Calibration by Proxy.

Calibration by Proxy (CbPx) is a matrix-matched calibration method that utilizes multiple internal standard species to build a calibration curve. The technique requires only two solutions: solution 1 containing a sample solution and a suite of internal standards at known concentrations, and solution 2 identical to solution 1, plus an aliquot of a standard containing all analytes and the internal standards at the same concentration. The calibration curve is prepared by plotting the signal measured for each internal standard in solution 1 divided by the signal arising due to the aliquot of internal standard added to solution 2 on the y-axis. In this ratio, the sensitivity for each element cancels, because the sample matrix is equal between the solutions. Therefore, the y-axis value measured for a specific internal standard is identical to the value that would be measured for any other element present at the same concentrations in the two solutions. Hence, each internal standard serves as a proxy for analyte values. The concentrations of internal standards in solution 1 are plotted on the x-axis, and these correspond to any analytes present in solution 1 at the same concentration. CbPx is applied to the analysis of five certified reference materials by inductively coupled plasma optical emission spectrometry (ICP-OES). Percent recoveries for analytes range from 89 to 106%, with relative standard deviations on the order of 1%. A recommended working range for the method is developed through both theoretical simulation and experimental results and then exhibited through the analysis of off-the-shelf vitamin tablets.

N - acetyl-transferases required for iron uptake and aminoglycoside resistance promote virulence lipid production in M. marinum.

Phagosomal lysis is a key aspect of mycobacterial infection of host macrophages. Acetylation is a protein modification mediated enzymatically by N-acetyltransferases (NATs) that impacts bacterial pathogenesis and physiology. To identify NATs required for lytic activity, we leveraged Mycobacterium marinum, a nontubercular pathogen and an established model for M. tuberculosis. M. marinum hemolysis is a proxy for phagolytic activity. We generated M. marinum strains with deletions in conserved NAT genes and screened for hemolytic activity. Several conserved lysine acetyltransferases (KATs) contributed to hemolysis. Hemolysis is mediated by the ESX-1 secretion system and by phthiocerol dimycocerosate (PDIM), a virulence lipid. For several strains, the hemolytic activity was restored by the addition of second copy of the ESX-1 locus. Using thin-layer chromatography (TLC), we found a single NAT required for PDIM and phenolic glycolipid (PGL) production. MbtK is a conserved KAT required for mycobactin siderophore synthesis and virulence. Mycobactin J exogenously complemented PDIM/PGL production in the Δ mbtK strain. The Δ mbtK M. marinum strain was attenuated in macrophage and Galleria mellonella infection models. Constitutive expression of either eis or papA5, which encode a KAT required for aminoglycoside resistance and a PDIM/PGL biosynthetic enzyme, rescued PDIM/PGL production and virulence of the Δ mbtK strain. Eis N-terminally acetylated PapA5 in vitro , supporting a mechanism for restored lipid production. Overall, our study establishes connections between the MbtK and Eis NATs, and between iron uptake and PDIM and PGL synthesis in M. marinum . Our findings underscore the multifunctional nature of mycobacterial NATs and their connection to key virulence pathways. Significance Statement: Acetylation is a modification of protein N-termini, lysine residues, antibiotics and lipids. Many of the enzymes that promote acetylation belong to the GNAT family of proteins. M. marinum is a well-established as a model to understand how M. tuberculosis causes tuberculosis. In this study we sought to identify conserved GNAT proteins required for early stages of mycobacterial infection. Using M. marinum, we determined that several GNAT proteins are required for the lytic activity of M. marinum. We uncovered previously unknown connections between acetyl-transferases required for iron uptake and antimicrobial resistance, and the production of the unique mycobacterial lipids, PDIM and PGLOur data support that acetyl-transferases from the GNAT family are interconnected, and have activities beyond those previously reported.

A simple phylogenetic approach to analyze hypermutated HIV proviruses reveals insights into their dynamics and persistence during antiretroviral therapy.

Hypermutated proviruses, which arise in a single HIV replication cycle when host antiviral APOBEC3 proteins introduce extensive G-to-A mutations throughout the viral genome, persist in all people living with HIV receiving antiretroviral therapy (ART). But, the within-host evolutionary origins of hypermutated sequences are incompletely understood because phylogenetic inference algorithms, which assume that mutations gradually accumulate over generations, incorrectly reconstruct their ancestor-descendant relationships. Using > 1400 longitudinal single-genome-amplified HIV env-gp120 sequences isolated from six women over a median 18 years of follow-up - including plasma HIV RNA sequences collected over a median 9 years between seroconversion and ART initiation, and > 500 proviruses isolated over a median 9 years on ART - we evaluated three approaches for removing hypermutation from nucleotide alignments. Our goals were to 1) reconstruct accurate phylogenies that can be used for molecular dating and 2) phylogenetically infer the integration dates of hypermutated proviruses persisting during ART. Two of the tested approaches (stripping all positions containing putative APOBEC3 mutations from the alignment, or replacing individual putative APOBEC3 mutations in hypermutated sequences with the ambiguous base R) consistently normalized tree topologies, eliminated erroneous clustering of hypermutated proviruses, and brought env-intact and hypermutated proviruses into comparable ranges with respect to multiple tree-based metrics. Importantly, these corrected trees produced integration date estimates for env-intact proviruses that were highly concordant with those from benchmark trees that excluded hypermutated sequences, indicating that the corrected trees can be used for molecular dating. Use of these trees to infer the integration dates of hypermutated proviruses persisting during ART revealed that these spanned a wide age range, with the oldest ones dating to shortly after infection. This indicates that hypermutated proviruses, like other provirus types, begin to be seeded into the proviral pool immediately following infection, and can persist for decades. In two of the six participants, hypermutated proviruses differed from env-intact ones in terms of their age distributions, suggesting that different provirus types decay at heterogeneous rates in some hosts. These simple approaches to reconstruct hypermutated provirus' evolutionary histories, allow insights into their in vivo origins and longevity, towards a more comprehensive understanding of HIV persistence during ART.

Random interval schedule of reinforcement influences punishment resistance for cocaine in rats.

In an animal model of compulsive drug use, a subset of rats continues to self-administer cocaine despite footshock consequences and is considered punishment resistant. We recently found that punishment resistance is associated with habits that persist under conditions that typically encourage a transition to goal-directed control. Given that random ratio (RR) and random interval (RI) schedules of reinforcement influence whether responding is goal-directed or habitual, we investigated the influence of these schedules on punishment resistance for cocaine or food. Male and female Sprague Dawley rats were trained to self-administer either intravenous cocaine or food pellets on a seeking-taking chained schedule of reinforcement, with the seeking lever requiring completion of either an RR20 or RI60 schedule. Rats were then given four days of punishment testing with footshock administered at the completion of seeking on a random one-third of trials. For cocaine-trained rats, the RI60 schedule led to greater punishment resistance (i.e., more trials completed) than the RR20 schedule in males and females. For food-trained rats, the RI60 schedule led to greater punishment resistance (i.e., higher reward rates) than the RR20 schedule in female rats, although male rats showed punishment resistance on both RR20 and RI60 schedules. For both cocaine and food, we found that seeking responses were suppressed to a greater degree than reward rate with the RI60 schedule, whereas response rate and reward rate were equally suppressed with the RR20 schedule. This dissociation between punishment effects on reward rate and response rate with the RI60 schedule can be explained by the nonlinear relation between these variables on RI schedules, but it does not account for the enhanced resistance to punishment. Overall, the results show greater punishment resistance with the RI60 schedule as compared to the RR20 schedule, indicating that schedules of reinforcement are an influencing factor on resistance to negative consequences.

Punishment resistance for cocaine is associated with inflexible habits in rats.

Addiction is characterized by continued drug use despite negative consequences. In an animal model, a subset of rats continues to self-administer cocaine despite footshock consequences, showing punishment resistance. We sought to test the hypothesis that punishment resistance arises from failure to exert goal-directed control over habitual cocaine seeking. While habits are not inherently permanent or maladaptive, continued use of habits under conditions that should encourage goal-directed control makes them maladaptive and inflexible. We trained male and female Sprague Dawley rats on a seeking-taking chained schedule of cocaine self-administration. We then exposed them to four days of punishment testing in which footshock was delivered randomly on one-third of trials. Before and after punishment testing (four days pre-punishment and ≥ four days post-punishment), we assessed whether cocaine seeking was goal-directed or habitual using outcome devaluation via cocaine satiety. We found that punishment resistance was associated with continued use of habits, whereas punishment sensitivity was associated with increased goal-directed control. Although punishment resistance for cocaine was not predicted by habitual responding pre-punishment, it was associated with habitual responding post-punishment. In parallel studies of food self-administration, we similarly observed that punishment resistance was associated with habitual responding post-punishment but not pre-punishment in males, although it was associated with habitual responding both pre- and post-punishment in females, indicating that punishment resistance was predicted by habitual responding in food-seeking females. These findings indicate that punishment resistance is related to habits that have become inflexible and persist under conditions that should encourage a transition to goal-directed behavior.

The loss of the PDIM/PGL virulence lipids causes differential secretion of ESX-1 substrates in Mycobacterium marinum.

The mycobacterial cell envelope is a major virulence determinant in pathogenic mycobacteria. Specific outer lipids play roles in pathogenesis, modulating the immune system and promoting the secretion of virulence factors. ESX-1 (ESAT-6 system-1) is a conserved protein secretion system required for mycobacterial pathogenesis. Previous studies revealed that mycobacterial strains lacking the outer lipid PDIM have impaired ESX-1 function during laboratory growth and infection. The mechanisms underlying changes in ESX-1 function are unknown. We used a proteo-genetic approach to measure phthiocerol dimycocerosate (PDIM)- and phenolic glycolipid (PGL)-dependent protein secretion in M. marinum, a non-tubercular mycobacterial pathogen that causes tuberculosis-like disease in ectothermic animals. Importantly, M. marinum is a well-established model for mycobacterial pathogenesis. Our findings showed that M. marinum strains without PDIM and PGL showed specific, significant reductions in protein secretion compared to the WT and complemented strains. We recently established a hierarchy for the secretion of ESX-1 substrates in four (I-IV) groups. Loss of PDIM differentially impacted secretion of Group III and IV ESX-1 substrates, which are likely the effectors of pathogenesis. Our data suggest that the altered secretion of specific ESX-1 substrates is responsible for the observed ESX-1-related effects in PDIM-deficient strains.IMPORTANCEMycobacterium tuberculosis, the cause of human tuberculosis, killed an estimated 1.3 million people in 2022. Non-tubercular mycobacterial species cause acute and chronic human infections. Understanding how these bacteria cause disease is critical. Lipids in the cell envelope are essential for mycobacteria to interact with the host and promote disease. Strains lacking outer lipids are attenuated for infection, but the reasons are unclear. Our research aims to identify a mechanism for attenuation of mycobacterial strains without the PDIM and PGL outer lipids in M. marinum. These findings will enhance our understanding of the importance of lipids in pathogenesis and how these lipids contribute to other established virulence mechanisms.

The Impact of a Pandemic on a Military Oral and Maxillofacial Pathology Biopsy Service.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) and the resulting societal reaction presented new challenges to the medical community by limiting patient access to care in 2020 and 2021. The Navy Postgraduate Dental School (NPDS) oral and maxillofacial pathology biopsy service is dependent on in-office physician or dentist appointments and patient biopsies. The purpose of this study was to understand the regulatory and societal impacts of COVID-19 restrictions on biopsy service submissions by assessing NPDS biopsy submission quantities and disease distribution. MATERIALS AND METHODS: All NPDS oral and maxillofacial pathology biopsy submissions from calendar years 2015 to 2016 and 2019 to 2021 were evaluated, and patient demographics and biopsy diagnoses were recorded in a biopsy registry. Data collected included age, sex, biopsy site, and diagnosis. Data from 2015, 2016, and 2019 were defined as pre-COVID and 2020 and 2021 as COVID. Biopsy reports for each year were organized in quarters. Diagnoses were categorized as malignant, pre-malignant, or benign. Categorical and continuous data were evaluated and presented as counts with percentages and means or medians with standard deviations, respectively. Significant differences in proportions or means were assessed using chi-square analysis or Student t-test, respectively. Cases were aggregated by quarter and year and assessed for temporal trends using linear regression analysis. RESULTS: The study evaluated 9,351 biopsy submission reports. The annual pre-COVID count mean (± standard deviation) and yearly counts for 2020 and 2021 were 2,063 ± 33.3, 1,421, and 1,742, respectively. The mean (± standard deviation) percentage of diagnoses classified as malignant from pre-COVID, 2020, and 2021 were 2.46 ± 0.005%, 3.59%, and 3.04%, respectively. Case counts and representation as a percentage of all biopsy diagnoses for Human Papillomavirus (HPV)-associated squamous cell carcinoma increased significantly during COVID compared to pre-COVID years (P < .05). CONCLUSIONS: Overall, preventative COVID-19 health measures and protocols resulted in a reduction in biopsy submission frequency, particularly during the second quarter (April to June) of 2020. However, case counts for malignant biopsies remained consistent between pre-COVID and COVID time intervals, suggesting that the identification and analysis of cases requiring follow-on care were unaffected by COVID-19 protocols.

The loss of the PDIM/PGL virulence lipids causes differential secretion of ESX-1 substrates in Mycobacterium marinum.

The mycobacterial cell envelope is a major virulence determinant in pathogenic mycobacteria. Specific outer lipids play roles in pathogenesis, modulating the immune system and promoting the secretion of virulence factors. ESX-1 (ESAT-6 system-1) is a conserved protein secretion system required for mycobacterial pathogenesis (1, 2). Previous studies revealed that mycobacterial strains lacking the outer lipid PDIM have impaired ESX-1 function during laboratory growth and infection (3-5). The mechanisms underlying changes in ESX-1 function are unknown. We used a proteo-genetic approach to measure PDIM and PGL-dependent protein secretion in M. marinum , a non-tubercular mycobacterial pathogen that causes tuberculosis-like disease in ectothermic animals (6, 7). Importantly, M. marinum is a well-established model for mycobacterial pathogenesis (8, 9). Our findings showed that M. marinum strains without PDIM and PGL showed specific, significant reductions in protein secretion compared to the WT and complemented strains. We recently established a hierarchy for the secretion of ESX-1 substrates in four (I-IV) groups (10). Loss of PDIM differentially impacted secretion of Groups III and IV ESX-1 substrates, which are likely the effectors of pathogenesis. Our data suggests that the altered secretion of specific ESX-1 substrates is responsible for the observed ESX-1-related effects in PDIM-deficient strains.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and this diversity persists within infected cell reservoirs during antiretroviral therapy (ART). Achieving a better understanding of on-ART proviral evolutionary dynamics, and a better appreciation of how the overall persisting pool of (largely genetically defective) proviruses differs from the much smaller replication-competent HIV reservoir, is critical to HIV cure efforts. We reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study who experienced HIV seroconversion, and used these data to characterize the diversity, lineage origins, and ages of proviral env-gp120 sequences sampled longitudinally up to 12 years on ART. We also studied HIV sequences emerging from the reservoir in two participants. We observed that proviral clonality generally increased over time on ART, with clones frequently persisting long term. While on-ART proviral integration dates generally spanned the duration of untreated infection, HIV emerging in plasma was exclusively younger (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained stable during ART in all but one participant, in whom there was evidence that younger proviruses had been preferentially eliminated after 12 years on ART. Analysis of the gag region in three participants corroborated our env-gp120-based observations, indicating that our observations are not influenced by the HIV region studied. Our results underscore the remarkable genetic stability of the distinct proviral sequences that persist in blood during ART. Our results also suggest that the replication-competent HIV reservoir is a genetically restricted, younger subset of this overall proviral pool.IMPORTANCECharacterizing the genetically diverse HIV sequences that persist in the reservoir despite antiretroviral therapy (ART) is critical to cure efforts. Our observations confirm that proviruses persisting in blood on ART, which are largely genetically defective, broadly reflect the extent of within-host HIV evolution pre-ART. Moreover, on-ART clonal expansion is not appreciably accompanied by the loss of distinct proviral lineages. In fact, on-ART proviral genetic composition remained stable in all but one participant, in whom, after 12 years on ART, proviruses dating to around near ART initiation had been preferentially eliminated. We also identified recombinant proviruses between parental sequence fragments of different ages. Though rare, such sequences suggest that reservoir cells can be superinfected with HIV from another infection era. Overall, our finding that the replication-competent reservoir in blood is a genetically restricted, younger subset of all persisting proviruses suggests that HIV cure strategies will need to eliminate a reservoir that differs in key respects from the overall proviral pool.

HIV reservoirs are dominated by genetically younger and clonally enriched proviruses.

IMPORTANCE: Characterizing the human immunodeficiency virus (HIV) reservoir that endures despite antiretroviral therapy (ART) is critical to cure efforts. We observed that the oldest proviruses persisting during ART were exclusively defective, while intact proviruses (and rebound HIV) dated to nearer ART initiation. This helps explain why studies that sampled sub-genomic proviruses on-ART (which are largely defective) routinely found sequences dating to early infection, whereas those that sampled replication-competent HIV found almost none. Together with our findings that intact proviruses were more likely to be clonal, and that on-ART low-level/isolated viremia originated from proviruses of varying ages (including possibly defective ones), our observations indicate that (i) on-ART and rebound viremia can have distinct within-host origins, (ii) intact proviruses have shorter lifespans than grossly defective ones and thus depend more heavily on clonal expansion for persistence, and (iii) an HIV reservoir predominantly "dating" to near ART initiation will be substantially adapted to within-host pressures, complicating immune-based cure strategies.

An N-acetyltransferase required for ESAT-6 N-terminal acetylation and virulence in Mycobacterium marinum.

IMPORTANCE: N-terminal acetylation is a protein modification that broadly impacts basic cellular function and disease in higher organisms. Although bacterial proteins are N-terminally acetylated, little is understood how N-terminal acetylation impacts bacterial physiology and pathogenesis. Mycobacterial pathogens cause acute and chronic disease in humans and in animals. Approximately 15% of mycobacterial proteins are N-terminally acetylated, but the responsible enzymes are largely unknown. We identified a conserved mycobacterial protein required for the N-terminal acetylation of 23 mycobacterial proteins including the EsxA virulence factor. Loss of this enzyme from M. marinum reduced macrophage killing and spread of M. marinum to new host cells. Defining the acetyltransferases responsible for the N-terminal protein acetylation of essential virulence factors could lead to new targets for therapeutics against mycobacteria.

The replication-competent HIV reservoir is a genetically restricted, younger subset of the overall pool of HIV proviruses persisting during therapy, which is highly genetically stable over time.

Within-host HIV populations continually diversify during untreated infection, and members of these diverse forms persist within infected cell reservoirs, even during antiretroviral therapy (ART). Characterizing the diverse viral sequences that persist during ART is critical to HIV cure efforts, but our knowledge of on-ART proviral evolutionary dynamics remains incomplete, as does our understanding of the differences between the overall pool of persisting proviral DNA (which is largely genetically defective) and the subset of intact HIV sequences capable of reactivating. Here, we reconstructed within-host HIV evolutionary histories in blood from seven participants of the Women's Interagency HIV Study (WIHS) who experienced HIV seroconversion. We measured diversity, lineage origins and ages of proviral sequences (env-gp120) sampled up to four times, up to 12 years on ART. We used the same techniques to study HIV sequences emerging from the reservoir in two participants. Proviral clonality generally increased over time on ART, with clones frequently persisting across multiple time points. The integration dates of proviruses persisting on ART generally spanned the duration of untreated infection (though were often skewed towards years immediately pre-ART), while in contrast, reservoir-origin viremia emerging in plasma was exclusively "younger" (i.e., dated to the years immediately pre-ART). The genetic and age distributions of distinct proviral sequences remained highly stable during ART in all but one participant in whom, after 12 years, there was evidence that "younger" proviruses had been preferentially eliminated. Analysis of within-host recombinant proviral sequences also suggested that HIV reservoirs can be superinfected with virus reactivated from an older era, yielding infectious viral progeny with mosaic genomes of sequences with different ages. Overall, results underscore the remarkable genetic stability of distinct proviral sequences that persist on ART, yet suggest that replication-competent HIV reservoir represents a genetically-restricted and overall "younger" subset of the overall persisting proviral pool in blood.

Inferring Human Immunodeficiency Virus 1 Proviral Integration Dates With Bayesian Inference.

Human immunodeficiency virus 1 (HIV) proviruses archived in the persistent reservoir currently pose the greatest obstacle to HIV cure due to their evasion of combined antiretroviral therapy and ability to reseed HIV infection. Understanding the dynamics of the HIV persistent reservoir is imperative for discovering a durable HIV cure. Here, we explore Bayesian methods using the software BEAST2 to estimate HIV proviral integration dates. We started with within-host longitudinal HIV sequences collected prior to therapy, along with sequences collected from the persistent reservoir during suppressive therapy. We built a BEAST2 model to estimate integration dates of proviral sequences collected during suppressive therapy, implementing a tip date random walker to adjust the sequence tip dates and a latency-specific prior to inform the dates. To validate our method, we implemented it on both simulated and empirical data sets. Consistent with previous studies, we found that proviral integration dates were spread throughout active infection. Path sampling to select an alternative prior for date estimation in place of the latency-specific prior produced unrealistic results in one empirical data set, whereas on another data set, the latency-specific prior was selected as best fitting. Our Bayesian method outperforms current date estimation techniques with a root mean squared error of 0.89 years on simulated data relative to 1.23-1.89 years with previously developed methods. Bayesian methods offer an adaptable framework for inferring proviral integration dates.

Punishment resistance for cocaine is associated with inflexible habits in rats.

Addiction is characterized by continued drug use despite negative consequences. In an animal model, a subset of rats continues to self-administer cocaine despite footshock consequences, showing punishment resistance. We sought to test the hypothesis that punishment resistance arises from failure to exert goal-directed control over habitual cocaine seeking. While habits are not inherently permanent or maladaptive, continued use of habits under conditions that should encourage goal-directed control makes them maladaptive and inflexible. We trained male and female Sprague Dawley rats on a seeking-taking chained schedule of cocaine self-administration (2 h/day). We then exposed them to 4 days of punishment testing, in which footshock (0.4 mA, 0.3 s) was delivered randomly on one-third of trials, immediately following completion of seeking and prior to extension of the taking lever. Before and after punishment testing (4 days pre-punishment and ≥4 days post-punishment), we assessed whether cocaine seeking was goal-directed or habitual using outcome devaluation via cocaine satiety. We found that punishment resistance was associated with continued use of habits, whereas punishment sensitivity was associated with increased goal-directed control. Although punishment resistance was not predicted by habitual responding pre-punishment, it was associated with habitual responding post-punishment. In parallel studies of food self-administration, we similarly observed that punishment resistance was associated with habitual responding post-punishment but not pre-punishment. These findings indicate that punishment resistance is related to habits that have become inflexible and persist under conditions that should encourage a transition to goal-directed behavior.

HIV reservoirs are dominated by genetically younger and clonally enriched proviruses.

In order to cure HIV, we need to better understand the within-host evolutionary origins of the small reservoir of genome-intact proviruses that persists within infected cells during antiretroviral therapy (ART). Most prior studies on reservoir evolutionary dynamics however did not discriminate genome-intact proviruses from the vast background of defective ones. We reconstructed within-host pre-ART HIV evolutionary histories in six individuals and leveraged this information to infer the ages of intact and defective proviruses sampled after an average >9 years on ART, along with the ages of rebound and low-level/isolated viremia occurring during this time. We observed that the longest-lived proviruses persisting on ART were exclusively defective, usually due to large deletions. In contrast, intact proviruses and rebound HIV exclusively dated to the years immediately preceding ART. These observations are consistent with genome-intact proviruses having shorter lifespans, likely due to the cumulative risk of elimination following viral reactivation and protein production. Consistent with this, intact proviruses (and those with packaging signal defects) were three times more likely to be genetically identical compared to other proviral types, highlighting clonal expansion as particularly important in ensuring their survival. By contrast, low-level/isolated viremia sequences were genetically heterogeneous and sometimes ancestral, where viremia may have originated from defective proviruses. Results reveal that the HIV reservoir is dominated by clonally-enriched and genetically younger sequences that date to the untreated infection period when viral populations had been under within-host selection pressures for the longest duration. Knowledge of these qualities may help focus strategies for reservoir elimination.

Mindfulness for voices: An experimental analogue study of the effect of manipulating response style to simulated voices in a non-clinical population.

OBJECTIVES: We aimed to investigate the effects of directly manipulating response style to simulated voice hearing on emotional and cognitive outcomes in a non-clinical population. DESIGN: A between-subjects design with one independent variable, response style (with two levels: mindful acceptance vs attentional avoidance). The dependent variables were subjective distress and anxiety (primary outcomes) and performance on a sustained attention task (secondary outcomes). METHODS: Participants were randomly assigned to one of two response styles (mindful acceptance vs. attentional avoidance). They completed a computerised attention task (continuous performance task) whilst listening to a simulation of voice hearing. Participants rated their experience of anxiety and distress before and after completing the sustained attention task which was used to measure their accuracy and response times. RESULTS: One hundred and one participants took part (mindful acceptance (n = 54); attentional avoidance (n = 47)). There were no statistically significant group differences on post-test distress and anxiety scores, correct response rate or response times on the computerised attention task. Participants reported a range of different response styles along the spectrum of avoidance to acceptance, but this had no association with their assigned experimental condition. Adherence to task instructions was therefore low. CONCLUSIONS: We are unable to conclude from this study whether experimentally inducing people to respond to voices under cognitively demanding conditions in an avoidant or accepting way has an impact on their emotional or cognitive outcomes. Further research should focus on the development of more robust and reliable procedures for inducing differences in response style under experimental conditions.

An N-acetyltransferase required for EsxA N-terminal protein acetylation and virulence in Mycobacterium marinum.

N-terminal protein acetylation is a ubiquitous post-translational modification that broadly impacts diverse cellular processes in higher organisms. Bacterial proteins are also N-terminally acetylated, but the mechanisms and consequences of this modification in bacteria are poorly understood. We previously quantified widespread N-terminal protein acetylation in pathogenic mycobacteria (C. R. Thompson, M. M. Champion, and P.A. Champion, J Proteome Res 17(9): 3246-3258, 2018, https:// doi: 10.1021/acs.jproteome.8b00373). The major virulence factor EsxA (ESAT-6, Early secreted antigen, 6kDa) was one of the first N-terminally acetylated proteins identified in bacteria. EsxA is conserved in mycobacterial pathogens, including Mycobacterium tuberculosis and Mycobacterium marinum, a non-tubercular mycobacterial species that causes tuberculosis-like disease in ectotherms. However, enzyme responsible for EsxA N-terminal acetylation has been elusive. Here, we used genetics, molecular biology, and mass-spectroscopy based proteomics to demonstrate that MMAR_1839 (renamed Emp1, ESX-1 modifying protein, 1) is the putative N-acetyl transferase (NAT) solely responsible for EsxA acetylation in Mycobacterium marinum. We demonstrated that ERD_3144, the orthologous gene in M. tuberculosis Erdman, is functionally equivalent to Emp1. We identified at least 22 additional proteins that require Emp1 for acetylation, demonstrating that this putative NAT is not dedicated to EsxA. Finally, we showed that loss of emp1 resulted in a significant reduction in the ability of M. marinum to cause macrophage cytolysis. Collectively, this study identified a NAT required for N-terminal acetylation in Mycobacterium and provided insight into the requirement of N-terminal acetylation of EsxA and other proteins in mycobacterial virulence in the macrophage.

"Community 101 for researchers": an online training program to build capacity for ethical community-engaged research with Native Hawaiians and Pacific Islanders.

To address the history of unethical research and community distrust in research among Native Hawaiian and Pacific Islander communities, we developed the "Community 101 for Researchers" training program, which was launched in 2014 to enhance the capacity of researchers to engage in ethical community-engaged research. The purpose of this paper is to describe the development of this training program as well as its reach and feedback from participants. The Community 101 training program is a self-paced, 2-h online training program featuring community-engaged researchers from the University of Hawai'i and their longstanding community partners. Throughout the five modules, we highlight the historical context of Native Hawaiians and Pacific Islander populations in Hawai'i related to research ethics and use examples from the community as well as our own research projects that integrate community ethics, relevance, benefits, and input. To determine reach and gather participant feedback on the training, we extracted data from the user accounts. The training has been completed by 697 users to-date since its launch. Despite very little advertisement, an average of nearly 70 users have completed the Community 101 Program each year. The majority of the participants were located in Hawai'i though participants were also from other states and territories in the US, and international locations. The majority of participants were from universities in Hawai'i in 51 different departments demonstrating multidisciplinary relevance of the program's training. The general feedback from the 96 participants who completed an optional anonymous evaluation survey given at the end of the training was positive. The "Community 101 for Researchers" Training program is an accessible and relevant tool that can be used to advance ethical community engaged research, specifically with Native Hawaiian and Pacific Islander communities.

Determining the Presence and Size of Shoulder Lesions in Sows Using Computer Vision.

Shoulder sores predominantly arise in breeding sows and often result in untimely culling. Reported prevalence rates vary significantly, spanning between 5% and 50% depending upon the type of crate flooring inside a farm, the animal's body condition, or an existing injury that causes lameness. These lesions represent not only a welfare concern but also have an economic impact due to the labor needed for treatment and medication. The objective of this study was to evaluate the use of computer vision techniques in detecting and determining the size of shoulder lesions. A Microsoft Kinect V2 camera captured the top-down depth and RGB images of sows in farrowing crates. The RGB images were collected at a resolution of 1920 × 1080. To ensure the best view of the lesions, images were selected with sows lying on their right and left sides with all legs extended. A total of 824 RGB images from 70 sows with lesions at various stages of development were identified and annotated. Three deep learning-based object detection models, YOLOv5, YOLOv8, and Faster-RCNN, pre-trained with the COCO and ImageNet datasets, were implemented to localize the lesion area. YOLOv5 was the best predictor as it was able to detect lesions with an mAP@0.5 of 0.92. To estimate the lesion area, lesion pixel segmentation was carried out on the localized region using traditional image processing techniques like Otsu's binarization and adaptive thresholding alongside DL-based segmentation models based on U-Net architecture. In conclusion, this study demonstrates the potential of computer vision techniques in effectively detecting and assessing the size of shoulder lesions in breeding sows, providing a promising avenue for improving sow welfare and reducing economic losses.

HIV proviral genetic diversity, compartmentalization and inferred dynamics in lung and blood during long-term suppressive antiretroviral therapy.

The lung is an understudied site of HIV persistence. We isolated 898 subgenomic proviral sequences (nef) by single-genome approaches from blood and lung from nine individuals on long-term suppressive antiretroviral therapy (ART), and characterized genetic diversity and compartmentalization using formal tests. Consistent with clonal expansion as a driver of HIV persistence, identical sequences comprised between 8% to 86% of within-host datasets, though their location (blood vs. lung) followed no consistent pattern. The majority (77%) of participants harboured at least one sequence shared across blood and lung, supporting the migration of clonally-expanded cells between sites. The extent of blood proviral diversity on ART was also a strong indicator of diversity in lung (Spearman's ρ = 0.98, p<0.0001). For three participants, insufficient lung sequences were recovered to reliably investigate genetic compartmentalization. Of the remainder, only two participants showed statistically significant support for compartmentalization when analysis was restricted to distinct proviruses per site, and the extent of compartmentalization was modest in both cases. When all within-host sequences (including duplicates) were considered, the number of compartmentalized datasets increased to four. Thus, while a subset of individuals harbour somewhat distinctive proviral populations in blood and lung, this can simply be due to unequal distributions of clonally-expanded sequences. For two participants, on-ART proviruses were also phylogenetically analyzed in context of plasma HIV RNA populations sampled up to 18 years prior, including pre-ART and during previous treatment interruptions. In both participants, on-ART proviruses represented the most ancestral sequences sampled within-host, confirming that HIV sequences can persist in the body for decades. This analysis also revealed evidence of re-seeding of the reservoir during treatment interruptions. Results highlight the genetic complexity of proviruses persisting in lung and blood during ART, and the uniqueness of each individual's proviral composition. Personalized HIV remission and cure strategies may be needed to overcome these challenges.