Combined Molnupiravir and Nirmatrelvir Treatment Improves the Inhibitory Effect on SARS-CoV-2 in Rhesus Macaques

The periodic emergence of SARS-CoV-2 variants of concern (VOCs) with unpredictable clinical severity and ability to escape preexisting immunity emphasizes the continued need for antiviral interventions. Two small molecule inhibitors, molnupiravir (MK-4482), a nucleoside analog, and nirmatrelvir (PF-07321332), a 3C-like protease inhibitor, have each recently been approved as monotherapy for use in high risk COVID-19 patients. As preclinical data are only available for rodent and ferret models, we originally assessed the efficacy of MK-4482 and PF-07321332 alone and then in combination Against infection with the SARS-CoV-2 Delta VOC in the rhesus macaque COVID-19 model. Notably, use of MK-4482 and PF-07321332 in combination improved the individual inhibitory effect of both drugs. Combined treatment resulted in milder disease progression, stronger reduction of virus shedding from mucosal tissues of the upper respiratory tract, stronger reduction of viral replication in the lower respiratory tract, and reduced lung pathology. Our data strongly indicate superiority of combined MK-4482 and PF-07321332 treatment of SARS-CoV-2 infections as demonstrated here in the closest COVID-19 surrogate model. One Sentence SummaryThe combination of molnupiravir and nirmatrelvir inhibits SARS-CoV-2 replication and shedding more effectively than individual treatments in the rhesus macaque model.

SARS-CoV-2 Omicron BA.1 and BA.2 are attenuated in rhesus macaques as compared to Delta

Since the emergence of SARS-CoV-2, five different variants of concern (VOCs) have been identified: Alpha, Beta, Gamma, Delta, and Omicron. Due to confounding factors in the human population, such as pre-existing immunity, comparing severity of disease caused by different VOCs is challenging. Here, we investigate disease progression in the rhesus macaque model upon inoculation with the Delta, Omicron BA.1, and Omicron BA.2 VOCs. Disease severity in rhesus macaques inoculated with Omicron BA.1 or BA.2 was lower than those inoculated with Delta and resulted in significantly lower viral loads in nasal swabs, bronchial cytology brush samples, and lung tissue in rhesus macaques. Cytokines and chemokines were upregulated in nasosorption samples of Delta animals compared to Omicron BA.1 and BA.2 animals. Overall, these data suggests that in rhesus macaques, Omicron replicates to lower levels than the Delta VOC, resulting in reduced clinical disease.

Replicating RNA platform enables rapid response to the SARS-CoV-2 Omicron variant and elicits enhanced protection in naïve hamsters compared to ancestral vaccine

In late 2021, the SARS-CoV-2 Omicron (B.1.1.529) variant of concern (VoC) was reported with many mutations in the viral spike protein that were predicted to enhance transmissibility and allow viral escape of neutralizing antibodies. Within weeks of the first report of B.1.1.529, this VoC has rapidly spread throughout the world, replacing previously circulating strains of SARS-CoV-2 and leading to a resurgence in COVID-19 cases even in populations with high levels of vaccine- and infection-induced immunity. Initial studies have shown that B.1.1.529 is less sensitive to protective antibody conferred by previous infections and vaccines developed against earlier lineages of SARS-CoV-2. The ability of B.1.1.529 to spread even among vaccinated populations has led to a global public health demand for updated vaccines that can confer protection against B.1.1.529. We report here the rapid development of a replicating RNA vaccine expressing the B.1.1.529 spike and show that this B.1.1.529-targeted vaccine is immunogenic in mice and hamsters. Interestingly, we found that mice previously immunized with A.1-specific vaccines failed to elevate neutralizing antibody titers against B.1.1.529 following B.1.1.529-targeted boosting, suggesting pre-existing immunity may impact the efficacy of B.1.1.529-targeted boosters. Furthermore, we found that our B.1.1.529-targeted vaccine provides superior protection compared to the ancestral A.1-targeted vaccine in hamsters challenged with the B.1.1.529 VoC after a single dose of each vaccine. One Sentence SummaryRapidly developed RNA vaccine protects against SARS-CoV-2 Omicron variant

Genetically diverse mouse models of SARS-CoV-2 infection model clinical variation and cytokine responses in COVID-19

Inflammation in response to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection drives severity of coronavirus disease 2019 (COVID-19), with effective versus dysregulated responses influenced by host genetics. To understand mechanisms of inflammation, animal models that reflect genetic diversity and clinical outcomes observed in humans are needed. We report a mouse panel comprising the diverse genetic backgrounds of the Collaborative Cross founder strains crossed to K18-hACE2 transgenic mice that confers high susceptibility to SARS-CoV-2. Infection of CC x K18-hACE2 F1 progeny resulted in a spectrum of weight loss, survival, viral replication kinetics, histopathology, and cytokine profiles, some of which were sex-specific. Importantly, survival was associated with early type I interferon (IFN) expression and a phased proinflammatory response distinct from mice with severe disease. Thus, dynamics of inflammatory responses observed in COVID-19 can be modeled in diverse mouse strains that provide a genetically tractable platform for understanding antiviral immunity and evaluating countermeasures. One Sentence SummaryGenetically diverse mice display a broad spectrum of clinically relevant responses to SARS-CoV-2 infection, reflecting variability in COVID-19 disease.

Antiviral metabolite 3'-Deoxy-3',4'-didehydro-cytidine is detectable in serum and identifies acute viral infections including COVID-19

There is a critical need for improved infectious disease diagnostics to enable rapid case identification in a viral pandemic and support targeted antimicrobial prescribing. Here we use high-resolution liquid chromatography coupled with mass spectrometry to compare the admission serum metabolome of patients attending hospital with a range of viral infections, including SARS-CoV-2, to those with bacterial infections, non-infected inflammatory conditions and healthy controls. We demonstrate for the first time that 3-Deoxy-3,4-didehydro-cytidine (ddhC), a free base of the only known human antiviral small molecule ddhC-triphosphate (ddhCTP), is detectable in serum. ddhC acts as an accurate biomarker for viral infections, generating an area under the receiver operating characteristic curve of 0.954 (95% confidence interval 0.923-0.986) when comparing viral to non-viral cases. Gene expression of viperin, the enzyme responsible for ddhCTP synthesis, is highly correlated with ddhC, providing a biological mechanism for its increase during viral infection. These findings underline a key future diagnostic role of ddhC in the context of pandemic preparedness and antimicrobial stewardship.

Efficacy of methylene blue in a murine model of amlodipine overdose.

INTRODUCTION: Amlodipine overdoses have significant cardiac toxicity and are difficult to treat. Methylene blue has potential as a treatment for overdoses. METHODS: A randomized controlled study of methylene blue as a treatment for amlodipine toxicity was conducted in C57Bl/6 mice. A baseline echocardiography was followed by gavage administration of amlodipine (90 mg/kg). Five minutes after gavage, animals received either vehicle solution (controls) or methylene blue (20 mg/kg) by intra-peritoneal injection. Animals were continuously monitored, and cardiac parameters were acquired every 15 min up to two hours. RESULTS: Only 50% of control animals survived to the two-hour endpoint compared to 83% that received methylene blue. Amlodipine delivery induced significant reduction in left ventricular ejection fraction (EF), fractional shortening (FS), stroke volume (SV), and cardiac output (CO) in the vehicle treated animals relative to animals in the treatment group (p < 0.05 vehicle versus Methylene blue for EF, FS, SV, CO, and HR). DISCUSSION: The amlodipine dose induced cardiotoxicity that were effects were more pronounced in the untreated group. 50% vehicle controls quickly progressed into heart failure (within 90 min of exposure) and did not survive the two h observation endpoint. Distinctly, only one animal from the Methylene blue treatment group did not survive (83% survival) the study. Additionally, the surviving animals from the Methylene blue group displayed significantly higher ejection fraction, fractional shortening, stroke volume, and cardiac output compared to vehicle group, indicating that methylene blue preserved cardiac function. CONCLUSION: In this mouse model of amlodipine overdose, methylene blue decreased cardiac toxicity.

Towards conserving natural diversity: A biotic inventory by observations, specimens, DNA barcoding and high-throughput sequencing methods.

The Kenai National Wildlife Refuge has been given a broad conservation mandate to conserve natural diversity. A prerequisite for fulfilling this purpose is to be able to identify the species and communities that make up that biodiversity. We tested a set of varied methods for inventory and monitoring of plants, birds and terrestrial invertebrates on a grid of 40 sites in a 938 ha study area in the Slikok Creek watershed, Kenai Peninsula, Alaska. We sampled plants and lichens through observation and specimen-based methods. We surveyed birds using bird call surveys on variable circular plots. We sampled terrestrial arthropods by sweep net sampling, processing samples with High Throughput Sequencing methods. We surveyed for earthworms, using the hot mustard extraction method and identified worm specimens by morphology and DNA barcoding. We examined community membership using clustering methods and Nonmetric Multidimensional Scaling. We documented a total of 4,764 occurrences of 984 species and molecular operational taxonomic units: 87 vascular plants, 51 mosses, 12 liverworts, 111 lichens, 43 vertebrates, 663 arthropods, 9 molluscs and 8 annelid worms. Amongst these records, 102 of the arthropod species appeared to be new records for Alaska. We found three non-native species: Deroceras agreste (Linnaeus, 1758) (Stylommatophora: Agriolimacidae), Dendrobaena octaedra (Savigny, 1826) (Crassiclitellata: Lumbricidae) and Heterarthrus nemoratus (Fallén, 1808) (Hymenoptera: Tenthredinidae). Both D. octaedra and H. nemoratus were found at sites distant from obvious human disturbance. The 40 sites were grouped into five community groups: upland mixed forest, black spruce forest, open deciduous forest, shrub-sedge bog and willow. We demonstrated that, at least for a subset of species that could be detected using these methods, we were able to document current species distributions and assemblages in a way that could be efficiently repeated for the purposes of biomonitoring. While our methods could be improved and additional methods and groups could be added, our combination of techniques yielded a substantial portion of the data necessary for fulfilling Kenai National Wildlife Refuge's broad conservation purposes.

Time to surgery for hip fracture patients in a rural orthopaedic referral hospital.

PROBLEM: It is well established that shorter surgical waiting time for hip fracture patients improves outcomes. We identify and quantify time to surgery for hip fracture patients in a rural hospital. DESIGN: Retrospective observational study. SETTING: : A sixty-bed rural referral hospital with an orthopaedic service. Data were collected for 57 patients 50 years and older who had surgery for Muller AO type 31-A and 31-B fractures at Bega Hospital in 2012. KEY MEASURES FOR IMPROVEMENT: Time to surgery from presentation was compared for patients who presented directly to Bega hospital to those that were transferred from a peripheral hospital. STRATEGIES FOR CHANGE: To quantify contributing factors to surgical delay will help identify areas for future improvement. EFFECTS OF CHANGE: Delay to surgery from presentation was significantly greater for transferred patients (58 hours), compared with direct presentations (41 hours). Mean time for patient transfer was 23 hours. Thirty-five per cent of patients had their operation within 36 hours from presentation. LESSONS LEARNT: The time to surgery for most transfer and direct presentation patients fell outside current guidelines. In our geographically large referral network, delay to surgery was significantly influenced by time to transfer. Based on previously published research, surgery for our hip fracture patients should be expedited. We therefore recommend priority transfer for these significantly injured patients and dedicated emergency operating lists to perform this surgery in a timely manner.

Ceramide-Initiated Protein Phosphatase 2A Activation Contributes to Arterial Dysfunction In Vivo.

Prior studies have implicated accumulation of ceramide in blood vessels as a basis for vascular dysfunction in diet-induced obesity via a mechanism involving type 2 protein phosphatase (PP2A) dephosphorylation of endothelial nitric oxide synthase (eNOS). The current study sought to elucidate the mechanisms linking ceramide accumulation with PP2A activation and determine whether pharmacological inhibition of PP2A in vivo normalizes obesity-associated vascular dysfunction and limits the severity of hypertension. We show in endothelial cells that ceramide associates with the inhibitor 2 of PP2A (I2PP2A) in the cytosol, which disrupts the association of I2PP2A with PP2A leading to its translocation to the plasma membrane. The increased association between PP2A and eNOS at the plasma membrane promotes dissociation of an Akt-Hsp90-eNOS complex that is required for eNOS phosphorylation and activation. A novel small-molecule inhibitor of PP2A attenuated PP2A activation, prevented disruption of the Akt-Hsp90-eNOS complex in the vasculature, preserved arterial function, and maintained normal blood pressure in obese mice. These findings reveal a novel mechanism whereby ceramide initiates PP2A colocalization with eNOS and demonstrate that PP2A activation precipitates vascular dysfunction in diet-induced obesity. Therapeutic strategies targeted to reducing PP2A activation might be beneficial in attenuating vascular complications that exist in the context of type 2 diabetes, obesity, and conditions associated with insulin resistance.

Growth, root formation, and nutrient value of triticale plants fertilized with biosolids.

Biosolids are utilized as nutrient rich fertilizer. Little material is available on benefits to forage crops resulting from fertilization with biosolids. This paper aimed to compare the effects of fertilization with biosolids versus commercial nitrogen fertilizer on growth, root formation, and nutrient value of triticale plants in a greenhouse experiment. Per treatment, five pots were seeded with five triticale seeds each. Treatments included a nonfertilized control, fertilization with 100, 200, 300, 400, and 500 ml biosolids per pot, and fertilization with a commercial nitrogen fertilizer at the recommended application rate and at double that rate. Biomass production, root length, root diameter, nitrogen, phosphorus, and potassium concentration were analyzed at harvest. Fertilization with biosolids increased triticale production (P < 0.001); production was similar for the 100 to 400 mL treatments. Root length, nitrogen, and phosphorus concentration increased, and potassium concentration decreased linearly with application rate. At the recommended rate, biomass production was similar between fertilization with biosolids and commercial fertilizer. However, plants fertilized with commercial fertilizer had considerably longer roots (P < 0.001), higher nitrogen concentration (P < 0.05), and lower potassium concentration (P < 0.01) than those fertilized with biosolids. Our results indicate that at the recommended application rate, biomass production was similar between fertilization with biosolids and with commercial nitrogen fertilizer, indicating the value of biosolids fertilization as a potential alternative.