Twelve-Month Follow-up of the Immune Response After COVID-19 Vaccination in Patients with Genitourinary Cancers: A Prospective Cohort Analysis.

BACKGROUND: Vaccinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have had a transformative impact on morbidity and mortality. However, the long-term impact of vaccination on patients with genitourinary cancers is currently unknown. MATERIALS AND METHODS: This study aimed to assess seroconversion rates in patients with genitourinary cancers receiving COVID-19 vaccination. Patients with prostate cancer, renal cell carcinoma, or urothelial cancer who had not been vaccinated for COVID-19 were included. Blood samples were obtained at baseline and after 2, 6, and 12 months of one dose of an FDA-approved COVID-19 vaccine. Antibody titer analysis was performed using the SCoV-2 Detect IgG ELISA assay, and the results were reported as immune status ratio (ISR). A paired t-test was used for comparison of ISR values between timepoints. In addition, T-cell receptor (TCR) sequencing was performed to assess for differences in TCR repertoire 2 months after vaccination. RESULTS: Out of 133 patients enrolled, 98 baseline blood samples were collected. At 2-, 6-, and 12-month time points 98, 70, and 50 samples were collected, respectively. Median age was 67 (IQR, 62-75), with the majority of patients diagnosed with prostate (55.1%) or renal cell carcinoma (41.8%). Compared to baseline (0.24 [95% CI, 0.19-0.31]) a significant increase in the geometric mean ISR values was observed at the 2-month timepoint (5.59 [4.76-6.55]) (P < .001). However, at the 6-month timepoint, a significant decrease in the ISR values was observed (4.66 [95% CI, 4.04-5.38]; P < .0001). Notably, at the 12-month timepoint, the addition of a booster dose resulted in an absolute increase in the ISR values compared to those who did not receive a booster dose (P = .04). CONCLUSIONS: Only a minority of patients with genitourinary cancers did not ultimately achieve satisfactory seroconversion after receiving commercial COVID-19 vaccination. Cancer type or treatment rendered did not appear to affect the immune response mounted after vaccination.

The K18-Human ACE2 Transgenic Mouse Model Recapitulates Non-severe and Severe COVID-19 in Response to an Infectious Dose of the SARS-CoV-2 Virus.

A comprehensive analysis and characterization of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection model that mimics non-severe and severe coronavirus disease 2019 (COVID-19) in humans is warranted for understating the virus and developing preventive and therapeutic agents. Here, we characterized the K18-hACE2 mouse model expressing human (h)ACE2 in mice, controlled by the human keratin 18 (K18) promoter, in the epithelia, including airway epithelial cells where SARS-CoV-2 infections typically start. We found that intranasal inoculation with higher viral doses (2 × 103 and 2 × 104 PFU) of SARS-CoV-2 caused lethality of all mice and severe damage of various organs, including lung, liver, and kidney, while lower doses (2 × 101 and 2 × 102 PFU) led to less severe tissue damage and some mice recovered from the infection. In this hACE2 mouse model, SARS-CoV-2 infection damaged multiple tissues, with a dose-dependent effect in most tissues. Similar damage was observed in postmortem samples from COVID-19 patients. Finally, the mice that recovered from infection with a low dose of virus survived rechallenge with a high dose of virus. Compared to other existing models, the K18-hACE2 model seems to be the most sensitive COVID-19 model reported to date. Our work expands the information available about this model to include analysis of multiple infectious doses and various tissues with comparison to human postmortem samples from COVID-19 patients. In conclusion, the K18-hACE2 mouse model recapitulates both severe and non-severe COVID-19 in humans being dose-dependent and can provide insight into disease progression and the efficacy of therapeutics for preventing or treating COVID-19. IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19) has reached nearly 240 million cases, caused nearly 5 million deaths worldwide as of October 2021, and has raised an urgent need for the development of novel drugs and therapeutics to prevent the spread and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, an animal model that recapitulates the features of human COVID-19 disease progress and pathogenesis is greatly needed. In this study, we have comprehensively characterized a mouse model of SARS-CoV-2 infection using K18-hACE2 transgenic mice. We infected the mice with low and high doses of SARS-CoV-2 to study the pathogenesis and survival in response to different infection patterns. Moreover, we compared the pathogenesis of the K18-hACE2 transgenic mice with that of the COVID-19 patients to show that this model could be a useful tool for the development of antiviral drugs and therapeutics.

Selecting Counterions to Improve Ionized Hydrophilic Drug Encapsulation in Polymeric Nanoparticles.

Hydrophobic ion pairing (HIP) can successfully increase the drug loading and control the release kinetics of ionizable hydrophilic drugs, addressing challenges that prevent these molecules from reaching the clinic. Nevertheless, polymeric nanoparticle (PNP) formulation development requires trial-and-error experimentation to meet the target product profile, which is laborious and costly. Herein, we design a preformulation framework (solid-state screening, computational approach, and solubility in PNP-forming emulsion) to understand counterion-drug-polymer interactions and accelerate the PNP formulation development for HIP systems. The HIP interactions between a small hydrophilic molecule, AZD2811, and counterions with different molecular structures were investigated. Cyclic counterions formed amorphous ion pairs with AZD2811; the 0.7 pamoic acid/1.0 AZD2811 complex had the highest glass transition temperature (Tg; 162 °C) and the greatest drug loading (22%) and remained as phase-separated amorphous nanosized domains inside the polymer matrix. Palmitic acid (linear counterion) showed negligible interactions with AZD2811 (crystalline-free drug/counterion forms), leading to a significantly lower drug loading despite having similar log P and pKa with pamoic acid. Computational calculations illustrated that cyclic counterions interact more strongly with AZD2811 than linear counterions through dispersive interactions (offset π-π interactions). Solubility data indicated that the pamoic acid/AZD2811 complex has a lower organic phase solubility than AZD2811-free base; hence, it may be expected to precipitate more rapidly in the nanodroplets, thus increasing drug loading. Our work provides a generalizable preformulation framework, complementing traditional performance-indicating parameters, to identify optimal counterions rapidly and accelerate the development of hydrophilic drug PNP formulations while achieving high drug loading without laborious trial-and-error experimentation.

Genomic characterization of Parengyodontium americanum sp. nov.

Modern genome analysis and phylogenomic methods have increased the number of fungal species, as well as enhanced appreciation of the degree of diversity within the fungal kingdom. In this context, we describe a new Parengyodontium species, P. americanum, which is phylogenetically related to the opportunistic human fungal pathogen P. album. Five unusual fungal isolates were recovered from five unique and confirmed coccidioidomycosis patients, and these isolates were subsequently submitted to detailed molecular and morphological identification procedures to determine identity. Molecular and morphological diagnostic analyses showed that the isolates belong to the Cordycipitaceae. Subsequently, three representative genomes were sequenced and annotated, and a new species, P. americanum, was identified. Using various genomic analyses, gene family expansions related to novel compounds and potential for ability to grow in diverse habitats are predicted. A general description of the genomic composition of this newly described species and comparison of genome content with Beauveria bassiana, Isaria fumosorosea and Cordyceps militaris shows a shared core genome of 6371 genes, and 148 genes that appear to be specific for P. americanum. This work provides the framework for future investigations of this interesting fungal species.

Characterizing in vitro spherule morphogenesis of multiple strains of both species of Coccidioides.

The disease San Joaquin Valley Fever (coccidioidomycosis) is caused by the inhalation of Coccidioides arthroconidia. In vivo, arthroconidia transform into pathogenic structures termed spherules. Exposure to the host milieu triggers spherule development; however, the molecular mechanisms responsible for the morphological shift are not well characterized. This study compared the morphogenesis of five strains of both species of Coccidioides in two media types to improve the in vitro model of dimorphism that can be easily reproduced, and is amenable to tissue culture. We also sought to establish a modern record of the morphological switch among commonly used lab strains through a detailed account of growth under various conditions. Spherules from five strains were grown in standard (Converse) and experimental media (RPMI-sph). Strain behavior was quantified by median spherule size and spherule concentration, beginning 3 days after inoculation and followed for 10 days of growth. There were significant differences observed among Coccidioides immitis and C. posadasii strains, as well as differences between the in vitro systems.

In vitro small molecule screening to inform novel candidates for use in fluconazole combination therapy in vivo against Coccidioides.

Identifying improved treatments for severe and refractory coccidioidomycosis (Valley fever) is needed. This endemic fungal disease is common in North and South America, and cases have increased substantially over the last 30 years. The current standard of care, oral daily fluconazole, often fails to completely eradicate Coccidioides infection; however, the high cost of identifying new compounds effective in treating Valley fever is a barrier to improving treatment. Therefore, repurposing existing pharmaceutical agents in combination with fluconazole therapy is an attractive option. We screened the Library of Pharmacologically Active Compounds (LOPAC) small molecule library for compounds that inhibited fungal growth in vitro and determined IC50 values for a subset of compounds. Based on these findings, we tested a small subset of these agents to validate the screen, as well as to test the performance of fluconazole in a combination therapy approach, as compared with fluconazole alone, in a murine model. We observed that combination therapy of tamoxifen:fluconazole and sertraline:fluconazole significantly reduced the burden of live fungus in the lung compared with fluconazole alone, and we observed reduced or nonexistent dissemination. These results suggest that tamoxifen and sertraline may be repurposed as adjunctive agents in the treatment of this important fungal disease. IMPORTANCE: Developing new drugs, especially for regional orphan diseases, such as Valley Fever, is a slow and costly endeavor. However, there is a wealth of FDA-approved drugs available for repurposing, offering a more economical and expedited approach to improve treatment. Those existing compounds with antifungal properties can become novel therapies with relative ease: a considerable advantage for patients in need of alternative treatment. Despite the scope of remaining tasks, our comprehensive screening of potential candidates has revealed promising combinations for further exploration. This effort outlines a practical pipeline for Valley fever drug screening and identifies viable drug combinations that could impact patients more rapidly than single drug development pathways.

Recurrent SARS-CoV-2 mutations at Spike D796 evade antibodies from pre-Omicron convalescent and vaccinated subjects.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lineages of the Omicron variant rapidly became dominant in early 2022 and frequently cause human infections despite vaccination or prior infection with other variants. In addition to antibody-evading mutations in the receptor-binding domain, Omicron features amino acid mutations elsewhere in the Spike protein; however, their effects generally remain ill defined. The Spike D796Y substitution is present in all Omicron sub-variants and occurs at the same site as a mutation (D796H) selected during viral evolution in a chronically infected patient. Here, we map antibody reactivity to a linear epitope in the Spike protein overlapping position 796. We show that antibodies binding this region arise in pre-Omicron SARS-CoV-2 convalescent and vaccinated subjects but that both D796Y and D796H abrogate their binding. These results suggest that D796Y contributes to the fitness of Omicron in hosts with pre-existing immunity to other variants of SARS-CoV-2 by evading antibodies targeting this site.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved substantially through the coronavirus disease 2019 (COVID-19) pandemic: understanding the drivers and consequences of this evolution is essential for projecting the course of the pandemic and developing new countermeasures. Here, we study the immunological effects of a particular mutation present in the Spike protein of all Omicron strains and find that it prevents the efficient binding of a class of antibodies raised by pre-Omicron vaccination and infection. These findings reveal a novel consequence of a poorly understood Omicron mutation and shed light on the drivers and effects of SARS-CoV-2 evolution.

Inferring the composition of a mixed culture of natural microbial isolates by deep sequencing.

Next generation sequencing has unlocked a wealth of genotype information for microbial populations, but phenotyping remains a bottleneck for exploiting this information, particularly for pathogens that are difficult to manipulate. Here, we establish a method for high-throughput phenotyping of mixed cultures, in which the pattern of naturally occurring single-nucleotide polymorphisms in each isolate is used as intrinsic barcodes which can be read out by sequencing. We demonstrate that our method can correctly deconvolute strain proportions in simulated mixed-strain pools. As an experimental test of our method, we perform whole genome sequencing of 66 natural isolates of the thermally dimorphic pathogenic fungus Coccidioides posadasii and infer the strain compositions for large mixed pools of these strains after competition at 37°C and room temperature. We validate the results of these selection experiments by recapitulating the temperature-specific enrichment results in smaller pools. Additionally, we demonstrate that strain fitness estimated by our method can be used as a quantitative trait for genome-wide association studies. We anticipate that our method will be broadly applicable to natural populations of microbes and allow high-throughput phenotyping to match the rate of genomic data acquisition.

Formulation-dependent stability mechanisms affecting dissolution performance of directly compressed griseofulvin tablets.

During drug product development, stability studies are used to ensure that the safety and efficacy of a product are not affected during storage. Any change in the dissolution performance of a product must be investigated, as this may indicate a change in the bioavailability. In this study, three different griseofulvin formulations were prepared containing microcrystalline cellulose (MCC) with either mannitol, lactose monohydrate, or dibasic calcium phosphate anhydrous (DCPA). The tensile strength, porosity, contact angle, disintegration time, and dissolution rate were measured after storage under five different accelerated temperature and humidity conditions for 1, 2, and 4 weeks. The dissolution rate was found to decrease after storage for all three batches, with the change in dissolution rate strongly correlating with the storage humidity. The changes in physical properties of each formulation were found to relate to either the premature swelling (MCC/DCPA, MCC/lactose) or dissolution (MCC/mannitol) of particles during storage. These results are also discussed with consideration of the performance- and stability-controlling mechanisms of placebo tablets of the same formulations (Maclean et al., 2021; Maclean et al., 2022).

B Cell Chronic Lymphocytic Leukemia Development in Mice with Chronic Lung Exposure to Coccidioides Fungal Arthroconidia.

Links between repeated microbial infections and B cell chronic lymphocytic leukemia (B-CLL) have been proposed but not tested directly. This study examines how prolonged exposure to a human fungal pathogen impacts B-CLL development in Eµ-hTCL1-transgenic mice. Monthly lung exposure to inactivated Coccidioides arthroconidia, agents of Valley fever, altered leukemia development in a species-specific manner, with Coccidioides posadasii hastening B-CLL diagnosis/progression in a fraction of mice and Coccidioides immitis delaying aggressive B-CLL development, despite fostering more rapid monoclonal B cell lymphocytosis. Overall survival did not differ significantly between control and C. posadasii-treated cohorts but was significantly extended in C. immitis-exposed mice. In vivo doubling time analyses of pooled B-CLL showed no difference in growth rates of early and late leukemias. However, within C. immitis-treated mice, B-CLL manifests longer doubling times, as compared with B-CLL in control or C. posadasii-treated mice, and/or evidence of clonal contraction over time. Through linear regression, positive relationships were noted between circulating levels of CD5+/B220low B cells and hematopoietic cells previously linked to B-CLL growth, albeit in a cohort-specific manner. Neutrophils were positively linked to accelerated growth in mice exposed to either Coccidioides species, but not in control mice. Conversely, only C. posadasii-exposed and control cohorts displayed positive links between CD5+/B220low B cell frequency and abundance of M2 anti-inflammatory monocytes and T cells. The current study provides evidence that chronic lung exposure to fungal arthroconidia affects B-CLL development in a manner dependent on fungal genotype. Correlative studies suggest that fungal species differences in the modulation of nonleukemic hematopoietic cells are involved.

Volatile Metabolites in Lavage Fluid Are Correlated with Cytokine Production in a Valley Fever Murine Model.

Coccidioides immitis and Coccidioides posadasii are soil-dwelling fungi of arid regions in North and South America that are responsible for Valley fever (coccidioidomycosis). Forty percent of patients with Valley fever exhibit symptoms ranging from mild, self-limiting respiratory infections to severe, life-threatening pneumonia that requires treatment. Misdiagnosis as bacterial pneumonia commonly occurs in symptomatic Valley fever cases, resulting in inappropriate treatment with antibiotics, increased medical costs, and delay in diagnosis. In this proof-of-concept study, we explored the feasibility of developing breath-based diagnostics for Valley fever using a murine lung infection model. To investigate potential volatile biomarkers of Valley fever that arise from host−pathogen interactions, we infected C57BL/6J mice with C. immitis RS (n = 6), C. posadasii Silveira (n = 6), or phosphate-buffered saline (n = 4) via intranasal inoculation. We measured fungal dissemination and collected bronchoalveolar lavage fluid (BALF) for cytokine profiling and for untargeted volatile metabolomics via solid-phase microextraction (SPME) and two-dimensional gas chromatography coupled with time-of-flight mass spectrometry (GC×GC-TOFMS). We identified 36 volatile organic compounds (VOCs) that were significantly correlated (p < 0.05) with cytokine abundance. These 36 VOCs clustered mice by their cytokine production and were also able to separate mice with moderate-to-high cytokine production by infection strain. The data presented here show that Coccidioides and/or the host produce volatile metabolites that may yield biomarkers for a Valley fever breath test that can detect coccidioidal infection and provide clinically relevant information on primary pulmonary disease severity.

Developing a robust in vitro release method for a polymeric nanoparticle: Challenges and learnings.

Nanomedicines have emerged as a promising approach for targeted therapeutic delivery and specifically as a beneficial alternative to conventional cancer therapies as they can deliver higher concentrations of chemotherapeutic agents at the tumour site compared to healthy tissue, thus providing improved drug efficacy and lower systemic toxicity. Long acting injectables are increasingly becoming the focus of pharmaceutical research, as they can reduce dosing frequency and improve the life quality of patients. Development of an in vitro release (IVR) method for modified release nanomedicines is challenging because of the uniqueness and range of different formulation design approaches, as well as the complex nature of drug release mechanisms which may result in inherent variability. Regulatory guidance on the development of dissolution or release methods for parenteral products is limited relative to oral products. This article details the extensive in vitro release method development work conducted on a polymeric nanoparticle to develop the release media composition and selection of suitable apparatus and sampling technique to separate the released drug from the formulation. The aim was to develop a suitably robust analytical method that generated clinically relevant in vitro release data.

"Sniffing" out SARS-CoV-2 in Arizona working dogs: an exploratory serosurvey.

Susceptibility to and infection with SARS-CoV-2 in companion animals has been well-documented throughout the COVID-19 pandemic. Surveillance for the virus in dogs has largely been focused on household pets; however, other canine populations may also be impacted. We partnered with a local veterinary hospital with a high working dog patient volume to conduct viral and neutralizing antibody testing in working dogs and identify potential risk factors in the dog's work and home environments. Surveillance of SARS-CoV-2 in law enforcement and security working dogs in Arizona found 24.81% (32/129) of dogs to be seropositive. Thirteen dogs presenting with clinical signs or with reported exposure to COVID-19 in the 30 days prior to sample collection were also tested by PCR; all samples were negative. 90.7% (n = 117) of dogs were reported to be asymptomatic or have no change in performance at the time of sampling. Two dogs (1.6%) had suspected anosmia as reported by their handlers; one of which was seropositive. Known exposure to the dog's COVID-19 positive handler or household member was identified as a significant risk factor. Demographics factors including sex, altered status, and type of work were not associated with canine seropositivity. Further work is warranted to understand the impact of SARS-CoV-2 and other infectious diseases in working dogs.

Virome-wide detection of natural infection events and the associated antibody dynamics using longitudinal highly-multiplexed serology.

Current methods for detecting infections either require a sample collected from an actively infected site, are limited in the number of agents they can query, and/or yield no information on the immune response. Here we present an approach that uses temporally coordinated changes in highly-multiplexed antibody measurements from longitudinal blood samples to monitor infection events at sub-species resolution across the human virome. In a longitudinally-sampled cohort of South African adolescents representing >100 person-years, we identify >650 events across 48 virus species and observe strong epidemic effects, including high-incidence waves of Aichivirus A and the D68 subtype of Enterovirus D earlier than their widespread circulation was appreciated. In separate cohorts of adults who were sampled at higher frequency using self-collected dried blood spots, we show that such events temporally correlate with symptoms and transient inflammatory biomarker elevations, and observe the responding antibodies to persist for periods ranging from ≤1 week to >5 years. Our approach generates a rich view of viral/host dynamics, supporting novel studies in immunology and epidemiology.

PepSeq: a fully in vitro platform for highly multiplexed serology using customizable DNA-barcoded peptide libraries.

PepSeq is an in vitro platform for building and conducting highly multiplexed proteomic assays against customizable targets by using DNA-barcoded peptides. Starting with a pool of DNA oligonucleotides encoding peptides of interest, this protocol outlines a fully in vitro and massively parallel procedure for synthesizing the encoded peptides and covalently linking each to a corresponding cDNA tag. The resulting libraries of peptide/DNA conjugates can be used for highly multiplexed assays that leverage high-throughput sequencing to profile the binding or enzymatic specificities of proteins of interest. Here, we describe the implementation of PepSeq for fast and cost-effective epitope-level analysis of antibody reactivity across hundreds of thousands of peptides from <1 µl of serum or plasma input. This protocol includes the design of the DNA oligonucleotide library, synthesis of DNA-barcoded peptide constructs, binding of constructs to sample, preparation for sequencing and data analysis. Implemented in this way, PepSeq can be used for a number of applications, including fine-scale mapping of antibody epitopes and determining a subject's pathogen exposure history. The protocol is divided into two main sections: (i) design and synthesis of DNA-barcoded peptide libraries and (ii) use of libraries for highly multiplexed serology. Once oligonucleotide templates are in hand, library synthesis takes 1-2 weeks and can provide enough material for hundreds to thousands of assays. Serological assays can be conducted in 96-well plates and generate sequencing data within a further ~4 d. A suite of software tools, including the PepSIRF package, are made available to facilitate the design of PepSeq libraries and analysis of assay data.

Factor Xa cleaves SARS-CoV-2 spike protein to block viral entry and infection.

Serine proteases (SP), including furin, trypsin, and TMPRSS2 cleave the SARS-CoV-2 spike (S) protein, enabling the virus to enter cells. Here, we show that factor (F) Xa, an SP involved in blood coagulation, is upregulated in COVID-19 patients. In contrast to other SPs, FXa exerts antiviral activity. Mechanistically, FXa cleaves S protein, preventing its binding to ACE2, and thus blocking viral entry and infection. However, FXa is less effective against variants carrying the D614G mutation common in all pandemic variants. The anticoagulant rivaroxaban, a direct FXa inhibitor, inhibits FXa-mediated S protein cleavage and facilitates viral entry, whereas the indirect FXa inhibitor fondaparinux does not. In the lethal SARS-CoV-2 K18-hACE2 model, FXa prolongs survival yet its combination with rivaroxaban but not fondaparinux abrogates that protection. These results identify both a previously unknown function for FXa and an associated antiviral host defense mechanism against SARS-CoV-2 and suggest caution in considering direct FXa inhibitors for preventing or treating thrombotic complications in COVID-19 patients.

Investigating the role of excipients on the physical stability of directly compressed tablets.

Stability studies are an integral part of the drug development process for any drug product. In addition to monitoring chemical degradation, the physical stability of a drug product must also be evaluated to ensure that the drug release and performance is not affected by storage. In this study, directly compressed tablets of 16 different formulations were exposed to an accelerated stability program to quantify changes in tablet breaking force, porosity, contact angle and disintegration time. Tablets were exposed to five different storage conditions from 37∘ C/30% relative humidity (RH) to 70∘ C/75%RH with testing after 2 and 4 weeks of storage. Each formulation contained two different fillers (47% w/w each), a disintegrant (5% w/w) and magnesium stearate (1% w/w). The results show that tablets stored at high humidity show increases in porosity and decreases in tensile strength, particularly if they contain a highly hygroscopic filler such as microcrystalline cellulose (MCC). For tablets stored at high temperature, the most commonly affected property was the tablet wettability, measured by sessile drop contact angle measurements. These results are considered in combination with the performance-controlling disintegration mechanism (Maclean et al., 2021) to identify the critical properties which influence the performance after storage.

Coccidioidomycosis in Northern Arizona: an Investigation of the Host, Pathogen, and Environment Using a Disease Triangle Approach.

Coccidioides immitis and Coccidioides posadasii are the etiological agents of coccidioidomycosis (Valley fever [VF]). Disease manifestation ranges from mild pneumonia to chronic or extrapulmonary infection. If diagnosis is delayed, the risk of severe disease increases. In this report, we investigated the intersection of pathogen, host, and environment for VF cases in Northern Arizona (NAZ), where the risk of acquiring the disease is much lower than in Southern Arizona. We investigated reported cases and assessed pathogen origin by comparing genomes of NAZ clinical isolates to isolates from other regions. Lastly, we surveyed regional soils for presence of Coccidioides. We found that cases of VF increased in NAZ in 2019, and Coccidioides NAZ isolates are assigned to Arizona populations using phylogenetic inference. Importantly, we detected Coccidioides DNA in NAZ soil. Given recent climate modeling of the disease that predicts that cases will continue to increase throughout the region, and the evidence presented in this report, we propose that disease awareness outreach to clinicians throughout the western United States is crucial for improving patient outcomes, and further environmental sampling across the western U.S. is warranted. IMPORTANCE Our work is the first description of the Valley fever disease triangle in Northern Arizona, which addresses the host, the pathogen, and the environmental source in the region. Our data suggest that the prevalence of diagnosed cases rose in 2019 in this region, and some severe cases necessitate hospitalization. We present the first evidence of Coccidioides spp. in Northern Arizona soils, suggesting that the pathogen is maintained in the local environment. Until disease prevention is an achievable option via vaccination, we predict that incidence of Valley fever will rise in the area. Therefore, enhanced awareness of and surveillance for coccidioidomycosis is vital to community health in Northern Arizona.

A chromosomal-level reference genome of the widely utilized Coccidioides posadasii laboratory strain "Silveira".

Coccidioidomycosis is a common fungal disease that is endemic to arid and semi-arid regions of both American continents. Coccidioides immitis and Coccidioides posadasii are the etiological agents of the disease, also known as Valley Fever. For several decades, the C. posadasii strain Silveira has been used widely in vaccine studies, is the source strain for production of diagnostic antigens, and is a widely used experimental strain for functional studies. In 2009, the genome was sequenced using Sanger sequencing technology, and a draft assembly and annotation were made available. In this study, the genome of the Silveira strain was sequenced using single molecule real-time sequencing PacBio technology, assembled into chromosomal-level contigs, genotyped, and the genome was reannotated using sophisticated and curated in silico tools. This high-quality genome sequencing effort has improved our understanding of chromosomal structure, gene set annotation, and lays the groundwork for identification of structural variants (e.g. transversions, translocations, and copy number variants), assessment of gene gain and loss, and comparison of transposable elements in future phylogenetic and population genomics studies.

Developing Clinically Relevant Dissolution Specifications (CRDSs) for Oral Drug Products: Virtual Webinar Series.

A webinar series that was organised by the Academy of Pharmaceutical Sciences Biopharmaceutics focus group in 2021 focused on the challenges of developing clinically relevant dissolution specifications (CRDSs) for oral drug products. Industrial scientists, together with regulatory and academic scientists, came together through a series of six webinars, to discuss progress in the field, emerging trends, and areas for continued collaboration and harmonisation. Each webinar also hosted a Q&A session where participants could discuss the shared topic and information. Although it was clear from the presentations and Q&A sessions that we continue to make progress in the field of CRDSs and the utility/success of PBBM, there is also a need to continue the momentum and dialogue between the industry and regulators. Five key areas were identified which require further discussion and harmonisation.