A Set of FMRI Quality Control Tools in AFNI: Systematic, in-depth, and interactive QC with afni_proc.py and more.

Quality control (QC) assessment is a vital part of FMRI processing and analysis, and a typically underdiscussed aspect of reproducibility. This includes checking datasets at their very earliest stages (acquisition and conversion) through their processing steps (e.g., alignment and motion correction) to regression modeling (correct stimuli, no collinearity, valid fits, enough degrees of freedom, etc.) for each subject. There are a wide variety of features to verify throughout any single-subject processing pipeline, both quantitatively and qualitatively. We present several FMRI preprocessing QC features available in the AFNI toolbox, many of which are automatically generated by the pipeline-creation tool, afni_proc.py. These items include a modular HTML document that covers full single-subject processing from the raw data through statistical modeling, several review scripts in the results directory of processed data, and command line tools for identifying subjects with one or more quantitative properties across a group (such as triaging warnings, making exclusion criteria, or creating informational tables). The HTML itself contains several buttons that efficiently facilitate interactive investigations into the data, when deeper checks are needed beyond the systematic images. The pages are linkable, so that users can evaluate individual items across a group, for increased sensitivity to differences (e.g., in alignment or regression modeling images). Finally, the QC document contains rating buttons for each "QC block," as well as comment fields for each, to facilitate both saving and sharing the evaluations. This increases the specificity of QC, as well as its shareability, as these files can be shared with others and potentially uploaded into repositories, promoting transparency and open science. We describe the features and applications of these QC tools for FMRI.

Structural basis of paramyxo- and pneumovirus polymerase inhibition by non-nucleoside small-molecule antivirals.

Small-molecule antivirals can be used as chemical probes to stabilize transitory conformational stages of viral target proteins, facilitating structural analyses. Here, we evaluate allosteric pneumo- and paramyxovirus polymerase inhibitors that have the potential to serve as chemical probes and aid the structural characterization of short-lived intermediate conformations of the polymerase complex. Of multiple inhibitor classes evaluated, we discuss in-depth distinct scaffolds that were selected based on well-understood structure-activity relationships, insight into resistance profiles, biochemical characterization of the mechanism of action, and photoaffinity-based target mapping. Each class is thought to block structural rearrangements of polymerase domains albeit target sites and docking poses are distinct. This review highlights validated druggable targets in the paramyxo- and pneumovirus polymerase proteins and discusses discrete structural stages of the polymerase complexes required for bioactivity.

The small molecule inhibitor of SARS-CoV-2 3CLpro EDP-235 prevents viral replication and transmission in vivo.

The COVID-19 pandemic has led to the deaths of millions of people and severe global economic impacts. Small molecule therapeutics have played an important role in the fight against SARS-CoV-2, the virus responsible for COVID-19, but their efficacy has been limited in scope and availability, with many people unable to access their benefits, and better options are needed. EDP-235 is specifically designed to inhibit the SARS-CoV-2 3CLpro, with potent nanomolar activity against all SARS-CoV-2 variants to date, as well as clinically relevant human and zoonotic coronaviruses. EDP-235 maintains potency against variants bearing mutations associated with nirmatrelvir resistance. Additionally, EDP-235 demonstrates a ≥ 500-fold selectivity index against multiple host proteases. In a male Syrian hamster model of COVID-19, EDP-235 suppresses SARS-CoV-2 replication and viral-induced hamster lung pathology. In a female ferret model, EDP-235 inhibits production of SARS-CoV-2 infectious virus and RNA at multiple anatomical sites. Furthermore, SARS-CoV-2 contact transmission does not occur when naïve ferrets are co-housed with infected, EDP-235-treated ferrets. Collectively, these results demonstrate that EDP-235 is a broad-spectrum coronavirus inhibitor with efficacy in animal models of primary infection and transmission.

Anti-SARS-CoV-2 antibodies in a nasal spray efficiently block viral transmission between ferrets.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to spread in the population. We recently reported the production of bovine colostrum-derived antibodies that can neutralize the virus. These have been formulated into a nasal spray. The immunoglobulin preparation is capable of blocking interaction of the trimeric spike protein (Tri S) of SARS-CoV-2 with the cellular receptor angiotensin-converting enzyme 2 (ACE2), entry of a pseudovirus carrying the Tri S into ACE2 over-expressing human embryonic kidney (HEK) cells, and entry of the virus into live Vero E6 cells. Using an ELISA assay, we demonstrate here that this holds true for different SARS-CoV-2 variants of concern. Using the ferret transmission model, we show that the nasal spray formulation of anti-SARS-CoV-2 immunoglobulins efficiently blocks transmission of SARS-CoV-2 from infected to uninfected ferrets. The results indicate that the use of the nasal spray in humans can add an effective additional layer of protection against the virus, and might be applicable for other viruses of the upper respiratory tract.

Design and Execution of In Vitro Polymerase Assays for Measles Virus and Related Mononegaviruses.

Morbilliviruses such as measles virus (MeV) are responsible for major morbidity and mortality worldwide, despite the availability of an effective vaccine and global vaccination campaigns. MeV belongs to the mononegavirus order of viral pathogens that store their genetic information in non-segmented negative polarity RNA genomes. Genome replication and viral gene expression are carried out by a virus-encoded RNA-dependent RNA polymerase (RdRP) complex that has no immediate host cell analog. To better understand the organization and regulation of the viral RdRP and mechanistically characterize antiviral candidates, biochemical RdRP assays have been developed that employ purified recombinant polymerase complexes and synthetic RNA templates to monitor the initiation of RNA synthesis and RNA elongation in vitro. In this article, we will discuss strategies for the efficient expression and preparation of mononegavirus polymerase complexes, provide detailed protocols for the execution and optimization of RdRP assays, evaluate alternative options for the choice of template and detection system, and describe the application of the assay for the characterization of inhibitor candidates. Although MeV RdRP assays are the focus of this article, the general strategies and experimental approaches are readily transferable to related viruses in the mononegavirus order.

A Set of FMRI Quality Control Tools in AFNI: Systematic, in-depth and interactive QC with afni_proc.py and more.

Quality control (QC) assessment is a vital part of FMRI processing and analysis, and a typically under-discussed aspect of reproducibility. This includes checking datasets at their very earliest stages (acquisition and conversion) through their processing steps (e.g., alignment and motion correction) to regression modeling (correct stimuli, no collinearity, valid fits, enough degrees of freedom, etc.) for each subject. There are a wide variety of features to verify throughout any single subject processing pipeline, both quantitatively and qualitatively. We present several FMRI preprocessing QC features available in the AFNI toolbox, many of which are automatically generated by the pipeline-creation tool, afni_proc.py. These items include: a modular HTML document that covers full single subject processing from the raw data through statistical modeling; several review scripts in the results directory of processed data; and command line tools for identifying subjects with one or more quantitative properties across a group (such as triaging warnings, making exclusion criteria or creating informational tables). The HTML itself contains several buttons that efficiently facilitate interactive investigations into the data, when deeper checks are needed beyond the systematic images. The pages are linkable, so that users can evaluate individual items across a group, for increased sensitivity to differences (e.g., in alignment or regression modeling images). Finally, the QC document contains rating buttons for each "QC block", as well as comment fields for each, to facilitate both saving and sharing the evaluations. This increases the specificity of QC, as well as its shareability, as these files can be shared with others and potentially uploaded into repositories, promoting transparency and open science. We describe the features and applications of these QC tools for FMRI.

Tetracistronic Minigenomes Elucidate a Functional Promoter for Ghana Virus and Unveils Cedar Virus Replicase Promiscuity for all Henipaviruses.

Batborne henipaviruses, such as Nipah virus and Hendra virus, represent a major threat to global health due to their propensity for spillover, severe pathogenicity, and high mortality rate in human hosts. Coupled with the absence of approved vaccines or therapeutics, work with the prototypical species and uncharacterized, emergent species is restricted to high biocontainment facilities. There is a scarcity of such specialized spaces for research, and often the scope and capacity of research which can be conducted at BSL-4 is limited. Therefore, there is a pressing need for innovative life-cycle modeling systems to enable comprehensive research within lower biocontainment settings. This work showcases tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, and Ghana virus, which encode viral proteins facilitating budding, fusion, and receptor binding. We validate the functionality of all encoded viral proteins and demonstrate a variety of applications to interrogate the viral life cycle. Notably, we found that the Cedar virus replicase exhibits remarkable promiscuity, efficiently rescuing minigenomes from all tested henipaviruses. We also apply this technology to GhV, an emergent species which has so far not been isolated in culture. We demonstrate that the reported sequence of GhV is incomplete, but that this missing sequence can be substituted with analogous sequences from other henipaviruses. Use of our GhV system establishes the functionality of the GhV replicase and identifies two antivirals which are highly efficacious against the GhV polymerase.

Influenza A virus resistance to 4'-fluorouridine coincides with viral attenuation in vitro and in vivo.

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.

Therapeutic mitigation of measles-like immune amnesia and exacerbated disease after prior respiratory virus infections in ferrets.

Measles cases have surged pre-COVID-19 and the pandemic has aggravated the problem. Most measles-associated morbidity and mortality arises from destruction of pre-existing immune memory by measles virus (MeV), a paramyxovirus of the morbillivirus genus. Therapeutic measles vaccination lacks efficacy, but little is known about preserving immune memory through antivirals and the effect of respiratory disease history on measles severity. We use a canine distemper virus (CDV)-ferret model as surrogate for measles and employ an orally efficacious paramyxovirus polymerase inhibitor to address these questions. A receptor tropism-intact recombinant CDV with low lethality reveals an 8-day advantage of antiviral treatment versus therapeutic vaccination in maintaining immune memory. Infection of female ferrets with influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks pre-CDV causes fatal hemorrhagic pneumonia with lung onslaught by commensal bacteria. RNAseq identifies CDV-induced overexpression of trefoil factor (TFF) peptides in the respiratory tract, which is absent in animals pre-infected with IAV. Severe outcomes of consecutive IAV/CDV infections are mitigated by oral antivirals even when initiated late. These findings validate the morbillivirus immune amnesia hypothesis, define measles treatment paradigms, and identify priming of the TFF axis through prior respiratory infections as risk factor for exacerbated morbillivirus disease.

Hydrogenation of functionalised pyridines with a rhodium oxide catalyst under mild conditions.

Piperidines are one of the most widely used building blocks in the synthesis of pharmaceutical and agrochemical compounds. The hydrogenation of pyridines is a convenient method to synthesise such compounds as it only requires reactant, catalyst, and a hydrogen source. However, this reaction still remains difficult for the reduction of functionalised and multi-substituted pyridines. Here we report the use of a stable, commercially available rhodium compound, Rh2O3, for the reduction of various unprotected pyridines. The reaction only requires mild conditions, and the substrate scope is broad, making it practically useful.

Effects of testosterone on urogenital tract morphology and androgen receptor expression in immature Eastern Fence lizards (Sceloporus undulatus).

In non-avian reptiles, the onset of sexual dimorphism of the major structures of the urogenital tract varies temporally relative to gonadal differentiation, more so than in other amniote lineages. In the current study, we used tonic-release implants to investigate the effects of exogenous testosterone (T) on postnatal development of the urogenital tract in juvenile Eastern Fence Lizards (Sceloporus undulatus) to better understand the mechanisms underlying the ontogeny of sexual differentiation in reptiles. We examined gonads, mesonephric kidneys and ducts (male reproductive tract primordia), paramesonephric ducts (oviduct primordia), sexual segments of the kidneys (SSKs), and hemiphalluses to determine which structures were sexually dimorphic independent of T treatment and which structures exhibited sexually dimorphic responses to T. To better understand tissue-level responsiveness to T treatment, we also characterized androgen receptor (AR) expression by immunohistochemistry. At approximately 4 months after hatching in control animals, gonads were well differentiated but quiescent; paramesonephric ducts had fully degenerated in males; mesonephric kidneys, mesonephric ducts, and SSKs remained sexually undifferentiated; and hemiphalluses could not be everted in either sex. Exogenous T caused enlargement, regionalization, and secretory activity of the mesonephric ducts and SSKs in both sexes; enlargement and regionalization of the oviducts in females; and enlargement of male hemipenes. The most responsive tissues exhibited moderate but diffuse staining for AR in control lizards and intense nuclear staining in T-treated lizards, suggestive of autoregulation of AR. The similarity between sexes in the responsiveness of the mesonephric ducts and SSK to T indicates an absence of sexually dimorphic organizational effects in these structures prior to treatment, which was initiated approximately 2 months after hatching. In contrast, the sex-specific responses in oviducts and hemipenes indicate that significant organization and/or differentiation had taken place prior to treatment.

Influenza A virus resistance to 4'-fluorouridine coincides with viral attenuation in vitro and in vivo.

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD 50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU. Author Summary: Reduced sensitivity to FDA-approved influenza drugs is a major obstacle to effective antiviral therapy. We have previously demonstrated oral efficacy of a novel clinical candidate drug, 4'-FlU, against seasonal, pandemic, and highly pathogenic avian influenza viruses. In this study, we have determined possible routes of influenza virus escape from 4'-FlU and addressed whether resistance imposes a viral fitness penalty, affecting pathogenicity or ability to transmit. We identified three distinct clusters of mutations that lead to moderately reduced viral sensitivity to the drug. Testing of resistant variants against two chemically unrelated nucleoside analog inhibitors of influenza virus, conditionally approved favipiravir and the broad-spectrum SARS-CoV-2 drug molnupiravir, revealed cross-resistance of one cluster with favipiravir, whereas no viral escape from molnupiravir was noted. We found that the resistant variants are severely attenuated in mice, impaired in their ability to invade the lower respiratory tract and cause viral pneumonia in ferrets, and transmission-defective or compromised. We could fully mitigate lethal infection of mice with the resistant variants with standard or 5-fold elevated oral dose of 4'-FlU. These results demonstrate that partial viral escape from 4'-FlU is feasible in principle, but escape mutation clusters are unlikely to reach clinical significance or persist in circulating influenza virus strains.

Design and Optimization of Novel Competitive, Non-peptidic, SARS-CoV-2 Mpro Inhibitors.

The SARS-CoV-2 main protease (Mpro) has been proven to be a highly effective target for therapeutic intervention, yet only one drug currently holds FDA approval status for this target. We were inspired by a series of publications emanating from the Jorgensen and Anderson groups describing the design of potent, non-peptidic, competitive SARS-CoV-2 Mpro inhibitors, and we saw an opportunity to make several design modifications to improve the overall pharmacokinetic profile of these compounds without losing potency. To this end, we created a focused virtual library using reaction-based enumeration tools in the Schrödinger suite. These compounds were docked into the Mpro active site and subsequently prioritized for synthesis based upon relative binding affinity values calculated by FEP+. Fourteen compounds were selected, synthesized, and evaluated both biochemically and in cell culture. Several of the synthesized compounds proved to be potent, competitive Mpro inhibitors with improved metabolic stability profiles.

Comparing molnupiravir and nirmatrelvir/ritonavir efficacy and the effects on SARS-CoV-2 transmission in animal models.

Therapeutic options against SARS-CoV-2 are underutilized. Two oral drugs, molnupiravir and paxlovid (nirmatrelvir/ritonavir), have received emergency use authorization. Initial trials suggested greater efficacy of paxlovid, but recent studies indicated comparable potency in older adults. Here, we compare both drugs in two animal models; the Roborovski dwarf hamster model for severe COVID-19-like lung infection and the ferret SARS-CoV-2 transmission model. Dwarf hamsters treated with either drug survive VOC omicron infection with equivalent lung titer reduction. Viral RNA copies in the upper respiratory tract of female ferrets receiving 1.25 mg/kg molnupiravir twice-daily are not significantly reduced, but infectious titers are lowered by >2 log orders and direct-contact transmission is stopped. Female ferrets dosed with 20 or 100 mg/kg nirmatrelvir/ritonavir twice-daily show 1-2 log order reduction of viral RNA copies and infectious titers, which correlates with low nirmatrelvir exposure in nasal turbinates. Virus replication resurges towards nirmatrelvir/ritonavir treatment end and virus transmits efficiently (20 mg/kg group) or partially (100 mg/kg group). Prophylactic treatment with 20 mg/kg nirmatrelvir/ritonavir does not prevent spread from infected ferrets, but prophylactic 5 mg/kg molnupiravir or 100 mg/kg nirmatrelvir/ritonavir block productive transmission. These data confirm reports of similar efficacy in older adults and inform on possible epidemiologic benefit of antiviral treatment.

Species differences in hormonally mediated gene expression underlie the evolutionary loss of sexually dimorphic coloration in Sceloporus lizards.

Phenotypic sexual dimorphism often involves the hormonal regulation of sex-biased expression for underlying genes. However, it is generally unknown whether the evolution of hormonally mediated sexual dimorphism occurs through upstream changes in tissue sensitivity to hormone signals, downstream changes in responsiveness of target genes, or both. Here, we use comparative transcriptomics to explore these possibilities in 2 species of Sceloporus lizards exhibiting different patterns of sexual dichromatism. Sexually dimorphic S. undulatus develops blue and black ventral coloration in response to testosterone, while sexually monomorphic S. virgatus does not, despite exhibiting similar sex differences in circulating testosterone levels. We administered testosterone implants to juveniles of each species and used RNAseq to quantify gene expression in ventral skin. Transcriptome-wide responses to testosterone were stronger in S. undulatus than in S. virgatus, suggesting species differences in tissue sensitivity to this hormone signal. Species differences in the expression of genes for androgen metabolism and sex hormone-binding globulin were consistent with this idea, but expression of the androgen receptor gene was higher in S. virgatus, complicating this interpretation. Downstream of androgen signaling, we found clear species differences in hormonal responsiveness of genes related to melanin synthesis, which were upregulated by testosterone in S. undulatus, but not in S. virgatus. Collectively, our results indicate that hormonal regulation of melanin synthesis pathways contributes to the development of sexual dimorphism in S. undulatus, and that changes in the hormonal responsiveness of these genes in S. virgatus contribute to the evolutionary loss of ventral coloration.

Metagenomics-enabled reverse-genetics assembly and characterization of myotis bat morbillivirus.

Morbilliviruses are among the most contagious viral pathogens of mammals. Although previous metagenomic surveys have identified morbillivirus sequences in bats, full-length morbilliviruses from bats are limited. Here we characterize the myotis bat morbillivirus (MBaMV) from a bat surveillance programme in Brazil, whose full genome was recently published. We demonstrate that the fusion and receptor binding protein of MBaMV utilize bat CD150 and not human CD150, as an entry receptor in a mammalian cell line. Using reverse genetics, we produced a clone of MBaMV that infected Vero cells expressing bat CD150. Electron microscopy of MBaMV-infected cells revealed budding of pleomorphic virions, a characteristic morbillivirus feature. MBaMV replication reached 103-105 plaque-forming units ml-1 in human epithelial cell lines and was dependent on nectin-4. Infection of human macrophages also occurred, albeit 2-10-fold less efficiently than measles virus. Importantly, MBaMV is restricted by cross-neutralizing human sera elicited by measles, mumps and rubella vaccination and is inhibited by orally bioavailable polymerase inhibitors in vitro. MBaMV-encoded P/V genes did not antagonize human interferon induction. Finally, we show that MBaMV does not cause disease in Jamaican fruit bats. We conclude that, while zoonotic spillover into humans may theoretically be plausible, MBaMV replication would probably be controlled by the human immune system.

Mechanisms underlying the antiarrhythmic effect of ARumenamide-787 in experimental models of the J wave syndromes and hypothermia.

BACKGROUND: Brugada (BrS) and early repolarization syndromes (ERS), the so-called J wave syndromes (JWS), are associated with life-threatening ventricular arrhythmias. Pharmacologic approaches to therapy are currently limited. In this study, we examine the effects of ARumenamide-787 (AR-787) to suppress the electrocardiographic and arrhythmic manifestations of JWS and hypothermia. METHODS: We studied the effects of AR-787 on INa and IKr in HEK-293 cells stably expressing the α- and ß1-subunits of the cardiac (NaV1.5) sodium channel and hERG channel, respectively. In addition, we studied its effect on Ito, INa and ICa in dissociated canine ventricular myocytes along with action potentials and ECG from coronary-perfused right (RV) and left (LV) ventricular wedge preparations. The Ito agonist, NS5806 (5-10 µM), ICa blocker, verapamil (2.5 µM), and INa blocker, ajmaline (2.5 µM), were used to mimic the genetic defects associated with JWS and to induce the electrocardiographic and arrhythmic manifestations of JWS (prominent J waves/ST segment elevation, phase 2 reentry and polymorphic VT/VF) in canine ventricular wedge preparations. RESULTS: AR-787 (1, 10 and 50 µM) exerted pleiotropic effects on cardiac ion channels. The predominant effect was inhibition of the transient outward current (Ito) and enhancement of the sodium channel current (INa), with lesser effects to inhibit IKr and augment calcium channel current (ICa). AR-787 diminished the electrocardiographic J wave and prevented and/or suppressed all arrhythmic activity in canine RV and LV experimental models of BrS, ERS and hypothermia. CONCLUSIONS: Our findings point to AR-787 as promising candidate for the pharmacologic treatment of JWS and hypothermia.

Copper-Catalyzed Racemization-Recycle of a Quaternary Center and Optimization Using a Combined Kinetics-DoE/MLR Modeling Approach.

The copper-catalyzed racemization of a complex, quaternary center of a key intermediate on route to lanabecestat has been identified. Optimization and mechanistic understanding were achieved through the use of an efficient, combined kinetic-multiple linear regression approach to experimental design and modeling. The use of a definitive screening design with mechanistically relevant factors and a mixture of fitted kinetic descriptors and empirical measurements facilitated the generation of a model that accurately predicted complex reaction time course behavior. The synergistic model was used to minimize the formation of dimer byproducts, determine optimal conditions for batch operation, and highlight superheated conditions that could be accessed in flow, leading to a further increase in yield which was predicted by the original model.

4'-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses.

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4'-Fluorouridine (4'-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4'-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar to sub-micromolar potency in HAE cells. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4'-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4'-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time window for effective intervention. Therapeutic oral 4'-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pdmCa09. Recoverees were protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4'-FlU and supports 4'-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.

Inadvertent, Self-Induced Pneumoparotitis Provoking Pneumomediastinum.

Pneumoparotid refers to the presence of air within the parotid gland and pneumoparotitis indicates overlying inflammation or infection. Several physiologic mechanisms exist to prevent the reflux of air and oral contents into the parotid gland, however, these safeguards can be overcome by high intraoral pressures, thus provoking pneumoparotid. Whereas the relationship between pneumomediastinum and air dissecting up into cervical tissues is well understood, the relationship between pneumoparotitis and free air traveling downwards through contiguous structures within the mediastinum is less defined. We present a case of a gentleman who experienced the sudden onset of facial swelling and crepitus in the context of inflating an air mattress with his mouth, who was ultimately found to have pneumoparotid with consequent pneumomediastinum. Discussion of this unusual presentation is important to facilitate recognition and treatment of this uncommon pathology.