Artificial and Natural Sweeteners Biased T1R2/T1R3 Taste Receptors Transactivate Glycosylated Receptors on Cancer Cells to Induce Epithelial-Mesenchymal Transition of Metastatic Phenotype.

Understanding the role of biased taste T1R2/T1R3 G protein-coupled receptors (GPCR) agonists on glycosylated receptor signaling may provide insights into the opposing effects mediated by artificial and natural sweeteners, particularly in cancer and metastasis. Sweetener-taste GPCRs can be activated by several active states involving either biased agonism, functional selectivity, or ligand-directed signaling. However, there are increasing arrays of sweetener ligands with different degrees of allosteric biased modulation that can vary dramatically in binding- and signaling-specific manners. Here, emerging evidence proposes the involvement of taste GPCRs in a biased GPCR signaling crosstalk involving matrix metalloproteinase-9 (MMP-9) and neuraminidase-1 (Neu-1) activating glycosylated receptors by modifying sialic acids. The findings revealed that most natural and artificial sweeteners significantly activate Neu-1 sialidase in a dose-dependent fashion in RAW-Blue and PANC-1 cells. To confirm this biased GPCR signaling crosstalk, BIM-23127 (neuromedin B receptor inhibitor, MMP-9i (specific MMP-9 inhibitor), and oseltamivir phosphate (specific Neu-1 inhibitor) significantly block sweetener agonist-induced Neu-1 sialidase activity. To assess the effect of artificial and natural sweeteners on the key survival pathways critical for pancreatic cancer progression, we analyzed the expression of epithelial-mesenchymal markers, CD24, ADLH-1, E-cadherin, and N-cadherin in PANC-1 cells, and assess the cellular migration invasiveness in a scratch wound closure assay, and the tunneling nanotubes (TNTs) in staging the migratory intercellular communication. The artificial and natural sweeteners induced metastatic phenotype of PANC-1 pancreatic cancer cells to promote migratory intercellular communication and invasion. The sweeteners also induced the downstream NFκB activation using the secretory alkaline phosphatase (SEAP) assay. These findings elucidate a novel taste T1R2/T1R3 GPCR functional selectivity of a signaling platform in which sweeteners activate downstream signaling, contributing to tumorigenesis and metastasis via a proposed NFκB-induced epigenetic reprogramming modeling.

The Biomechanical, Biochemical, and Morphological Properties of 19 Human Cadaveric Lower Limb Tendons and Ligaments: An Open-Access Data Set.

BACKGROUND: Methodological heterogeneity hinders data comparisons across isolated studies of tendon and ligament properties, limiting clinical understanding and affecting the development and evaluation of replacement materials. PURPOSE: To create an open-access data set on the morphological, biomechanical, and biochemical properties of clinically important tendons and ligaments of the lower limb, using consistent methodologies, to enable direct tendon/ligament comparisons. STUDY DESIGN: Descriptive laboratory study. METHODS: Nineteen distinct lower limb tendons and ligaments were retrieved from 8 fresh-frozen human cadavers (5 male, 3 female; aged 49-65 years) including Achilles, tibialis posterior, tibialis anterior, fibularis (peroneus) longus, fibularis (peroneus) brevis, flexor hallucis longus, extensor hallucis longus, plantaris, flexor digitorum longus, quadriceps, patellar, semitendinosus, and gracilis tendons; anterior cruciate, posterior cruciate, medial collateral, and lateral collateral ligaments; and 10 mm-wide grafts from the contralateral quadriceps and patellar tendons. Outcomes included morphology (tissue length, ultrasound-quantified cross-sectional area [CSAUS], and major and minor axes), biomechanics (failure load, ultimate tensile strength [UTS], failure strain, and elastic modulus), and biochemistry (sulfated glycosaminoglycan [sGAG] and hydroxyproline contents). Tissue differences were analyzed using mixed-model regression. RESULTS: There was a range of similarities and differences between tendons and ligaments across outcomes. A key finding relating to potential graft tissue suitability was the comparable failure loads, UTS, CSAUS, sGAG, and hydroxyproline present between hamstring tendons (a standard graft source) and 5 tendons not typically used for grafting: fibularis (peroneus) longus and brevis, flexor and extensor hallucis longus, and flexor digitorum longus tendons. CONCLUSION: This study of lower limb tendons and ligaments has enabled direct comparison of morphological, biomechanical, and biochemical human tissue properties-key factors in the selection of suitable graft tissues. This analysis has identified 6 potential new donor tissues with properties comparable to currently used grafts. CLINICAL RELEVANCE: This extensive data set reduces the need to utilize data from incompatible sources, which may aid surgical decisions (eg, evidence to expand the range of tendons considered suitable for use as grafts) and may provide congruent design inputs for new biomaterials and computational models. The complete data set has been provided to facilitate further investigations, with the capacity to expand the resource to include additional outcomes and tissues.

Broad antibody and T cell responses to Ebola, Sudan, and Bundibugyo ebolaviruses using mono- and multi-valent adjuvanted glycoprotein vaccines.

Currently, there are two approved vaccine regimens designed to prevent Ebola virus (EBOV) disease (EVD). Both are virus-vectored, and concerns about cold-chain storage and pre-existing immunity to the vectors warrant investigating additional vaccine strategies. Here, we have explored the utility of adjuvanted recombinant glycoproteins (GPs) from ebolaviruses Zaire (EBOV), Sudan (SUDV), and Bundibugyo (BDBV) for inducing antibody (Ab) and T cell cross-reactivity. Glycoproteins expressed in insect cells were administered to C57BL/6 mice as free protein or bound to the surface of liposomes, and formulated with toll-like receptor agonists CpG and MPLA (agonists for TLR 9 and 4, respectively), with or without the emulsions AddaVax or TiterMax. The magnitude of Ab cross-reactivity in binding and neutralization assays, and T cell cross-reactivity in antigen recall assays, correlated with phylogenetic relatedness. While most adjuvants screened induced IgG responses, a combination of CpG, MPLA and AddaVax emulsion ("IVAX-1") was the most potent and polarized in an IgG2c (Th1) direction. Breadth was also achieved by combining GPs into a trivalent (Tri-GP) cocktail with IVAX-1, which did not compromise antibody responses to individual components in binding and neutralizing assays. Th1 signature cytokines in T cell recall assays were undetectable after Tri-GP/IVAX-1 administration, despite a robust IgG2c response, although administration of Tri-GP on lipid nanoparticles in IVAX-1 elevated Th1 cytokines to detectable levels. Overall, the data indicate an adjuvanted trivalent recombinant GP approach may represent a path toward a broadly reactive, deployable vaccine against EVD.

Considerations for Glycoprotein Production.

This chapter is intended to provide insights for researchers aiming to choose an appropriate expression system for the production of recombinant glycoproteins. Producing glycoproteins is complex, as glycosylation patterns are determined by the availability and abundance of specific enzymes rather than a direct genetic blueprint. Furthermore, the cell systems often employed for protein production are evolutionarily distinct, leading to significantly different glycosylation when utilized for glycoprotein production. The selection of an appropriate production system depends on the intended applications and desired characteristics of the protein. Whether the goal is to produce glycoproteins mimicking native conditions or to intentionally alter glycan structures for specific purposes, such as enhancing immunogenicity in vaccines, understanding glycosylation present in the different systems and in different growth conditions is essential. This chapter will cover Escherichia coli, baculovirus/insect cell systems, Pichia pastoris, as well as different mammalian cell culture systems including Chinese hamster ovary (CHO) cells, human endothelial kidney (HEK) cell lines, and baby hamster kidney (BHK) cells.

Tendon biomechanical properties are altered by storage duration but not freeze-thaw temperatures or cycles.

Tendon allograft and xenograft processing often involves one or more steps of freezing and thawing. As failure strength is an important graft consideration, this study aimed to evaluate effects on failure properties when varying freeze-thaw conditions. Kangaroo tendons, a potential xenograft source, were used to evaluate changes in ultimate tensile strength (UTS), failure strain and elastic modulus after exposure to different freezer-storage temperatures (-20°C vs. -80°C), storage durations (1, 3, 6, 9, or 12 months), number of freeze-thaw cycles (1, 2, 3, 4, 5, or 10), or freeze-thaw temperature ranges (including freezing in liquid nitrogen to thawing at 37°C). Tendons stored for 6 or more months had significantly increased UTS and elastic modulus compared with 1 or 3 months of storage. This increase occurred irrespective of the freezing temperature (-20°C vs. -80°C) or the number of freeze-thaw cycles (1 vs. 10). In contrast, UTS, failure strain and the elastic modulus were no different between storage temperatures, number of freeze-thaw cycles and multiple freeze-thaw cycles across a range of freeze and thaw temperatures. Common freeze-thaw protocols did not negatively affect failure properties, providing flexibility for graft testing, storage, transportation and decellularisation procedures. However, the change in properties with the overall storage duration has implications for assessing the consistent performance of grafts stored for short versus extended periods of time (<6 months vs. >6 months), and the interpretation of data obtained from tissues of varying or unknown storage durations.

Dorsal Plating, Lateral Plating, and Intramedullary Screw Fixation of Extra-Articular Proximal Phalangeal Fractures: A Cadaveric Biomechanical Comparison.

PURPOSE: To provide a biomechanical comparison of dorsal plating, lateral plating and intramedullary screw [IMS] fixation for extra-articular proximal phalangeal fractures. METHODS: Midshaft osteotomies were performed on 36 cadaveric proximal phalanges. The phalanges were fixed by dorsal plating, lateral plating or IMS fixation, and subjected to a four-point bending force. Force was applied to achieve displacement of 1 mm/s, until construct failure or to a maximum of 10 mm of displacement. Clinical failure was defined as 2 mm of displacement, and force required to result in 1 mm and 2 mm of displacement was recorded, as was mode of failure. RESULTS: Dorsal plating [127.5 N ± 52.6; 46.51-229.17] and lateral plating [77.1 N ± 25.1; 48.3-113.8] required significantly greater force to achieve 1 mm of displacement when compared to IMS [41.2 N ± 12.4; 20.6-62.3]. Dorsal plating [339.2 N ± 91.8; 158.5-538.6] required significantly greater force than lateral plating [154.5 N ± 33.8; 99.0 -204.4] and intramedullary screw fixation [110.0 ± 38.6; 51.1-189.3] to result in 2 mm of displacement. Lateral and dorsal plating constructs failed through plate bending, screw cut-out or plate failure, whilst IMS failed via implant deformity. All three constructs required greater force to result in even 1 mm of displacement than what is likely subjected through rehabilitation via active motion. CONCLUSIONS: Lateral plating and IMS fixation offer sufficient stiffness to withstand the likely forces subjected via early active motion without displacement. CLINICAL RELEVANCE: Dorsal plating required significantly greater force than lateral plating and intramedullary screw fixation to achieve 1 mm of displacement when used in extra-articular proximal phalangeal fractures in an in vitro setting. However, all three modalities confer enough stability to likely withstand the forces associated with active range of motion.

Streamlining quantitative joint-wide medial femoro-tibial histopathological scoring of mouse post-traumatic knee osteoarthritis models.

OBJECTIVES: Histological scoring remains the gold-standard for quantifying post-traumatic osteoarthritis (ptOA) in animal models, allowing concurrent evaluation of numerous joint tissues. Available systems require scoring multiple sections/joint making analysis laborious and expensive. We investigated if a single section allowed equivalent quantitation of pathology in different joint tissues and disease stages, in three ptOA models. METHOD: Male 10-12-week-old C57BL/6 mice underwent surgical medial-meniscal-destabilization, anterior-cruciate-ligament (ACL) transection, non-invasive-ACL-rupture, or served as sham-surgical, non-invasive-ACL-strain, or naïve/non-operated controls. Mice (n = 12/group) were harvested 1-, 4-, 8-, and 16-week post-intervention. Serial sagittal toluidine-blue/fast-green stained sections of the medial-femoro-tibial joint (n = 7/joint, 84 µm apart) underwent blinded scoring of 40 histology-outcomes. We evaluated agreement between single-slide versus entire slide-set maximum or median scores (weighted-kappa), and sensitivity/specificity of single-slide versus median/maximum to detect OA pathology. RESULTS: A single optimal mid-sagittal section showed excellent agreement with median (weighted-kappa 0.960) and maximum (weighted-kappa 0.926) scores. Agreement for individual histology-outcomes was high with only 19/240 median and 15/240 maximum scores having a weighted-kappa ≤0.4, the majority of these (16/19 and 11/15) in control groups. Statistically-significant histology-outcome differences between ptOA models and their controls detected with the entire slide-set were reliably reproduced using a single slide (sensitivity >93.15%, specificity >93.10%). The majority of false-negatives with single-slide scoring were meniscal and subchondral bone histology-outcomes (89%) and occurred in weeks 1-4 post-injury (84%). CONCLUSION: A single mid-sagittal slide reduced the time needed to score diverse histopathological changes by 87% without compromising the sensitivity or specificity of the analysis, across a variety of ptOA models and time-points.

A method for mapping the linear epitopes targeted by the natural antibody response to Zika virus infection using a VLP platform technology.

Zika virus (ZIKV), a mosquito-borne pathogen, is associated with neurological complications in adults and congenital abnormalities in newborns. There are no vaccines or treatments for ZIKV infection. Understanding the specificity of natural antibody responses to ZIKV could help inform vaccine efforts. Here, we used a technology called Deep Sequence-Coupled Biopanning to map the targets of the human antibody responses to ZIKV infection. A bacteriophage virus-like particle (VLP) library displaying overlapping linear peptides derived from the ZIKV polyprotein was generated. The library was panned using IgG from 23 ZIKV-infected patients from Panama and deep sequencing identified common targets of anti-ZIKV antibodies within the ZIKV envelope glycoprotein. These included epitopes within the fusion loop within domain II and four epitopes within domain III. Additionally, we showed that VLPs displaying selected epitopes elicited antibodies that bound to native ZIKV envelope protein but failed to prevent infection in a mouse challenge model.

Challenging the Perceptions of Human Tendon Allografts: Influence of Donor Age, Sex, Height, and Tendon on Biomechanical Properties.

BACKGROUND: The use of allograft tendons has increased for primary and revision anterior cruciate ligament reconstruction, but allograft supply is currently limited to a narrow range of tendons and donors up to the age of 65 years. Expanding the range of donors and tendons could help offset an increasing clinical demand. PURPOSE: To investigate the effects of donor age, sex, height, and specific tendon on the mechanical properties of a range of human lower leg tendons. STUDY DESIGN: Descriptive laboratory study. METHODS: Nine tendons were retrieved from 39 fresh-frozen human cadaveric lower legs (35 donors [13 female, 22 male]; age, 49-99 years; height, 57-85 inches [145-216 cm]) including: Achilles tendon, tibialis posterior and anterior, fibularis longus and brevis, flexor and extensor hallucis longus, plantaris, and flexor digitorum longus. Tendons underwent tensile loading to failure measuring cross-sectional area (CSA), maximum load, strain at failure, ultimate tensile strength, and elastic modulus. Results from 332 tendons were analyzed using mixed-effects linear regression, accounting for donor age, sex, height, and weight. RESULTS: Mechanical properties were significantly different among tendons and were substantially greater than the effects of donor characteristics. Significant effects of donor sex, age, and height were limited to specific tendons: Achilles tendon, tibialis posterior, and tibialis anterior. All other tendons were unaffected. The Achilles tendon was most influenced by donor variables: greater CSA in men (ß = 15.45 mm2; Sidák adjusted P < .0001), decreased maximum load with each year of increased age (ß = -17.20 N per year; adjusted P = .0253), and increased CSA (ß = 1.92 mm2 per inch; adjusted P < .0001) and maximum load (ß = 86.40 N per inch; adjusted P < .0001) with each inch of increased height. CONCLUSION: Mechanical properties vary significantly across different human tendons. The effects of donor age, sex, and height are relatively small, are limited to specific tendons, and affect different tendons uniquely. The findings indicate that age negatively affected only the Achilles tendon (maximum load) and challenge the exclusion of donors aged >65 years across all tendon grafts. CLINICAL RELEVANCE: The findings support including a broader range of tendons for use as allografts for anterior cruciate ligament reconstruction and reviewing the current exclusion criterion of donors aged >65 years.

Rapid on-site molecular Point of Care Testing during influenza outbreaks in aged care facilities improves antiviral use and reduces hospitalisation.

OBJECTIVE: Western Sydney Local Health District (WSLHD) measured the utility and validity of rapid molecular point-of-care testing (POCT) in aged care facilities (ACFs) experiencing influenza-like illness (ILI) outbreaks against routine laboratory testing. METHODS: A descriptive epidemiological study into 82 respiratory outbreaks reported across 63 ACFs within WSLHD supporting approximately 6,500 residents aged ≥65 years and staffed by ∼6,500 employees, from 1 August 2018 to 31 December 2019. RESULTS: WSLHD Public Health Unit performed on-site testing at 27 ACF outbreaks (34%), while 53(66%) ACFs conducted only routine laboratory testing. The Xpert®Xpress Flu/RSV molecular PCR provided a sensitivity and specificity of 100%. Those with on-site testing, antiviral prophylaxis was prescribed at 75% of facilities within 24 hours of testing, as opposed to 32% of those using laboratory testing (p<0.01). There were 24 of 181 ACF residents hospitalised in the POCT group compared to 76 of 357 in the laboratory-only group (OR=0.57; p=0.02). CONCLUSIONS: On-site ACF testing is reliable and practical for early identification of influenza, enabling timely use of antiviral treatment and prophylaxis, and was associated with decreased hospitalisation. PUBLIC HEALTH IMPLICATIONS: Enhanced respiratory surveillance and on-site testing should be strongly considered as part of routine management of respiratory outbreaks in ACFs and may reduce outbreak severity.

Animal models of osteoarthritis : the benefits and costs of reducing variability.

Cite this article: Bone Joint Res 2022;11(8):514-517.

Outbreaks of Human Metapneumovirus in Western Sydney Aged-Care Facilities in 2018.

Background: Human metapneumovirus (hMPV), first described in 2001, is a cause of acute respiratory tract infection in the elderly, with symptoms ranging from mild to severe, including pneumonia, but outbreaks are rarely described. Methods: Between 1 July and 31 December 2018, there were three outbreaks of Influenza-like Illness (ILI) where hMPV was the primary pathogen observed, among 64 aged-care facilities (ACFs) in Western Sydney. These outbreaks were investigated by the Western Sydney Local Health District (WSLHD) Public Health Unit (PHU); multiplex polymerase chain reaction (PCR) testing was performed on nasopharyngeal swabs collected by the ACF. Results: hMPV was the main causative pathogen in three outbreaks (27, 28, and 15 symptomatic cases, respectively) in late winter and early spring. Fifty-five residents and 15 staff cases (70 total cases) were identified; hMPV was detected in 12 of 63 specimens submitted. Of the cases in residents, eight were hospitalised (8/63; 15%), including five with confirmed hMPV and a further one epidemiologically linked to a positive case. Six residents died (6/63; 11%) during the hMPV outbreaks; four of these had laboratory-confirmed hMPV, with a further case epidemiologically linked to a hMPV case, with a primary diagnosis of pneumonia/viral pneumonia. The sixth death was not epidemiologically linked, nor did this case exhibit any respiratory symptoms during the outbreak; however, it was reported in line with public health guidelines. Conclusion: A major challenge in 2018 was the incompleteness of testing for, and awareness of, hMPV as a viral cause of ILI by facilities, laboratories, and emergency departments, which generally opted for rapid testing for influenza and RSV only. There is no licensed vaccine or approved treatment for hMPV, so efficient infection control measures are most important.

Temporal trends in the management of metacarpal and phalangeal fractures in the 21st century: an analysis of Australian population-based data.

BACKGROUND: Fractures of the hand, specifically the metacarpals and phalanges, are a common injury. Whilst many of these fractures can be treated non-operatively, a number of advances have led to the increase in popularity of surgical intervention. The aim of this study was to assess and describe trends in management of phalangeal and metacarpal fractures in Australia over the last two decades. METHODS: A review was conducted of the Medicare Benefits Scheme (MBS), specifically querying the item numbers pertaining to the management of metacarpal and phalanx fractures. Data was recorded as the incidence per 100 000 patients. RESULTS: Overall, there was a statistically significant decrease in the incidence of closed reduction of metacarpal and phalanx fractures, with a converse statistically significant increase in open reduction internal fixation. CONCLUSION: This study demonstrates that over the last 20 years, there has been a decrease in closed reduction of intra- and extra-articular phalangeal and metacarpal fractures, with a converse but smaller increase in open reduction and fixation. These trends are likely multi-factorial in aetiology, and should be monitored to guide resource allocation and health provision in the future.

Long-term Effect of a Single Subcritical Knee Injury: Increasing the Risk of Anterior Cruciate Ligament Rupture and Osteoarthritis.

BACKGROUND: Rupture of the anterior cruciate ligament (ACL) is a well-known risk factor for the development of posttraumatic osteoarthritis (PTOA), but patients with the "same injury" can have vastly different trajectories for the onset and progression of disease. Minor subcritical injuries preceding the critical injury event may drive this disparity through preexisting tissue pathologies and sensory changes. PURPOSE: To investigate the role of subcritical injury on ACL rupture risk and PTOA through the evaluation of pain behaviors, joint mechanics, and tissue structural change in a mouse model of knee injury. STUDY DESIGN: Controlled laboratory study. METHODS: Ten-week-old male C57BL/6J mice were allocated to naïve control and subcritical knee injury groups. Injury was induced by a single mechanical compression to the right hindlimb, and mice were evaluated using joint histopathology, anteroposterior joint biomechanics, pain behaviors (mechanical allodynia and hindlimb weightbearing), and isolated ACL tensile testing to failure at 1, 2, 4, or 8 weeks after injury. RESULTS: Subcritical knee injury produced focal osteochondral lesions in the patellofemoral and lateral tibiofemoral compartments with no resolution for the duration of the study (8 weeks). These lesions were characterized by focal loss of proteoglycan staining, cartilage structural change, chondrocyte pathology, microcracks, and osteocyte cell loss. Injury also resulted in the rapid onset of allodynia (at 1 week), which persisted over time and reduced ACL failure load (P = .006; mean ± SD, 7.91 ± 2.01 N vs 9.37 ± 1.01 N in naïve controls at 8 weeks after injury), accompanied by evidence of ACL remodeling at the femoral enthesis. CONCLUSION: The present study in mice establishes a direct effect of a single subcritical knee injury on the development of specific joint tissue pathologies (osteochondral lesions and progressive weakening of the ACL) and allodynic sensitization. These findings demonstrate a predisposition for secondary critical injuries (eg, ACL rupture) and an increased risk of PTOA onset and progression (structurally and symptomatically). CLINICAL RELEVANCE: Subcritical knee injuries are a common occurrence and, based on this study, can cause persistent sensory and structural change. These findings have important implications for the understanding of risk factors of ACL injury and subsequent PTOA, particularly with regard to prevention and management strategies following an often underreported event.

Sex- and injury-based differences in knee biomechanics in mouse models of post-traumatic osteoarthritis.

Sex and joint injury are risk factors implicated in the onset and progression of osteoarthritis (OA). In mouse models of post-traumatic OA (ptOA), the pathogenesis of disease is notably impacted by sex (often worse in males) and injury model (e.g. meniscal versus ligament injury). Increasing ptOA progression and severity is often associated with greater relative instability of the joint but few studies have directly quantified changes in joint mechanics after injury and compared outcomes across multiple models in both male and female mice. Passive anterior-posterior knee biomechanics were evaluated in 10-week-old, male and female C57BL/6J mice. PtOA injury models included destabilisation of the medial meniscus (DMM), anterior cruciate ligament transection (ACLT) or mechanical rupture (ACLR), and combined DMM and ACLT (DMM + ACLT). Sham operated and non-operated controls (NOC) were included for baseline comparisons. The test apparatus loaded hindlimbs at 60° flexion between ± 1 N at 0.5 mm/s (build specifications available for download: https://doi.org/10.17632/z754455x3c.1). Measures of joint laxity (range of motion, neutral zone) and stiffness were calculated. Joint laxity was comparable between male and female mice while joint stiffness was greater in females (P ≤ 0.002, correcting for body-mass and injury-model). Anterior-posterior joint mechanics were minimally altered by DMM but significantly affected by loss of the ACL (P < 0.001), with equivalent changes between ACL-injury models despite different injury mechanisms and adjacent meniscal damage. These findings suggest that despite the important role of joint injury; sex- and model-specific differences in ptOA progression and severity are not primarily driven by altered anterior-posterior knee biomechanics.

The iminosugars celgosivir, castanospermine and UV-4 inhibit SARS-CoV-2 replication.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic poses an unprecedented challenge for health care and the global economy. Repurposing drugs that have shown promise in inhibiting other viral infections could allow for more rapid dispensation of urgently needed therapeutics. The Spike protein of SARS-CoV-2 is extensively glycosylated with 22 occupied N glycan sites and is required for viral entry. In other glycosylated viral proteins, glycosylation is required for interaction with calnexin and chaperone-mediated folding in the endoplasmic reticulum, and prevention of this interaction leads to unfolded viral proteins and thus inhibits viral replication. As such, we investigated two iminosugars, celgosivir, a prodrug of castanospermine, and UV-4, or N-(9-methoxynonyl)-1-deoxynojirimycin, a deoxynojirimycin derivative. Iminosugars are known inhibitors of the α-glucosidase I and II enzymes and were effective at inhibiting authentic SARS-CoV-2 viral replication in a cell culture system. Celgosivir prevented SARS-CoV-2-induced cell death and reduced viral replication and Spike protein levels in a dose-dependent manner in culture with Vero E6 cells. Castanospermine, the active form of celgosivir, was also able to inhibit SARS-CoV-2, confirming the canonical castanospermine mechanism of action of celgosivir. The monocyclic UV-4 also prevented SARS-CoV-2-induced death and reduced viral replication after 24 h of treatment, although the reduction in viral copies was lost after 48 h. Our findings suggest that iminosugars should be urgently investigated as potential SARS-CoV-2 inhibitors.

Ambroxol and Ciprofloxacin Show Activity Against SARS-CoV2 in Vero E6 Cells at Clinically-Relevant Concentrations.

We studied the activity of a range of weakly basic and moderately lipophilic drugs against SARS CoV2 in Vero E6 cells, using Vero E6 survival, qPCR of viral genome and plaque forming assays. No clear relationship between their weakly basic and hydrophobic nature upon their activity was observed. However, the approved drugs ambroxol and ciprofloxacin showed potent activity at concentrations that are clinically relevant and within their known safety profiles, and so may provide potentially useful agents for preclinical and clinical studies in COVID-19.

The use of mice lacking type I or both type I and type II interferon responses in research on hemorrhagic fever viruses. Part 1: Potential effects on adaptive immunity and response to vaccination.

For more than 20 years, researchers have used laboratory mice lacking type I or both type I and type II interferon (IFN) responses to study viruses that cause hemorrhagic fever (HF) in humans. Whereas immunocompetent mice do not become ill when infected with Ebola, Lassa, dengue and other HF viruses, IFN-deficient mice typically develop severe or fatal disease when inoculated with these pathogens. The ease of employment of these "mouse models" has led to their extensive use in biocontainment laboratories to assess the efficacy of novel vaccines, often without consideration of whether adaptive immune responses in IFN-deficient mice accurately mirror those in humans. Failure to consider these questions may lead to inappropriate expectations of the predictive value of mouse experiments. In two invited articles, we investigate this question. This paper examines how the lack of type I or both type I and type II IFN signaling may affect the development of adaptive immune responses in mice and the outcome of vaccine studies. A second article reviews the published literature on the use of IFN-deficient mice for the assessment of novel vaccines against HF viruses.

Behind the Wheel: Process Evaluation of a Safe-Transport Program for Older Drivers Delivered in a Randomized Controlled Trial.

This process evaluation explores relationships between program outcomes and intervention implementation in a trial evaluating "Behind the Wheel," an education-based safe-transport program for older drivers. Participants (intervention group) were 190 Sydney drivers aged ⩾75 years (M = 80 ± 4years). Process measures included fidelity, dose delivered, and received. Outcomes were self-reported driving regulation and objectively measured driving exposure. Relationships were explored using regression models. Older drivers who took ownership of driving retirement and self-regulation by developing plans were more likely to reduce their weekly driving, (ß = 38 km, 95% confidence interval (CI) = [7.5,68.7]), and night driving (ß = 7 km, 95% CI = [3.5, 10.4]). Drivers of older age (odds ratio [OR] = 1.1/year older, 95% CI = [1.05, 1.3]) had greater odds of developing driving retirement plans. Female drivers (OR = 2.7,95% CI = [1.1, 6.9]), drivers with poorer function (OR = 1.2/5-point decrease on DriveSafe, 95% CI = [1.04, 1.4]), and worse health (OR = 1.2/additional medication, 95% CI = [1.02, 1.5]) had greater odds of developing safe mobility plans. This program had greatest impact with older, lower functioning drivers. A stronger message was delivered and received, as intended, to older drivers with lower function and poorer health. Our logic model can help channel resources to drivers who benefit most.