Anthoteibinenes A-E from the Irish Deep-Sea Coral Anthothela grandiflora: An Amination Puzzle.

Chemical investigation of extracts from the Irish deep-sea soft coral Anthothela grandiflora revealed cadinene-like sesquiterpenes, anthoteibinenes A-E, bearing unusual dimethylamine substitution. Structure elucidation was accomplished using 1D/2D NMR spectroscopy and high-resolution mass spectrometry, while NOESY NMR experiments, gauge invariant atomic orbital (GIAO) NMR calculations coupled with DP4+ probabilities measures, and ECD comparisons were incorporated to propose their relative and absolute configurations. Anthoteibinene B (2) exhibited 49% inhibition of respiratory syncytial virus (RSV) at 3.1 µM with no cytotoxicity.

Extracellular ISG15 triggers ISGylation via a type-I interferon independent non-canonical mechanism to regulate host response during virus infection.

Type-I interferons (IFN) induce cellular proteins with antiviral activity. One such protein is Interferon Stimulated Gene 15 (ISG15). ISG15 is conjugated to proteins during ISGylation to confer antiviral activity and regulate cellular activities associated with inflammatory and neurodegenerative diseases and cancer. Apart from ISGylation, unconjugated free ISG15 is also released from cells during various conditions, including virus infection. The role of extracellular ISG15 during virus infection was unknown. We show that extracellular ISG15 triggers ISGylation and acts as a soluble antiviral factor to restrict virus infection via an IFN-independent mechanism. Specifically, extracellular ISG15 acts post-translationally to markedly enhance the stability of basal intracellular ISG15 protein levels to support ISGylation. Furthermore, extracellular ISG15 interacts with cell surface integrin (α5ß1 integrins) molecules via its RGD-like motif to activate the integrin-FAK (Focal Adhesion Kinase) pathway resulting in IFN-independent ISGylation. Thus, our studies have identified extracellular ISG15 protein as a new soluble antiviral factor that confers IFN-independent non-canonical ISGylation via the integrin-FAK pathway by post-translational stabilization of intracellular ISG15 protein.

Consistency in Reporting of Loss of Righting Reflex for Assessment of General Anesthesia in Rats and Mice: A Systematic Review.

General anesthesia induces a reversible loss of consciousness (LOC), a state that is characterized by the inability to feel pain. Identifying LOC in animals poses unique challenges, because the method most commonly used in humans, responding to questions, cannot be used in animals. For over a century, loss of righting reflex (LORR) has been used to assess LOC in animals. This is the only animal method that correlates directly with LOC in humans and has become the standard proxy measure used in research. However, the reporting of how LORR is assessed varies extensively. This systematic literature review examined the consistency and completeness of LORR methods used in rats and mice. The terms 'righting reflex,' 'anesthesia,' 'conscious,' 'rats,' 'mice,' and their derivatives were used to search 5 electronic databases. The abstracts of the 985 articles identified were screened for indications that the study assessed LORR in mice or rats. Full texts of selected articles were reviewed for LORR methodological completeness, with reported methods categorized by 1) animal placement method, 2) behavioral presence of righting reflex, 3) duration of LORR testing, 4) behavioral LORR, and 5) animal position for testing LORR. Only 22 papers reported on all 5 methodological categories. Of the 22 papers, 21 used unique LORR methodologies, with descriptions of LORR methods differing in at least one category as compared with all other studies. This variability indicates that even papers that included all 5 categories still had substantial differences in their methodological descriptions. These findings reveal substantial inconsistencies in LORR methodology and reporting in the biomedical literature likely compromising study replicability and data interpretation.

Analysis of the evolution of COVID-19 disease understanding through temporal knowledge graphs.

The COVID-19 pandemic highlighted two critical barriers hindering rapid response to novel pathogens. These include inefficient use of existing biological knowledge about treatments, compounds, gene interactions, proteins, etc. to fight new diseases, and the lack of assimilation and analysis of the fast-growing knowledge about new diseases to quickly develop new treatments, vaccines, and compounds. Overcoming these critical challenges has the potential to revolutionize global preparedness for future pandemics. Accordingly, this article introduces a novel knowledge graph application that functions as both a repository of life science knowledge and an analytics platform capable of extracting time-sensitive insights to uncover evolving disease dynamics and, importantly, researchers' evolving understanding. Specifically, we demonstrate how to extract time-bounded key concepts, also leveraging existing ontologies, from evolving scholarly articles to create a single temporal connected source of truth specifically related to COVID-19. By doing so, current knowledge can be promptly accessed by both humans and machines, from which further understanding of disease outbreaks can be derived. We present key findings from the temporal analysis, applied to a subset of the resulting knowledge graph known as the temporal keywords knowledge graph, and delve into the detailed capabilities provided by this innovative approach.

The choice of region of interest after spinal procedures alters bone mineral density measurements.

PURPOSE: Vertebrae affected by artifacts, such as metallic implants or bone cement, should be excluded when measuring the spine bone mineral density (BMD) by dual-energy X-ray absorptiometry (DXA). Exclusion may be performed using two methods: first, the affected vertebrae are included in the region of interest (ROI) and subsequently excluded from the analysis; second, the affected vertebrae are completely excluded from the ROI. This study aimed to investigate the influence of metallic implants and bone cement on BMD with and without the inclusion of artifact-affected vertebrae in the ROI. METHODS: DXA images of 285 patients, including 144 with spinal metallic implants and 141 who had undergone spinal vertebroplasty from 2018 to 2021, were retrospectively reviewed. Spine BMD measurements were performed when the images were evaluated using two different ROIs for each patient during the same examination. In the first measurement, the affected vertebrae were included in the ROI; however, the affected vertebrae were excluded from the BMD analysis. In the second measurement, the affected vertebrae were excluded from the ROI. Differences between the two measurements were evaluated using a paired t-test. RESULTS: Among 285 patients (average age, 73 years; 218 women), spinal metallic implants led to an overestimation of bone mass in 40 of 144 patients, whereas bone cement resulted in an underestimation of bone mass in 30 of 141 patients when the first measurement was compared with the second measurement. The opposite effect occurred in 5 and 7 patients, respectively. Differences in results between the inclusion and exclusion of the affected vertebrae in the ROI were statistically significant (p<0.001). Spinal implants or cemented vertebrae included in the ROI might significantly alter BMD measurements. Additionally, different materials were associated with varying modifications in BMD. CONCLUSION: The inclusion of affected vertebrae in the ROI may notably alter BMD measurements, even when they are excluded from the analysis. This study suggests that the vertebrae affected by spinal metallic implants or bone cement should be excluded from the ROI.

Neosuberitenone, a New Sesterterpenoid Carbon Skeleton; New Suberitenones; and Bioactivity against Respiratory Syncytial Virus, from the Antarctic Sponge Suberites sp.

Respiratory syncytial virus (RSV) is a highly contagious human pathogen that poses a significant threat to children under the age of two, and there is a current need for new small molecule treatments. The Antarctic sponge Suberites sp. is a known source of sesterterpenes, and following an NMR-guided fractionation procedure, it was found to produce several previously unreported metabolites. Neosuberitenone (1), with a new carbon scaffold herein termed the 'neosuberitane' backbone, six suberitenone derivatives (2-7), an ansellane-type terpenoid (8), and a highly degraded sesterterpene (9), as well as previously reported suberitenones A (10) and B (11), were characterized. The structures of all of the isolated metabolites including absolute configurations are proposed on the basis of NMR, HRESIMS, optical rotation, and XRD data. The biological activities of the metabolites were evaluated in a range of infectious disease assays. Suberitenones A, B, and F (3) were found to be active against RSV, though, along with other Suberites sp. metabolites, they were inactive in bacterial and fungal screens. None of the metabolites were cytotoxic for J774 macrophages or A549 adenocarcinoma cells. The selectivity of suberitenones A, B, and F for RSV among other infectious agents is noteworthy.

Bioengineered peptibodies as blockers of ion channels.

We engineered and produced an ion channel blocking peptibody, that targets the acetylcholine-activated inwardly rectifying potassium current (IKACh). Peptibodies are chimeric proteins generated by fusing a biologically active peptide with the fragment crystallizable (Fc) region of the human immunoglobulin G (IgG). The IKACh blocking peptibody was engineered as a fusion between the human IgG1 Fc fragment and the IKACh inhibitor tertiapinQ (TP), a 21-amino acid synthetic peptidotoxin, originally isolated from the European honey bee venom. The peptibody was purified from the culture supernatant of human embryonic kidney (HEK) cells transfected with the peptibody construct. We tested the hypothesis that the bioengineered peptibody is bioactive and a potent blocker of IKACh. In HEK cells transfected with Kir3.1 and Kir3.4, the molecular correlates of IKACh, patch clamp showed that the peptibody was ~300-fold more potent than TP. Molecular dynamics simulations suggested that the increased potency could be due to an increased stabilization of the complex formed by peptibody-Kir3.1/3.4 channels compared to tertiapin-Kir3.1/3.4 channels. In isolated mouse myocytes, the peptibody blocked carbachol (Cch)-activated IKACh in atrial cells but did not affect the potassium inwardly rectifying background current in ventricular myocytes. In anesthetized mice, the peptibody abrogated the bradycardic effects of intraperitoneal Cch injection. Moreover, in aged mice, the peptibody reduced the inducibility of atrial fibrillation, likely via blocking constitutively active IKACh. Bioengineered anti-ion channel peptibodies can be powerful and highly potent ion channel blockers, with the potential to guide the development of modulators of ion channels or antiarrhythmic modalities.

Nuclear Transport of Respiratory Syncytial Virus Matrix Protein Is Regulated by Dual Phosphorylation Sites.

Respiratory syncytial virus (RSV) is a major cause of respiratory infections in infants and the elderly. Although the RSV matrix (M) protein has key roles in the nucleus early in infection, and in the cytoplasm later, the molecular basis of switching between the nuclear and cytoplasmic compartments is not known. Here, we show that protein kinase CK2 can regulate M nucleocytoplasmic distribution, whereby inhibition of CK2 using the specific inhibitor 4,5,6,7-tetrabromobenzo-triazole (TBB) increases M nuclear accumulation in infected cells as well as when ectopically expressed in transfected cells. We use truncation/mutagenic analysis for the first time to show that serine (S) 95 and threonine (T) 205 are key CK2 sites that regulate M nuclear localization. Dual alanine (A)-substitution to prevent phosphorylation abolished TBB- enhancement of nuclear accumulation, while aspartic acid (D) substitution to mimic phosphorylation at S95 increased nuclear accumulation. D95 also induced cytoplasmic aggregate formation, implying that a negative charge at S95 may modulate M oligomerization. A95/205 substitution in recombinant RSV resulted in reduced virus production compared with wild type, with D95/205 substitution resulting in an even greater level of attenuation. Our data support a model where unphosphorylated M is imported into the nucleus, followed by phosphorylation of T205 and S95 later in infection to facilitate nuclear export and cytoplasmic retention of M, respectively, as well as oligomerization/virus budding. In the absence of widely available, efficacious treatments to protect against RSV, the results raise the possibility of antiviral strategies targeted at CK2.

Association of possible sarcopenic obesity with osteoporosis and fragility fractures in postmenopausal women.

Possible sarcopenic obese women had a decreased likelihood of osteoporosis but an increased likelihood of fragility fractures compared with non-sarcopenic non-obese and sarcopenia-only women. Furthermore, possible sarcopenic obese women had lower values of trabecular bone score than non-sarcopenic non-obese and sarcopenia-only women. PURPOSE: The coexistence of possible sarcopenia and obesity may have opposing effects on osteoporosis. This study aimed to investigate whether possible sarcopenic obesity is associated with osteoporosis or fragility fracture. METHODS: In this cross-sectional study of 1007 postmenopausal women from Taiwan, bone mineral density of the spine and hips was evaluated using dual-energy X-ray absorptiometry (DXA), and bone microarchitecture was evaluated using the trabecular bone score (TBS) derived from a lumbar spine image acquired by DXA. According to the definition of sarcopenia by the 2019 Asian Working Group for Sarcopenia, possible sarcopenia was defined by either low muscle strength or reduced physical performance. Obesity was defined as a body mass index of ≥ 27 kg/m2. Based on the presence of possible sarcopenia and obesity, study participants were classified as follows: control (non-sarcopenic non-obese), sarcopenic (non-obese), obese (non-sarcopenic), and sarcopenic obese. Prevalent fragility fractures were determined by retrospectively reviewing medical records. RESULTS: In this study, 10.1% of participants were classified as sarcopenic obese, 9.1% as obese, 35.2% as sarcopenic, and 45.6% as control. Relative to the control group, the sarcopenic obese group (OR, 0.28; 95% CI 0.18, 0.46) and obese group (OR, 0.38; 95% CI 0.23, 0.61) had a decreased likelihood of osteoporosis. However, the sarcopenic obese group (OR, 2.29; 95% CI 1.31, 4.00) and obese group (OR, 1.94; 95% CI 1.04, 3.62) had an increased likelihood of fragility fractures than with the control group. In addition, the sarcopenic obese group had a higher likelihood of fragility fractures than the sarcopenic group. Possible sarcopenic obese women also had significantly lower TBS values than those in the control and sarcopenic groups. CONCLUSIONS: Possible sarcopenic obese women had a lower likelihood of osteoporosis but a higher likelihood of fragility fractures than non-sarcopenic non-obese and sarcopenia-only women. Furthermore, possible sarcopenic obese individuals had lower values of TBS than non-sarcopenic non-obese and sarcopenia-only women.

Human Respiratory Syncytial Virus NS2 Protein Induces Autophagy by Modulating Beclin1 Protein Stabilization and ISGylation.

Paramyxoviruses such as respiratory syncytial virus (RSV) are the leading cause of pneumonia in infants, the elderly, and immunocompromised individuals. Understanding host-virus interactions is essential for the development of effective interventions. RSV induces autophagy to modulate the immune response. The viral factors and mechanisms underlying RSV-induced autophagy are unknown. Here, we identify the RSV nonstructural protein NS2 as the virus component mediating RSV-induced autophagy. We show that NS2 interacts and stabilizes the proautophagy mediator Beclin1 by preventing its degradation by the proteasome. NS2 further impairs interferon-stimulated gene 15 (ISG15)-mediated Beclin1 ISGylation and generates a pool of "hypo-ISGylated" active Beclin1 to engage in functional autophagy. Studies with NS2-deficient RSV revealed that NS2 contributes to RSV-mediated autophagy during infection. The present study is the first report to show direct activation of autophagy by a paramyxovirus nonstructural protein. We also report a new viral mechanism for autophagy induction wherein the viral protein NS2 promotes hypo-ISGylation of Beclin1 to ensure availability of active Beclin1 to engage in the autophagy process. IMPORTANCE Understanding host-virus interactions is essential for the development of effective interventions against respiratory syncytial virus (RSV), a paramyxovirus that is a leading cause of viral pneumonia in infants. RSV induces autophagy following infection, although the viral factors involved in this mechanism are unknown. Here, we identify the RSV nonstructural protein 2 (NS2) as the virus component involved in autophagy induction. NS2 promotes autophagy by interaction with and stabilization of the proautophagy mediator Beclin1 and by impairing its ISGylation to overcome autophagy inhibition. To the best of our knowledge, this is the first report of a viral protein regulating the autophagy pathway by modulating ISGylation of autophagy mediators. Our studies highlight a direct role of a paramyxovirus nonstructural protein in activating autophagy by interacting with the autophagy mediator Beclin1. NS2-mediated regulation of the autophagy and ISGylation processes is a novel function of viral nonstructural proteins to control the host response against RSV.

Respiratory Syncytial Virus Matrix Protein-Chromatin Association Is Key to Transcriptional Inhibition in Infected Cells.

The morbidity and mortality caused by the globally prevalent human respiratory pathogen respiratory syncytial virus (RSV) approaches that world-wide of influenza. We previously demonstrated that the RSV matrix (M) protein shuttles, in signal-dependent fashion, between host cell nucleus and cytoplasm, and that this trafficking is central to RSV replication and assembly. Here we analyze in detail the nuclear role of M for the first time using a range of novel approaches, including quantitative analysis of de novo cell transcription in situ in the presence or absence of RSV infection or M ectopic expression, as well as in situ DNA binding. We show that M, dependent on amino acids 110-183, inhibits host cell transcription in RSV-infected cells as well as cells transfected to express M, with a clear correlation between nuclear levels of M and the degree of transcriptional inhibition. Analysis of bacterially expressed M protein and derivatives thereof mutated in key residues within M's RNA binding domain indicates that M can bind to DNA as well as RNA in a cell-free system. Parallel results for point-mutated M derivatives implicate arginine 170 and lysine 172, in contrast to other basic residues such as lysine 121 and 130, as critically important residues for inhibition of transcription and DNA binding both in situ and in vitro. Importantly, recombinant RSV carrying arginine 170/lysine 172 mutations shows attenuated infectivity in cultured cells and in an animal model, concomitant with altered inflammatory responses. These findings define an RSV M-chromatin interface critical for host transcriptional inhibition in infection, with important implications for anti-RSV therapeutic development.

Respiratory Syncytial Virus Phosphoprotein Residue S156 Plays a Role in Regulating Genome Transcription and Replication.

Respiratory syncytial virus (RSV) is a single-stranded, negative-sense RNA virus in the family Pneumoviridae and genus Orthopneumovirus that can cause severe disease in infants, immunocompromised adults, and the elderly. The RSV viral RNA-dependent RNA polymerase (vRdRp) complex is composed of the phosphoprotein (P) and the large polymerase protein (L). The P protein is constitutively phosphorylated by host kinases and has 41 serine (S) and threonine (T) residues as potential phosphorylation sites. To identify important phosphorylation residues in the P protein, we systematically and individually mutated all S and T residues to alanine (A) and analyzed their effects on genome transcription and replication by using a minigenome system. We found that the mutation of eight residues resulted in minigenome activity significantly lower than that of wild-type (WT) P. We then incorporated these mutations (T210A, S203A, T151A, S156A, T160A, S23A, T188A, and T105A) into full-length genome cDNA to rescue recombinant RSV. We were able to recover four recombinant viruses (with T151A, S156A, T160A, or S23A), suggesting that RSV-P residues T210, S203, T188, and T105 are essential for viral RNA replication. Among the four recombinant viruses rescued, rRSV-T160A caused a minor growth defect relative to its parental virus while rRSV-S156A had severely restricted replication due to decreased levels of genomic RNA. During infection, P-S156A phosphorylation was decreased, and when passaged, the S156A virus acquired a known compensatory mutation in L (L795I) that enhanced both WT-P and P-S156A minigenome activity and was able to partially rescue the S156A viral growth defect. This work demonstrates that residues T210, S203, T188, and T105 are critical for RSV replication and that S156 plays a critical role in viral RNA synthesis. IMPORTANCE RSV-P is a heavily phosphorylated protein that is required for RSV replication. In this study, we identified several residues, including P-S156, as phosphorylation sites that play critical roles in efficient viral growth and genome replication. Future studies to identify the specific kinase(s) that phosphorylates these residues can lead to kinase inhibitors and antiviral drugs for this important human pathogen.

Heparan Sulfate Facilitates Spike Protein-Mediated SARS-CoV-2 Host Cell Invasion and Contributes to Increased Infection of SARS-CoV-2 G614 Mutant and in Lung Cancer.

The severe acute respiratory syndrome (SARS)-like coronavirus disease (COVID-19) is caused by SARS-CoV-2 and has been a serious threat to global public health with limited treatment. Cellular heparan sulfate (HS) has been found to bind SARS-CoV-2 spike protein (SV2-S) and co-operate with cell surface receptor angiotensin-converting enzyme 2 (ACE2) to mediate SARS-CoV-2 infection of host cells. In this study, we determined that host cell surface SV2-S binding depends on and correlates with host cell surface HS expression. This binding is required for SARS-Cov-2 virus to infect host cells and can be blocked by heparin lyase, HS antagonist surfen, heparin, and heparin derivatives. The binding of heparin/HS to SV2-S is mainly determined by its overall sulfation with potential, minor contribution of specific SV2-S binding motifs. The higher binding affinity of SV2-S G614 mutant to heparin and upregulated HS expression may be one of the mechanisms underlying the higher infectivity of the SARS-CoV-2 G614 variant and the high vulnerability of lung cancer patients to SARS-CoV-2 infection, respectively. The higher host cell infection by SARS-CoV-2 G614 variant pseudovirus and the increased infection caused by upregulated HS expression both can be effectively blocked by heparin lyase and heparin, and possibly surfen and heparin derivatives too. Our findings support blocking HS-SV2-S interaction may provide one addition to achieve effective prevention and/treatment of COVID-19.

The Impact of the "Osteo" Component of Osteosarcopenia on Fragility Fractures in Post-Menopausal Women.

Osteosarcopenia, the coexistence of bone and muscle loss, is common in older adults, but its definition lacks international consensus. This cross-sectional study (n = 1199 post-menopausal women) aimed to determine the association between osteosarcopenia and fragility fractures and to investigate the impact of the definition of the "osteo" component. Bone mineral density and bone microarchitecture were measured by dual-energy X-ray absorptiometry and the trabecular bone score (TBS), respectively. The "osteo" component of osteosarcopenia was classified as osteoporosis (T-score ≤ -2.5 SD), osteopenia/osteoporosis (T-score < -1 SD), and high-fracture-risk osteopenia (-2.5 SD < T-score < -1 SD)/osteoporosis (T-score ≤ -2.5 SD). The Fracture Risk Assessment Tool was used to identify high-fracture-risk osteopenia. Altogether, 30.3%, 32.2%, 14.4%, and 23.1% of participants had osteosarcopenia, osteoporosis alone, sarcopenia alone, and neither condition, respectively. The odds ratios between osteosarcopenia and fragility fractures were 3.70 (95% CI: 1.94-7.04) for osteosarcopenia, 2.48 (95% CI: 1.30-4.71) for osteoporosis alone, and 1.87 (95% CI: 0.84-4.14) for sarcopenia alone. Women with osteosarcopenia also had lower TBS, indicating worse bone microarchitecture. In conclusion, women with osteosarcopenia were more likely to have previously sustained a fracture compared to those without osteosarcopenia, with sarcopenia alone, and with osteoporosis alone. The relationship between osteosarcopenia and fracture risk may be best identified when considering high-fracture-risk osteopenia and osteoporosis.

Hand grip strength in predicting the risk of osteoporosis in Asian adults.

INTRODUCTION: There is a need for a cost-effective method to identify individuals with a high risk of osteoporosis. This study aimed to investigate the suitability of hand grip strength in predicting the risk of osteoporosis in Asian adults. MATERIALS AND METHODS: In this cross-sectional, hospital-based study of 1007 participants, the bone mineral density of the spine and hips was evaluated using dual-energy X-ray absorptiometry according to the 2019 International Society for Clinical Densitometry official positions. Bone microarchitecture was evaluated using the trabecular bone score, and hand grip strength was measured in the dominant hand using a hand digital dynamometer. RESULTS: Hand grip strength was significantly related to bone density and bone microarchitecture. Moreover, hand grip strength was a significant predictor of osteoporosis in both women and men. For osteoporosis prediction in women, a threshold of 21.9 kg of hand grip strength had a sensitivity of 59%, specificity of 59%, and area under the curve (AUC) of 0.61. In men, a threshold of 28.7 kg had a sensitivity of 66%, specificity of 78%, and AUC of 0.75. The optimal cutoff strengths for osteoporosis depended on age and sex. CONCLUSION: The measurement of hand grip strength is a simple, cost-effective and an easy assessment method for identifying individuals at a high risk of osteoporosis. The cutoff strength for evaluating osteoporosis in adults is age and sex specific.

3-Dimensional Printed Alternative to the Standard Synthetic Flocked Nasopharyngeal Swabs Used for Coronavirus Disease 2019 Testing.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019 (COVID-19), can be detected in respiratory samples by real-time reverse transcriptase polymerase chain reaction (RT-PCR) or other molecular methods. Accessibility of diagnostic testing for COVID-19 has been limited by intermittent shortages of supplies required for testing, including flocked nasopharyngeal (FLNP) swabs. METHODS: We developed a 3-dimensional printed nasopharyngeal (3DP) swab as a replacement of the FLNP swab. The performance of 3DP and FLNP swabs were compared in a clinical trial of symptomatic patients at 3 clinical sites (n = 291) using 3 SARS-CoV-2 emergency use authorization tests: a modified version of the Centers for Disease Control and Prevention (CDC) RT-PCR Diagnostic Panel and 2 commercial automated formats, Roche Cobas and NeuMoDx. RESULTS: The cycle threshold-C(t)-values from the gene targets and the RNase P gene control in the CDC assay showed no significant differences between swabs for both gene targets (P = .152 and P = .092), with the RNase P target performing significantly better in the 3DP swabs (P < .001). The C(t) values showed no significant differences between swabs for both viral gene targets in the Roche cobas assay (P = .05 and P = .05) as well as the NeuMoDx assay (P = .401 and P = .484). The overall clinical correlation of COVID-19 diagnosis between all methods was 95.88% (Kappa 0.901). CONCLUSIONS: The 3DP swabs were equivalent to standard FLNP in 3 testing platforms for SARS-CoV-2. Given the need for widespread testing, 3DP swabs printed onsite are an alternate to FLNP that can rapidly scale in response to acute needs when supply chain disruptions affect availability of collection kits.

A Novel Live Attenuated Respiratory Syncytial Virus Vaccine Candidate with Mutations in the L Protein SAM Binding Site and the G Protein Cleavage Site Is Protective in Cotton Rats and a Rhesus Macaque.

Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infections in children of <5 years of age worldwide, infecting the majority of infants in their first year of life. Despite the widespread impact of this virus, no vaccine is currently available. For more than 50 years, live attenuated vaccines (LAVs) have been shown to protect against other childhood viral infections, offering the advantage of presenting all viral proteins to the immune system for stimulation of both B and T cell responses and memory. The RSV LAV candidate described here, rgRSV-L(G1857A)-G(L208A), contains two modifications: an attenuating mutation in the S-adenosylmethionine (SAM) binding site of the viral mRNA cap methyltransferase (MTase) within the large (L) polymerase protein and a mutation in the attachment (G) glycoprotein that inhibits its cleavage during production in Vero cells, resulting in virus with a "noncleaved G" (ncG). RSV virions containing the ncG have an increased ability to infect primary well-differentiated human bronchial epithelial (HBE) cultures which model the in vivo site of immunization, the ciliated airway epithelium. This RSV LAV candidate is produced efficiently in Vero cells, is highly attenuated in HBE cultures, efficiently induces neutralizing antibodies that are long lasting, and provides protection against an RSV challenge in the cotton rat, without causing enhanced disease. Similar results were obtained in a rhesus macaque.IMPORTANCE Globally, respiratory syncytial virus (RSV) is a major cause of death in children under 1 year of age, yet no vaccine is available. We have generated a novel RSV live attenuated vaccine candidate containing mutations in the L and G proteins. The L polymerase mutation does not inhibit virus yield in Vero cells, the cell type required for vaccine production, but greatly reduces virus spread in human bronchial epithelial (HBE) cultures, a logical in vitro predictor of in vivo attenuation. The G attachment protein mutation reduces its cleavage in Vero cells, thereby increasing vaccine virus yield, making vaccine production more economical. In cotton rats, this RSV vaccine candidate is highly attenuated at a dose of 105 PFU and completely protective following immunization with 500 PFU, 200-fold less than the dose usually used in such studies. It also induced long-lasting antibodies in cotton rats and protected a rhesus macaque from RSV challenge. This mutant virus is an excellent RSV live attenuated vaccine candidate.

Comparing self-assessment, functional, and anthropometric techniques in predicting osteoporosis.

The osteoporosis self-assessment tool was more accurate than hand grip strength, gait speed, and calf circumference in predicting osteoporosis in women. Hand grip strength was more accurate than the osteoporosis self-assessment tool, gait speed, and calf circumference in predicting osteoporosis in men. PURPOSE: The osteoporosis self-assessment tool, functional assessment, and anthropometric measurement are different techniques to identify those at risk of osteoporosis. This study aimed to compare the performance of these techniques in predicting osteoporosis. METHODS: In this cross-sectional, hospital-based study including 1109 participants, the bone mineral density of the spine and hips was evaluated using the dual-energy X-ray absorptiometry. The Osteoporosis Self-Assessment Tool was used as a simple clinical risk assessment tool to screen for osteoporosis. Gait speed and hand grip strength were used as functional assessments to predict osteoporosis. Calf circumference was used as an anthropometric measurement to predict osteoporosis risk. RESULTS: In women, the Osteoporosis Self-Assessment Tool was better than hand grip strength, gait speed, and calf circumference in predicting osteoporosis. In contrast, in men, hand grip strength was better than the Osteoporosis Self-Assessment Tool, gait speed, and calf circumference. CONCLUSION: The application of simple, cost-effective techniques for the identification of osteoporosis risk will be beneficial for both screening and patient care when dual-energy X-ray absorptiometry is not available. We suggest that the Osteoporosis Self-Assessment Tool can be used to identify the risk of osteoporosis in women and hand grip strength measurement can be used for men.

Non-gradient and genotype-dependent patterns of RSV gene expression.

Respiratory syncytial virus (RSV) is a nonsegmented negative-strand RNA virus (NSV) and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3' promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription extending across the genome, with the highest level of mRNA coming from the most promoter-proximal gene, the first nonstructural (NS1) gene, and mRNA levels from subsequent genes dropping until reaching a minimum at the most promoter-distal gene, the polymerase (L) gene. However, recent reports show non-gradient levels of mRNA, with higher than expected levels from the attachment (G) gene. It is unknown to what extent different transcript stabilities might shape measured mRNA levels. It is also unclear whether patterns of RSV gene expression vary, or show strain- or genotype-dependence. To address this, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and in cotton rats infected with RSV isolates belonging to four genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were non-gradient and genotype-specific, where mRNA levels from the G gene exceeded those from the more promoter-proximal nucleocapsid (N) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. Our results indicate that gene expression from a small but diverse set of RSV genotypes is non-gradient and genotype-dependent. We propose novel models of RSV transcription that can account for non-gradient transcription.