Ultrastructural insights into the microsporidian infection apparatus reveal the kinetics and morphological transitions of polar tube and cargo during host cell invasion.

During host cell invasion, microsporidian spores translocate their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the Vairimorpha necatrix polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and uncovers new aspects of ribosome regulation and transport.

Phenotype and fate of liver-resident CD8 T cells during acute and chronic hepacivirus infection.

Immune correlates of hepatitis C virus (HCV) clearance and control remain poorly defined due to the lack of an informative animal model. We recently described acute and chronic rodent HCV-like virus (RHV) infections in lab mice. Here, we developed MHC class I and class II tetramers to characterize the serial changes in RHV-specific CD8 and CD4 T cells during acute and chronic infection in C57BL/6J mice. RHV infection induced rapid expansion of T cells targeting viral structural and nonstructural proteins. After virus clearance, the virus-specific T cells transitioned from effectors to long-lived liver-resident memory T cells (TRM). The effector and memory CD8 and CD4 T cells primarily produced Th1 cytokines, IFN-γ, TNF-α, and IL-2, upon ex vivo antigen stimulation, and their phenotype and transcriptome differed significantly between the liver and spleen. Rapid clearance of RHV reinfection coincided with the proliferation of virus-specific CD8 TRM cells in the liver. Chronic RHV infection was associated with the exhaustion of CD8 T cells (Tex) and the development of severe liver diseases. Interestingly, the virus-specific CD8 Tex cells continued proliferation in the liver despite the persistent high-titer viremia and retained partial antiviral functions, as evident from their ability to degranulate and produce IFN-γ upon ex vivo antigen stimulation. Thus, RHV infection in mice provides a unique model to study the function and fate of liver-resident T cells during acute and chronic hepatotropic infection.

A highly efficacious live attenuated mumps virus-based SARS-CoV-2 vaccine candidate expressing a six-proline stabilized prefusion spike.

With the rapid increase in SARS-CoV-2 cases in children, a safe and effective vaccine for this population is urgently needed. The MMR (measles/mumps/rubella) vaccine has been one of the safest and most effective human vaccines used in infants and children since the 1960s. Here, we developed live attenuated recombinant mumps virus (rMuV)-based SARS-CoV-2 vaccine candidates using the MuV Jeryl Lynn (JL2) vaccine strain backbone. The soluble prefusion SARS-CoV-2 spike protein (preS) gene, stablized by two prolines (preS-2P) or six prolines (preS-6P), was inserted into the MuV genome at the P-M or F-SH gene junctions in the MuV genome. preS-6P was more efficiently expressed than preS-2P, and preS-6P expression from the P-M gene junction was more efficient than from the F-SH gene junction. In mice, the rMuV-preS-6P vaccine was more immunogenic than the rMuV-preS-2P vaccine, eliciting stronger neutralizing antibodies and mucosal immunity. Sera raised in response to the rMuV-preS-6P vaccine neutralized SARS-CoV-2 variants of concern, including the Delta variant equivalently. Intranasal and/or subcutaneous immunization of IFNAR1-/- mice and golden Syrian hamsters with the rMuV-preS-6P vaccine induced high levels of neutralizing antibodies, mucosal immunoglobulin A antibody, and T cell immune responses, and were completely protected from challenge by both SARS-CoV-2 USA-WA1/2020 and Delta variants. Therefore, rMuV-preS-6P is a highly promising COVID-19 vaccine candidate, warranting further development as a tetravalent MMR vaccine, which may include protection against SARS-CoV-2.

SARS-CoV-2 prefusion spike protein stabilized by six rather than two prolines is more potent for inducing antibodies that neutralize viral variants of concern.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the main target for neutralizing antibodies (NAbs). The S protein trimer is anchored in the virion membrane in its prefusion (preS) but metastable form. The preS protein has been stabilized by introducing two or six proline substitutions, to generate stabilized, soluble 2P or HexaPro (6P) preS proteins. Currently, it is not known which form is the most immunogenic. Here, we generated recombinant vesicular stomatitis virus (rVSV) expressing preS-2P, preS-HexaPro, and native full-length S, and compared their immunogenicity in mice and hamsters. The rVSV-preS-HexaPro produced and secreted significantly more preS protein compared to rVSV-preS-2P. Importantly, rVSV-preS-HexaPro triggered significantly more preS-specific serum IgG antibody than rVSV-preS-2P in both mice and hamsters. Antibodies induced by preS-HexaPro neutralized the B.1.1.7, B.1.351, P.1, B.1.427, and B.1.617.2 variants approximately two to four times better than those induced by preS-2P. Furthermore, preS-HexaPro induced a more robust Th1-biased cellular immune response than preS-2P. A single dose (104 pfu) immunization with rVSV-preS-HexaPro and rVSV-preS-2P provided complete protection against challenge with mouse-adapted SARS-CoV-2 and B.1.617.2 variant, whereas rVSV-S only conferred partial protection. When the immunization dose was lowered to 103 pfu, rVSV-preS-HexaPro induced two- to sixfold higher antibody responses than rVSV-preS-2P in hamsters. In addition, rVSV-preS-HexaPro conferred 70% protection against lung infection whereas only 30% protection was observed in the rVSV-preS-2P. Collectively, our data demonstrate that both preS-2P and preS-HexaPro are highly efficacious but preS-HexaPro is more immunogenic and protective, highlighting the advantages of using preS-HexaPro in the next generation of SARS-CoV-2 vaccines.

Functional annotation of a divergent genome using sequence and structure-based similarity.

BACKGROUND: Microsporidia are a large taxon of intracellular pathogens characterized by extraordinarily streamlined genomes with unusually high sequence divergence and many species-specific adaptations. These unique factors pose challenges for traditional genome annotation methods based on sequence similarity. As a result, many of the microsporidian genomes sequenced to date contain numerous genes of unknown function. Recent innovations in rapid and accurate structure prediction and comparison, together with the growing amount of data in structural databases, provide new opportunities to assist in the functional annotation of newly sequenced genomes. RESULTS: In this study, we established a workflow that combines sequence and structure-based functional gene annotation approaches employing a ChimeraX plugin named ANNOTEX (Annotation Extension for ChimeraX), allowing for visual inspection and manual curation. We employed this workflow on a high-quality telomere-to-telomere sequenced tetraploid genome of Vairimorpha necatrix. First, the 3080 predicted protein-coding DNA sequences, of which 89% were confirmed with RNA sequencing data, were used as input. Next, ColabFold was used to create protein structure predictions, followed by a Foldseek search for structural matching to the PDB and AlphaFold databases. The subsequent manual curation, using sequence and structure-based hits, increased the accuracy and quality of the functional genome annotation compared to results using only traditional annotation tools. Our workflow resulted in a comprehensive description of the V. necatrix genome, along with a structural summary of the most prevalent protein groups, such as the ricin B lectin family. In addition, and to test our tool, we identified the functions of several previously uncharacterized Encephalitozoon cuniculi genes. CONCLUSION: We provide a new functional annotation tool for divergent organisms and employ it on a newly sequenced, high-quality microsporidian genome to shed light on this uncharacterized intracellular pathogen of Lepidoptera. The addition of a structure-based annotation approach can serve as a valuable template for studying other microsporidian or similarly divergent species.

Size Matters: Computational Insights into the Crowning of Noble Gas Trioxides.

In pursuit of enhancing the stability of the highly explosive and shock-sensitive compound XeO3, we performed quantum chemical calculations to investigate its possible complexation with electron-rich crown ethers, including 9-crown-3, 12-crown-4, 15-crown-5, 18-crown-6, and 21-crown-7, as well as their thio analogues. Furthermore, we expanded our study to other noble gas trioxides (NgO3), namely, KrO3 and ArO3. The basis set superposition error (BSSE) corrected interaction energies for these adducts range from -13.0 kcal/mol to -48.2 kcal/mol, which is notably high for σ-hole-mediated noncovalent interactions. The formation of these adducts was observed to be more favorable with the increase in the ring size of the crowns and less favorable while going from XeO3 to ArO3. A comprehensive analysis by various computational tools such as the mapping of the electrostatic potential (ESP), Wiberg bond indices (WBIs), Bader's theory of atoms-in-molecules (AIM), natural bond orbital (NBO) analysis, noncovalent interaction (NCI) plots, and energy decomposition analysis (EDA) revealed that the C-H···O interactions, as well as dispersion interactions, play a pivotal role in stabilizing adducts involving larger crowns. A noteworthy outcome of our study is the revelation of a coordination number of 9 for xenon in the complex formed between XeO3 and the thio analogue of 18-crown-6, which is higher than the largest number reported to date.

Brønsted acid- and Ni(II)-catalyzed C-H oxidation/rearrangement of cyclotriveratrylenes (CTVs) to cyclic and acyclic quinones as potential anti-cancer agents.

This paper describes a simple and practical protocol for the direct synthesis of acyclic and cyclic quinone derivatives via an acid-promoted nickel(II)-catalyzed inner rim C-H oxidation of cyclotriveratrylene (CTV) and its analogues. The cyclic quinone derivatives resulted from trimethoxy-cyclotriveratrylene (TCTV) through C-C bond formation via intramolecular ipso substitution followed by subsequent anionic rearrangement containing stereo-vicinal quaternary centers. The DFT calculations strongly support the experimental findings and reveal the role of Brønsted acids in the C-H bond activation of CTV. All the newly synthesized compounds were screened for their in vitro anti-cancer activity using colorimetric SRB assay analysis. Among them, compounds 3a, 3d, 3h, 4a, 4b, 4c and 4e exhibited moderate anticancer activity against A549, HCT-116, PC-3, MDA-MB-231, HEK-293 and SW620 human cancer cell lines.

A computer vision-based system for real-time component identification from waste printed circuit boards.

With an exponential increase in consumers' need for electronic products, the world is facing an ever-increasing economic and environmental threat of electronic waste (e-waste). To minimize their adverse effects, e-waste recycling is one of the pivotal factors that can help in minimizing the environmental pollution andto increase recovery of valuable materials. For instance, Printed Circuit Boards (PCBs), while they have several valuable elements, they are hazardous too; and therefore, they form a large chunk of e-waste being generated today. Thus, in recycling PCBs, Electronic Components (ECs) are segregated at first, and separately processed for recovering key elements that could be re-used. However, in the current recycling process, especially in developing nations, humans manually screen ECs, which goes on to affect their health. It also causes losses of valuable materials. Therefore, automated solutions need to be adopted for both to classify and to segregate ECs from waste PCBs. The study proposes a robust EC identification system based on computer vision and deep learning algorithms (YOLOv3) to automate sorting process which would help in further processing. The study uses a publicly available dataset, and a PCB dataset which reflect challenging recycling environments like lighting conditions, cast shadows, orientations, viewpoints, and different cameras/resolutions. The outcome of YOLOv3 detection model based on training of both datasets presents satisfactory classification accuracy and capability of real-time competent identification, which in turn, could help in automatically segregating ECs, while leading towards effective e-waste recycling.

Recombinant measles virus expressing prefusion spike protein stabilized by six rather than two prolines is more efficacious against SARS-CoV-2 infection.

Measles virus (MeV) has been an excellent vector platform for delivering vaccines against many pathogens because of its high safety and efficacy, and induction of long-lived immunity. Early in the COVID-19 pandemic, a recombinant MeV (rMeV) expressing the prefusion full-length spike protein stabilized by two prolines (TMV-083) was developed and tested in phase 1 and 1/2 clinical trials but was discontinued because of insufficient immunogenicity and a low seroconversion rate in adults. Here, we compared the immunogenicity of rMeV expressing a soluble prefusion spike (preS) protein stabilized by two prolines (rMeV-preS-2P) with a rMeV expressing a soluble preS protein stabilized by six prolines (rMeV-preS-6P). We found that rMeV-preS-6P expressed approximately five times more preS than rMeV-preS-2P in cell culture. Importantly, rMeV-preS-6P induced 30-60 and six times more serum immunoglobulin G and neutralizing antibody than rMeV-preS-2P, respectively, in IFNAR-/- mice. IFNAR-/- mice immunized with rMeV-preS-6P were completely protected from challenge with a mouse-adapted SARS-CoV-2, whereas those immunized with rMeV-preS-2P were partially protected. In addition, hamsters immunized with rMeV-preS-6P were completely protected from the challenge with a Delta variant of SARS-CoV-2. Our results demonstrate that rMeV-preS-6P is significantly more efficacious than rMeV-preS-2P, highlighting the value of using preS-6P as the antigen for developing vaccines against SARS-CoV-2.

Differences in structure and hibernation mechanism highlight diversification of the microsporidian ribosome.

Assembling and powering ribosomes are energy-intensive processes requiring fine-tuned cellular control mechanisms. In organisms operating under strict nutrient limitations, such as pathogenic microsporidia, conservation of energy via ribosomal hibernation and recycling is critical. The mechanisms by which hibernation is achieved in microsporidia, however, remain poorly understood. Here, we present the cryo-electron microscopy structure of the ribosome from Paranosema locustae spores, bound by the conserved eukaryotic hibernation and recycling factor Lso2. The microsporidian Lso2 homolog adopts a V-shaped conformation to bridge the mRNA decoding site and the large subunit tRNA binding sites, providing a reversible ribosome inactivation mechanism. Although microsporidian ribosomes are highly compacted, the P. locustae ribosome retains several rRNA segments absent in other microsporidia, and represents an intermediate state of rRNA reduction. In one case, the near complete reduction of an expansion segment has resulted in a single bound nucleotide, which may act as an architectural co-factor to stabilize a protein-protein interface. The presented structure highlights the reductive evolution in these emerging pathogens and sheds light on a conserved mechanism for eukaryotic ribosome hibernation.

Seizure outcomes after resection of primary brain tumors in pediatric patients: a systematic review and meta-analysis.

PURPOSE: Primary brain neoplasms are the most common solid tumors in pediatric patients and seizures are a common presenting symptom. Surgical intervention improves oncologic outcomes and seizure burden. A better understanding of factors that influence seizure outcomes in the surgical management of primary brain tumors of childhood can guide treatment approach thereby improving patient quality of life. METHODS: We performed a systematic analysis using articles queried from PubMed, EMBASE, and Cochrane published from January 1990 to August 2022 to determine predictors of seizure outcomes in pediatric patients undergoing resection of primary brain tumors. RESULTS: We identified 24 retrospective cohort studies, one prospective cohort study, and one mixed retrospective and prospective study for the systematic analysis. A total of 831 pediatric patients were available for analysis. 668 (80.4%) patients achieved seizure freedom after surgery. Complete tumor resection increased the likelihood of a seizure-free (Engel I) outcome compared to subtotal resection (OR 7.1, 95% CI 2.3-21.9). Rates of Engel I seizure outcomes did not significantly differ based on factors such as age at seizure onset, duration of epilepsy, gender, tumor laterality, or age at surgery, but trended towards significance for improved outcomes in temporal lobe tumors. CONCLUSION: Primary brain tumors in the pediatric population are commonly associated with seizures. Resection of these lesions reduces seizure burden and is associated with high rates of seizure freedom. Complete resection, compared to subtotal resection, significantly increases the likelihood of seizure-free outcomes.

Behavioral Improvements following Lesion Resection for Pediatric Epilepsy: Pediatric Psychosurgery?

INTRODUCTION: Resection of brain lesions associated with refractory epilepsy to achieve seizure control is well accepted. However, concurrent behavioral effects of these lesions such as changes in mood, personality, and cognition and the effects of surgery on behavior have not been well characterized. We describe 5 such children with epileptogenic lesions and significant behavioral abnormalities which improved after surgery. CASE DESCRIPTIONS: Five children (ages 3-14 years) with major behavioral abnormalities and lesional epilepsy were identified and treated at our center. Behavioral problems included academic impairment, impulsivity, self-injurious behavior, and decreased social interaction with diagnoses of ADHD, oppositional defiant disorder, and autism. Pre-operative neuropsychiatric testing was performed in 4/5 patients and revealed low-average cognitive and intellectual abilities for their age, attentional difficulties, and poor memory. Lesions were located in the temporal (2 gangliogliomas, 1 JPA, 1 cavernoma) and parietal (1 DNET) lobes. Gross total resection was achieved in all cases. At mean 1-year follow-up, seizure freedom (Engel 1a in 3 patients, Engel 1c in 2 patients) and significant behavioral improvements (academic performance, attention, socialization, and aggression) were achieved in all. Two patients manifested violence pre-operatively; one had extreme behavior with violence toward teachers and peers despite low seizure burden. Since surgery, his behavior has normalized. CONCLUSION: We identified 5 patients with severe behavioral disorders in the setting of lesional epilepsy, all of whom demonstrated improvement after surgery. The degree of behavioral abnormality was disproportionate to epilepsy severity, suggesting a more complicated mechanism by which lesional epilepsy impacts behavior. We propose a novel paradigm in which lesionectomy may offer behavioral benefit even when seizures are not refractory. Thus, behavioral improvement may be an important novel goal for neurosurgical resection in children with epileptic brain lesions.

Evaluating patient and neurosurgeon perspectives on virtualisation of neurosurgery clinics in the COVID-19 era: a prospective mirror survey study.

BACKGROUND: The COVID-19 pandemic has compelled a global shift in healthcare service delivery towards virtualisation, including in Neurosurgery. Our study aims to elucidate the patient and neurosurgeon perceptions of virtual neurosurgery consultations (VNCs) and formulate a guidance algorithm based on our experience. METHODS: Between June 2020 and December 2020, we prospectively surveyed patients and neurosurgeons following their VNCs using a 10-item survey (four qualitative and six five-point Likert scale questions). Non-parametric hypothesis testing and grounded coding with inter-coder agreement was used to analyse quantitative and qualitative data, respectively. RESULTS: 106 patients and 10 neurosurgeons completed the survey. Wilcoxon rank-sum test revealed a statistically significant difference between the neurosurgeon and patient responses (p < 0.001). Patients perceived VNCs benefits to be enhanced efficiency (n = 142) and communication (28); and VNCs drawbacks as safety (46), technological (32), and administration (15) issues. Neurosurgeons perceived VNCs benefits to be enhanced efficiency (13), reduced COVID-19 exposure (2); and VNCs drawbacks as examination practicality (14), technological (6), and concerns for patients (6). Neurosurgeons perceived the relative indications for VNCs as postoperative follow-up clinics, and scan result discussions; and relative contraindications as neuro-oncology, new patients, and patients with worsening neurological symptoms. CONCLUSIONS: Our mirror-survey study provides preliminary evidence that VNCs render increased efficiency, communication, and safety in the current COVID-19 era. Going forward, we believe that further improvements in technology and administration are necessary, greater neurosurgeon appreciation of the patient-perceived benefits of VNCs is required, and neurosurgeons are to exercise clinical discernment on when to use VNCs.Key PointsWhat are the perceptions of patient and consultant neurosurgeons towards virtual neurosurgery consultations (VNCs)?Patient-perceived benefits of VNCs: enhanced efficiency/communication; VNC drawbacks: safety, technological, and administration issues. Neurosurgeon-perceived VNCs benefits of VNCs: enhanced efficiency, reduced COVID-19 exposure; VNC drawbacks: examination practicality, technological, and concerns for patients.Post-operative reviews and scan result discussions are perceived relative indications for VNCs; whereas new patient consultations, neuro-oncology and patients with new-onset neurological deficits are perceived relative contraindications for VNCs.Improvement in technology and administration is necessary; greater neurosurgeon appreciation of patient-perceived VNCs benefits is required, and neurosurgeons are to exercise clinical discernment on when to use VNCs.

A Low-Power Wireless System for Predicting Early Signs of Sudden Cardiac Arrest Incorporating an Optimized CNN Model Implemented on NVIDIA Jetson.

The survival rate for sudden cardiac arrest (SCA) is low, and patients with long-term risks of SCA are not adequately alerted. Understanding SCA's characteristics will be key to developing preventive strategies. Many lives could be saved if SCA's early onset could be detected or predicted. Monitoring heart signals continuously is essential for diagnosing sporadic cardiac dysfunction. An electrocardiogram (ECG) can be used to continuously monitor heart function without having to go to the hospital. A zeolite-based dry electrode can provide safe on-skin ECG acquisition while the subject is out-of-hospital and facilitate long-term monitoring. To the ECG signal, a low-power 1 µW read-out circuit was designed and implemented in our prior work. However, having long-term ECG monitoring outside the hospital, i.e., high battery life, and low power consumption while transmission and reception of ECG signal are crucial. This paper proposes a prototype with a 10-bit resolution ADC and nRF24L01 transceivers placed 5 m apart. The system uses the 2.4 GHz worldwide ISM frequency band with GFSK modulation to wirelessly transmit digitized ECG bits at 250 kbps data rate to a physician's computer (or similar) for continuous monitoring of ECG signals; the power consumption is only 11.2 mW and 4.62 mW during transmission and reception, respectively, with a low bit error rate of ≤0.1%. Additionally, a subject-wise cross-validated, three-fold, optimized convolutional neural network (CNN) model using the Physionet-SCA dataset was implemented on NVIDIA Jetson to identify the irregular heartbeats yielding an accuracy of 89% with a run time of 5.31 s. Normal beat classification has an F1 score of 0.94 and a ROC score of 0.886. Thus, this paper integrates the ECG acquisition and processing unit with low-power wireless transmission and CNN model to detect irregular heartbeats.

Decanoic acid mitigates ischemia reperfusion injury by modulating neuroprotective, inflammatory and oxidative pathways in middle cerebral artery occlusion model of stroke in rats.

OBJECTIVE: Amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) is an ionotropic transmembrane receptor for glutamate. AMPA receptor blockers have been reported to prevent neurological damage and enhance the post stroke recovery in rats. Decanoic acid, a medium-chain fatty acid, has been reported to exhibit non-competitive AMPA receptor antagonism. This study evaluated the effect of decanoic acid administered before and after ischemia reperfusion injury on neurological damage and post stroke recovery in rats. METHODS: Middle cerebral artery occlusion (MCAo) was performed by using the intraluminal method to induce focal cerebral ischemia. Decanoic acid (120 mg/kg) was administered orally for 1 day (5-10 min post reperfusion) in one group and for 2 days (24 h pre and 5-10 min post reperfusion) in the other group. Effect on neurological damage and post stroke recovery was assessed by neurobehavioral parameters, MRI and TTC staining along with inflammatory, oxidative, apoptotic, and neuroprotective biomarkers. RESULTS: Decanoic acid significantly reduced the MCAo induced neurological damage and infarct size. Decanoic acid treatment increased the motor coordination and grip strength. Furthermore, levels of inflammatory (TNFα, IL-1ß and IL-6), oxidative stress (MDA), apoptotic (TUNEL positive cells) and neurological injury (GFAP) biomarkers were reduced after decanoic acid treatment. Anti-inflammatory cytokine (IL-10) and neuroprotective markers (NT-3, BDNF and TrkB) were found to be significantly increased with decanoic acid treatment. CONCLUSION: This study showed protective effects of decanoic acid against ischemia reperfusion injury in rats. Anti-inflammatory, antioxidant, neuroprotective, and anti-apoptotic properties may be responsible for the beneficial effects of decanoic acid observed in the study.

Protein targeting to starch 1, a functional protein of starch biosynthesis in wheat (Triticum aestivum L.).

KEY MESSAGE: TaPTST1, a wheat homolog of AtPTST1 containing CBM can interact with GBSSI and regulate starch metabolism in wheat endosperm. In cereal endosperm, native starch comprising amylose and amylopectin is synthesized by the coordinated activities of several pathway enzymes. Amylose in starch influences its physio-chemical properties resulting in several human health benefits. The Granule-Bound Starch Synthase I (GBSSI) is the most abundant starch-associated protein. GBSSI lacks dedicated Carbohydrate-binding module (CBM). Previously, Protein Targeting To Starch 1 (PTST1) was identified as a crucial protein for the localization of GBSSI to the starch granules in Arabidopsis. The function of its homologous protein in the wheat endosperm is not known. In this study, TaPTST1, an AtPTST1 homolog, containing a CBM and a coiled-coil domain was identified in wheat. Protein-coding nucleotide sequence of TaPTST1 from Indian wheat variety 'C 306' was cloned and characterized. Homology modelling and molecular docking suggested the potential interaction of TaPTST1 with glucans and GBSSI. The TaPTST1 expression was higher in wheat grain than the other tissues, suggesting a grain-specific function. In vitro binding assays demonstrated different binding affinities of TaPTST1 for native starch, amylose, and amylopectin. Furthermore, the immunoaffinity pull-down assay revealed that TaPTST1 directly interacts with GBSSI, and the interaction is mediated by a coiled-coil domain. The direct protein-protein interaction was further confirmed by bimolecular fluorescence complementation assay (BiFC) in planta. Based on our findings we postulate a functional role for TaPTST1 in starch metabolism by targeting GBSSI to starch granules in wheat endosperm.

Analyzing and Reanalyzing the Genome: Findings from the MedSeq Project.

Although genome sequencing is increasingly available in clinical and research settings, many questions remain about the interpretation of sequencing data. In the MedSeq Project, we explored how much effort is required to evaluate and report on more than 4,500 genes reportedly associated with monogenic conditions, as well as pharmacogenomic (PGx) markers, blood antigen serotyping, and polygenic risk scores in 100 individuals (50 with cardiomyopathy and 50 healthy) randomized to the sequencing arm. We defined the quality thresholds for determining the need for Sanger confirmation. Finally, we examined the effort needed and new findings revealed by reanalyzing each genome (6-23 months after initial analysis; mean 13 months). Monogenic disease risk and carrier status were reported in 21% and 94% of participants, respectively. Only two participants had no monogenic disease risk or carrier status identified. For the PGx results (18 genotypes in six genes for five drugs), the identified diplotypes prompted recommendation for non-standard dosing of at least one of the analyzed drugs in 95% of participants. For blood antigen studies, we found that 31% of participants had a rare blood antigen genotype. In the cardiomyopathy cohort, an explanation for disease was identified in 48% of individuals. Over the course of the study, 14 variants were reclassified and, upon reanalysis, 18 new variants met criteria for reporting. These findings highlight the quantity of medically relevant findings from a broad analysis of genomic sequencing data as well as the need for periodic reinterpretation and reanalysis of data for both diagnostic indications and secondary findings.

A Methyltransferase-Defective Vesicular Stomatitis Virus-Based SARS-CoV-2 Vaccine Candidate Provides Complete Protection against SARS-CoV-2 Infection in Hamsters.

The current pandemic of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to dramatic economic and health burdens. Although the worldwide SARS-CoV-2 vaccination campaign has begun, exploration of other vaccine candidates is needed due to uncertainties with the current approved vaccines, such as durability of protection, cross-protection against variant strains, and costs of long-term production and storage. In this study, we developed a methyltransferase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidate. We generated mtdVSVs expressing SARS-CoV-2 full-length spike (S) protein, S1, or its receptor-binding domain (RBD). All of these recombinant viruses grew to high titers in mammalian cells despite high attenuation in cell culture. The SARS-CoV-2 S protein and its truncations were highly expressed by the mtdVSV vector. These mtdVSV-based vaccine candidates were completely attenuated in both immunocompetent and immunocompromised mice. Among these constructs, mtdVSV-S induced high levels of SARS-CoV-2-specific neutralizing antibodies (NAbs) and Th1-biased T-cell immune responses in mice. In Syrian golden hamsters, the serum levels of SARS-CoV-2-specific NAbs triggered by mtdVSV-S were higher than the levels of NAbs in convalescent plasma from recovered COVID-19 patients. In addition, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 replication in lung and nasal turbinate tissues, cytokine storm, and lung pathology. Collectively, our data demonstrate that mtdVSV expressing SARS-CoV-2 S protein is a safe and highly efficacious vaccine candidate against SARS-CoV-2 infection. IMPORTANCE Viral mRNA cap methyltransferase (MTase) is essential for mRNA stability, protein translation, and innate immune evasion. Thus, viral mRNA cap MTase activity is an excellent target for development of live attenuated or live vectored vaccine candidates. Here, we developed a panel of MTase-defective recombinant vesicular stomatitis virus (mtdVSV)-based SARS-CoV-2 vaccine candidates expressing full-length S, S1, or several versions of the RBD. These mtdVSV-based vaccine candidates grew to high titers in cell culture and were completely attenuated in both immunocompetent and immunocompromised mice. Among these vaccine candidates, mtdVSV-S induces high levels of SARS-CoV-2-specific neutralizing antibodies (Nabs) and Th1-biased immune responses in mice. Syrian golden hamsters immunized with mtdVSV-S triggered SARS-CoV-2-specific NAbs at higher levels than those in convalescent plasma from recovered COVID-19 patients. Furthermore, hamsters immunized with mtdVSV-S were completely protected against SARS-CoV-2 challenge. Thus, mtdVSV is a safe and highly effective vector to deliver SARS-CoV-2 vaccine.

Equine pegiviruses cause persistent infection of bone marrow and are not associated with hepatitis.

Pegiviruses frequently cause persistent infection (as defined by >6 months), but unlike most other Flaviviridae members, no apparent clinical disease. Human pegivirus (HPgV, previously GBV-C) is detectable in 1-4% of healthy individuals and another 5-13% are seropositive. Some evidence for infection of bone marrow and spleen exists. Equine pegivirus 1 (EPgV-1) is not linked to disease, whereas another pegivirus, Theiler's disease-associated virus (TDAV), was identified in an outbreak of acute serum hepatitis (Theiler's disease) in horses. Although no subsequent reports link TDAV to disease, any association with hepatitis has not been formally examined. Here, we characterized EPgV-1 and TDAV tropism, sequence diversity, persistence and association with liver disease in horses. Among more than 20 tissue types, we consistently detected high viral loads only in serum, bone marrow and spleen, and viral RNA replication was consistently identified in bone marrow. PBMCs and lymph nodes, but not liver, were sporadically positive. To exclude potential effects of co-infecting agents in experimental infections, we constructed full-length consensus cDNA clones; this was enabled by determination of the complete viral genomes, including a novel TDAV 3' terminus. Clone derived RNA transcripts were used for direct intrasplenic inoculation of healthy horses. This led to productive infection detectable from week 2-3 and persisting beyond the 28 weeks of study. We did not observe any clinical signs of illness or elevation of circulating liver enzymes. The polyprotein consensus sequences did not change, suggesting that both clones were fully functional. To our knowledge, this is the first successful extrahepatic viral RNA launch and the first robust reverse genetics system for a pegivirus. In conclusion, equine pegiviruses are bone marrow tropic, cause persistent infection in horses, and are not associated with hepatitis. Based on these findings, it may be appropriate to rename the group of TDAV and related viruses as EPgV-2.

Pathogenesis, MicroRNA-122 Gene-Regulation, and Protective Immune Responses After Acute Equine Hepacivirus Infection.

BACKGROUND AND AIMS: Equine hepacivirus (EqHV) is phylogenetically the closest relative of HCV and shares genome organization, hepatotropism, transient or persistent infection outcome, and the ability to cause hepatitis. Thus, EqHV studies are important to understand equine liver disease and further as an outbred surrogate animal model for HCV pathogenesis and protective immune responses. Here, we aimed to characterize the course of EqHV infection and associated protective immune responses. APPROACH AND RESULTS: Seven horses were experimentally inoculated with EqHV, monitored for 6 months, and rechallenged with the same and, subsequently, a heterologous EqHV. Clearance was the primary outcome (6 of 7) and was associated with subclinical hepatitis characterized by lymphocytic infiltrate and individual hepatocyte necrosis. Seroconversion was delayed and antibody titers waned slowly. Clearance of primary infection conferred nonsterilizing immunity, resulting in shortened duration of viremia after rechallenge. Peripheral blood mononuclear cell responses in horses were minimal, although EqHV-specific T cells were identified. Additionally, an interferon-stimulated gene signature was detected in the liver during EqHV infection, similar to acute HCV in humans. EqHV, as HCV, is stimulated by direct binding of the liver-specific microRNA (miR), miR-122. Interestingly, we found that EqHV infection sequesters enough miR-122 to functionally affect gene regulation in the liver. This RNA-based mechanism thus could have consequences for pathology. CONCLUSIONS: EqHV infection in horses typically has an acute resolving course, and the protective immune response lasts for at least a year and broadly attenuates subsequent infections. This could have important implications to achieve the primary goal of an HCV vaccine; to prevent chronicity while accepting acute resolving infection after virus exposure.