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Cigarette smoking remains one of the leading causes of preventable death worldwide. A worldwide study by the World Health Organization concluded that more than 8 million people die every year from smoking, tobacco consumption, and secondhand smoke. The most effective tobacco cessation programs require personalized human intervention combined with costly pharmaceutical supplementation, making them unaffordable or inaccessible to most tobacco users. Thus, digital interventions offer a promising alternative to these traditional methods. However, the leading smartphone apps available in the market today have either not been studied in a clinical setting or are unable to match the smoking cessation success rates of their expensive offline counterparts. We would like to understand whether QuitSure, a novel smoking cessation app built by Rapidkart Online Private Limited, is able to bridge this efficacy gap and deliver affordable and effective smoking cessation at scale. Our objective was to do an initial exploration into the engagement, efficacy, and safety of QuitSure based on the self-reported experiences of its users. Outcomes measured were program completion, the effect of program completion on smoking behavior, including self-reported cessation outcomes, and negative health events from using the app. All QuitSure registered users who created their accounts on the QuitSure app between April 1, 2021, and February 28, 2022, were sent an anonymized web-based survey. The survey results were added to their engagement data on the app to evaluate the feasibility and efficacy of the app as a smoking cessation intervention. The data were analyzed using descriptive statistics (frequencies and percentages) and the χ test of independence. In total, 1299 users who had completed the QuitSure program submitted the survey and satisfied the inclusion criteria of the study. Of these, 1286 participants had completed the program more than 30 days before filling out the survey, and 1040 (80.1%, 95% CI 79.1%-82.6%) of them had maintained prolonged abstinence for at least 30 days after program completion. A majority of participants (770/891, 86.4%) who were still maintaining abstinence at the time of submitting the survey did not experience any severe nicotine withdrawal symptoms, while 41.9% (373/891) experienced no mild withdrawal symptoms either. Smoking quantity prior to completing the program significantly affected quit rates (P<.001), with heavy smokers (>20 cigarettes per day) having a lower 30-day prolonged abstinence rate (relative risk=0.91; 95% CI 90.0%-96.2%) compared to lighter smokers. No additional adverse events outside of known nicotine withdrawal symptoms were reported. The nature of web-based surveys and cohort selection allows for extensive unknown biases. However, the efficacy rates of survey respondents who completed the program were high and provide a case for further investigation in the form of randomized controlled trials on the QuitSure tobacco cessation program.

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4 . We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase ( ScPMA1 ) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace Sc Pma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits Sc Pma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a Sc Pma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae . This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.

A direct transfer of bacterial genomic DNA to yeast enables genome engineering in genetically intractable bacteria. Transfer of genomes into yeast facilitates genome engineering for genetically intractable organisms, but this process has been hampered by the need for cumbersome isolation of intact genomes before transfer. Here we demonstrate direct cell-to-cell transfer of bacterial genomes as large as 1.8 megabases (Mb) into yeast under conditions that promote cell fusion. Moreover, we discovered that removal of restriction endonucleases from donor bacteria resulted in the enhancement of genome transfer.

Given that many antifungal medications are susceptible to evolved resistance, there is a need for novel drugs with unique mechanisms of action. Inhibiting the essential proton pump Pma1p, a P-type ATPase, is a potentially effective therapeutic approach that is orthogonal to existing treatments. We identify NSC11668 and hitachimycin as structurally distinct antifungals that inhibit yeast Sc Pma1p. These compounds provide new opportunities for drug discovery aimed at this important target.

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum , the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum . Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium , IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing. Imidazolopiperazines (IZPs) are a class of compounds under clinical development for malaria, but their mechanism of action is unclear. Here, the authors show that IZPs inhibit the parasite’s secretory pathway, affecting protein trafficking and export.

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae . Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn 2 C 6 transcription factors YRR1 and YRM1 ( p  < 1 × 10 −100 ). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency. Ottilie et al. employ an experimental evolution approach to investigate the role of transcription factors in yeast chemical resistance. Most emergent mutations in resistant strains were enriched in transcription factor coding genes, highlighting their importance in drug resistance.

Given the limited availability of serological testing to date, the seroprevalence of SARS-CoV-2-specific antibodies in different populations has remained unclear. Here, we report very low SARS-CoV-2 seroprevalence in two San Francisco Bay Area populations. Seroreactivity was 0.26% in 387 hospitalized patients admitted for non-respiratory indications and 0.1% in 1,000 blood donors in early April 2020. We additionally describe the longitudinal dynamics of immunoglobulin-G (IgG), immunoglobulin-M (IgM), and in vitro neutralizing antibody titers in COVID-19 patients. The median time to seroconversion ranged from 10.3–11.0 days for these 3 assays. Neutralizing antibodies rose in tandem with immunoglobulin titers following symptom onset, and positive percent agreement between detection of IgG and neutralizing titers was >93%. These findings emphasize the importance of using highly accurate tests for surveillance studies in low-prevalence populations, and provide evidence that seroreactivity using SARS-CoV-2 anti-nucleocapsid protein IgG and anti-spike IgM assays are generally predictive of in vitro neutralizing capacity. Highly accurate antibody tests for SARS-CoV-2 are needed for surveillance in low-prevalence populations. Here, the authors find seroprevalence of less than 1% in two San Francisco Bay Area populations at the beginning of April, and that seroreactivity is generally predictive of in vitro neutralising activity.

Morphology-based classification of cells in the bone marrow aspirate (BMA) is a key step in the diagnosis and management of hematologic malignancies. However, it is time-intensive and must be performed by expert hematopathologists and laboratory professionals. We curated a large, high-quality dataset of 41,595 hematopathologist consensus-annotated single-cell images extracted from BMA whole slide images (WSIs) containing 23 morphologic classes from the clinical archives of the University of California, San Francisco. We trained a convolutional neural network, DeepHeme, to classify images in this dataset, achieving a mean area under the curve (AUC) of 0.99. DeepHeme was then externally validated on WSIs from Memorial Sloan Kettering Cancer Center, with a similar AUC of 0.98, demonstrating robust generalization. When compared to individual hematopathologists from three different top academic medical centers, the algorithm outperformed all three. Finally, DeepHeme reliably identified cell states such as mitosis, paving the way for image-based quantification of mitotic index in a cell-specific manner, which may have important clinical applications.

Summary In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in the model microbe, Saccharomyces cerevisiae. Analysis of 355 curated, laboratory-evolved clones, resistant to 80 different compounds, demonstrates differences in the types of mutations that are identified in selected versus neutral evolution and reveals numerous new, compound-target interactions. Through enrichment analysis we further identify a set of 137 genes strongly associated with or conferring drug resistance as indicated by CRISPR-Cas9 engineering. The set of 25 most frequently mutated genes was enriched for transcription factors and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function, single nucleotide variants found in 170-amino-acid domains in two Zn2C6 transcription factors, YRR1 and YRM1 (p < 1x 10 −100). This remarkable enrichment for transcription factors as drug resistance genes may explain why it is challenging to develop effective antifungal killing agents and highlights their important role in evolution. Competing Interest Statement L.E.C. and L.W. are co-founders and shareholders in Bright Angel Therapeutics, a platform company for development of novel antifungal therapeutics. L.E.C. is a consultant for Boragen, a small-molecule development company focused on leveraging the unique chemical properties of boron chemistry for crop protection and animal health. The other author(s) declare no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Footnotes * ↵12 Current affiliation: Aspen Neuroscience

One of the most promising new compound classes in clinical development for the treatment of malaria is the imidazolopiperazines (IZPs) class. Human trials have demonstrated that members of the IZP series, which includes KAF156 (Ganaplacide) and GNF179, are potent and effective against Plasmodium symptomatic asexual blood stage infections. Unlike other commonly used antimalarials, they also prevent transmission and block future infection in animal models. Despite the identification of several Plasmodium falciparum resistance mechanisms including mutations in ER-localized PfCARL (PfEMP65), Acetyl-coA transporter, and PfUGT transporter, IZPs mechanism of action remains unknown. To investigate, we combined in vitro evolution and whole-genome analysis in the model organism Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods, in P. falciparum. S. cerevisiae clones that resist IZP activity carry multiple mutations in genes that encode endoplasmic reticulum(ER)-based lipid homeostasis and autophagy including elo2, elo3, sur2, atg15 and lcb4, as well as ER-based sec66. In Plasmodium, IZPs cause inhibition of protein trafficking, block the establishment of new permeation pathways and result in ER expansion. We also observe sensitization with other secretion inhibitors such as brefeldin A and golgicidin as well as synthetic lethality with PfSEC62. Our data show that IZPs target the secretory pathway and highlight a novel mechanism for blocking parasite growth and development that is distinct from those of standard compounds used to treat malaria. In addition, we provide physiological signatures and hallmarks for inhibitors that work through this mechanism of action and show that IZPs are tool compounds for studying ER-dependent protein processing in different species.