suggest that persistent modest elevations in TDP-43 expression can lead to ALS and other neurological disorders involving TDP-43 proteinopathy. Because of the predictable and progressive clinical paralytic phenotype, this transgenic mouse model will be useful in preclinical trial of therapeutics targeting neurological disorders associated with elevated levels of TDP-43.

genetics↗

bioRxiv2021

Metabolism is the major driver of hydrogen isotope fractionation recorded in tree-ring glucose of Pinus nigra

- Stable isotope abundances convey valuable information about plant physiological processes and underlying environmental controls. Central gaps in our mechanistic understanding of hydrogen isotope abundances impede their widespread application within the plant and biogeosciences. - To address these gaps, we analysed intramolecular deuterium abundances in glucose of Pinus nigra extracted from an annually resolved tree-ring series (1961 to 1995). - We found fractionation signals (i.e., temporal variability in deuterium abundance) at glucose H1 and H2 introduced by closely related metabolic processes. Regression analysis indicates that these signals (and thus metabolism) respond to drought and atmospheric CO2 concentration beyond a response change point. They explain {approx}60% of the whole-molecule deuterium variability. Altered metabolism is associated with below-average yet not exceptionally low growth. - We propose the signals are introduced at the leaf-level by changes in sucrose-to-starch carbon partitioning and anaplerotic carbon flux into the Calvin-Benson cycle. In conclusion, metabolism can be the main driver of hydrogen isotope variation in plant glucose.

biophysics↗

bioRxiv2021

Switching Promotor Recognition of Phage RNA Polymerase in Silico Following Path along Lab Directed Evolution

In this work we computationally investigated how a viral RNA polymerase (RNAP) from bacteriophage T7 evolves into RNAP variants under lab-directed evolution to switch recognition from T7 promoter to T3 promoter in transcription initiation. We first constructed a closed initiation complex for the wild-type T7 RNAP, and then for six mutant RNAPs discovered from phage assisted continuous evolution experiments. All-atom molecular dynamics (MD) simulations up to one microsecond each were conducted on these RNAPs in complex with T7/T3 promoter. Our simulations show notably that protein-DNA electrostatic interactions or stabilities at the RNAP-DNA promoter interface well dictate the promoter recognition preference of the RNAP and variants. Key residues and structural elements that contribute significantly to switching the promoter recognition were identified. Followed by a first point mutation N748D on the specificity loop to slightly disengage the RNAP from the promoter to hinder the original recognition, we found an auxiliary helix (206-225) that takes over switching the promoter recognition upon further mutations (E222K and E207K), by forming additional charge interactions with the promoter DNA and reorientating differently on the T7 and T3 promoter. Further mutations on the AT-rich loop and the specificity loop can fully switch the RNAP-promoter recognition to the T3 promoter. Overall, our studies reveal energetics and structural dynamics details along an exemplary directed evolutionary path of the phage RNAP variants for a rewired promoter recognition function. The findings demonstrate underlying physical mechanisms and are expected to assist knowledge/data learning or rational redesign of the protein enzyme structure-function.

biophysics↗

bioRxiv2021

Structural model of PORCN illuminates disease-associated variants and drug binding sites

Wnt signaling is essential for normal development and is a therapeutic target in cancer. The enzyme PORCN, or porcupine, is a membrane-bound O-acyltransferase (MBOAT) that is required for the post-translational modification of all Wnts, adding an essential mono-unsaturated palmitoleic acid to a serine on the tip of Wnt hairpin 2. Inherited mutations in PORCN cause focal dermal hypoplasia, and therapeutic inhibition of PORCN slows the growth of Wnt-dependent cancers. Here, based on homology to mammalian MBOAT proteins we develop and validate a molecular structural model of PORCN. The model accommodates palmitoleoyl-CoA and Wnt hairpin 2 in two tunnels in the conserved catalytic core, shedding light on the catalytic mechanism. The model predicts how previously uncharacterized human variants of uncertain significance can alter PORCN function. Drugs including ETC-159, IWP-L6 and LGK-974 dock in the PORCN catalytic site, providing insights into PORCN pharmacologic inhibition. This structural model provides mechanistic insights into PORCN substrate recognition and catalysis as well as the inhibition of its enzymatic activity and can facilitate the development of improved inhibitors and the understanding of disease relevant PORCN mutants.

molecular biology↗

bioRxiv2021

A modular protein subunit vaccine candidate produced in yeast confers protection against SARS-CoV-2 in non-human primates

Vaccines against SARS-CoV-2 have been distributed at massive scale in developed countries, and have been effective at preventing COVID-19. Access to vaccines is limited, however, in low- and middle-income countries (LMICs) due to insufficient supply, high costs, and cold storage requirements. New vaccines that can be produced in existing manufacturing facilities in LMICs, can be manufactured at low cost, and use widely available, proven, safe adjuvants like alum, would improve global immunity against SARS-CoV-2. One such protein subunit vaccine is produced by the Serum Institute of India Pvt. Ltd. and is currently in clinical testing. Two protein components, the SARS-CoV-2 receptor binding domain (RBD) and hepatitis B surface antigen virus-like particles (VLPs), are each produced in yeast, which would enable a low-cost, high-volume manufacturing process. Here, we describe the design and preclinical testing of the RBD-VLP vaccine in cynomolgus macaques. We observed titers of neutralizing antibodies (>104) above the range of protection for other licensed vaccines in non-human primates. Interestingly, addition of a second adjuvant (CpG1018) appeared to improve the cellular response while reducing the humoral response. We challenged animals with SARS-CoV-2, and observed a ~3.4 and ~2.9 log10 reduction in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, compared to sham controls. These results inform the design and formulation of current clinical COVID-19 vaccine candidates like the one described here, and future designs of RBD-based vaccines against variants of SARS-CoV-2 or other betacoronaviruses.

immunology↗