Transcriptomic Signatures of Human Immunodeficiency Virus Post-Treatment Control.

Posttreatment controllers (PTCs) are rare HIV-infected individuals who can limit viral rebound after antiretroviral therapy interruption (ATI), but the mechanisms of this remain unclear. To investigate these mechanisms, we quantified various HIV RNA transcripts (via reverse transcription droplet digital PCR [RT-ddPCR]) and cellular transcriptomes (via RNA-seq) in blood cells from PTCs and noncontrollers (NCs) before and two time points after ATI. HIV transcription initiation did not significantly increase after ATI in PTCs or in NCs, whereas completed HIV transcripts increased at early ATI in both groups and multiply-spliced HIV transcripts increased only in NCs. Compared to NCs, PTCs showed lower levels of HIV DNA, more cell-associated HIV transcripts per total RNA at all times, no increase in multiply-spliced HIV RNA at early or late ATI, and a reduction in the ratio of completed/elongated HIV RNA after early ATI. NCs expressed higher levels of the IL-7 pathway before ATI and expressed higher levels of multiple cytokine, inflammation, HIV transcription, and cell death pathways after ATI. Compared to the baseline, the NCs upregulated interferon and cytokine (especially TNF) pathways during early and late ATI, whereas PTCs upregulated interferon and p53 pathways only at early ATI and downregulated gene translation during early and late ATI. In NCs, viral rebound after ATI is associated with increases in HIV transcriptional completion and splicing, rather than initiation. Differences in HIV and cellular transcription may contribute to posttreatment control, including an early limitation of spliced HIV RNA, a delayed reduction in completed HIV transcripts, and the differential expression of the IL-7, p53, and TNF pathways. IMPORTANCE The findings presented here provide new insights into how HIV and cellular gene expression change after stopping ART in both noncontrollers and posttreatment controllers. Posttreatment control is associated with an early ability to limit increases in multiply-spliced HIV RNA, a delayed (and presumably immune-mediated) ability to reverse an initial rise in processive/completed HIV transcripts, and multiple differences in cellular gene expression pathways. These differences may represent correlates or mechanisms of posttreatment control and may provide insight into the development and/or monitoring of therapeutic strategies that are aimed at a functional HIV cure.

New Assay Reveals Vast Excess of Defective over Intact HIV-1 Transcripts in Antiretroviral Therapy-Suppressed Individuals.

Most of the HIV DNA in infected individuals is noninfectious because of deleterious mutations. However, it is unclear how much of the transcribed HIV RNA is potentially infectious or defective. To address this question, we developed and validated a novel intact viral RNA assay (IVRA) that uses droplet digital reverse transcriptase PCR (dd-RT-PCR) for the commonly mutated packaging signal (Psi) and Rev response element (RRE) regions (from the intact proviral DNA assay [IPDA]) to quantify likely intact (Psi+ RRE+), 3' defective (Psi+ RRE-), and 5' defective (Psi- RRE+) HIV RNA. We then applied the IPDA and IVRA to quantify intact and defective HIV DNA and RNA from peripheral CD4+ T cells from 9 antiretroviral therapy (ART)-suppressed individuals. Levels of 3' defective HIV DNA were not significantly different from those of 5' defective HIV DNA, and both were higher than intact HIV DNA. In contrast, 3' defective HIV RNA (median 86 copies/106 cells; 94% of HIV RNA) was much more abundant than 5' defective (2.1 copies/106 cells; 5.6%) or intact (0.6 copies/106 cells; <1%) HIV RNA. Likewise, the frequency of CD4+ T cells with 3' defective HIV RNA was greater than the frequency with 5' defective or intact HIV RNA. Intact HIV RNA was transcribed by a median of 0.018% of all proviruses and 2.2% of intact proviruses. The vast excess of 3' defective RNA over 5' defective or intact HIV RNA, which was not observed for HIV DNA, suggests that HIV transcription is completely blocked prior to the RRE in most cells with intact proviruses and/or that cells transcribing intact HIV RNA are cleared at very high rates. IMPORTANCE We developed a new assay that can distinguish and quantify intact (potentially infectious) as well as defective HIV RNA. In ART-treated individuals, we found that the vast majority of all HIV RNA is defective at the 3' end, possibly due to incomplete transcriptional processivity. Only a very small percentage of all HIV RNA is intact, and very few total or intact proviruses transcribe intact HIV RNA. Though rare, this intact HIV RNA is tremendously important because it is necessary to serve as the genome of infectious virions that allow transmission and spread, including rebound after stopping ART. Moreover, intact viral RNA may contribute disproportionately to the immune activation, inflammation, and organ damage observed with untreated and treated HIV infection. The intact viral RNA assay can be applied to many future studies aimed at better understanding HIV pathogenesis and barriers to HIV cure.

The VA National Teleneurology Program (NTNP): Implementing Teleneurology to Improve Equitable Access to Outpatient Neurology Care.

BACKGROUND: Telehealth is increasingly utilized in many healthcare systems to improve access to specialty care and better allocate limited resources, especially for rurally residing persons who face unique barriers to care. OBJECTIVES: The VHA sought to address critical gaps in access to neurology care by developing and implementing the first outpatient National Teleneurology Program (NTNP). DESIGN: Pre-post evaluation of intervention and control sites. PARTICIPANTS: NTNP sites and VA control sites; Veterans completing an NTNP consult and their referring providers. INTERVENTION: Implementation of the NTNP at participating sites. MAIN MEASURES: NTNP and community care neurology (CCN) volume of consults before and after implementation; time to schedule and complete consults; Veteran satisfaction. KEY RESULTS: In FY2021, the NTNP was implemented at 12 VA sites; 1521 consults were placed and 1084 (71.3%) were completed. NTNP consults were scheduled (10.1 vs 29.0 days, p < 0.001) and completed (44.0 vs 96.9 days, p < 0.001) significantly faster than CCN consults. Post-implementation, monthly CCN consult volume was unchanged at NTNP sites compared to pre-implementation (mean change of 4.6 consults per month, [95% CI - 4.3, 13.6]), but control sites had a significant increase (mean change of 24.4 [5.2, 43.7]). The estimated difference in mean change in CCN consults between NTNP and control sites persisted after adjusting for local neurology availability (p < 0.001). Veterans (N = 259) were highly satisfied with NTNP care (mean (SD) overall satisfaction score 6.3 (1.2) on a 7-point Likert scale). CONCLUSIONS: Implementation of NTNP resulted in more timely neurologic care than care in the community. The observed significant increase in monthly CCN consults at non-participating sites during the post-implementation period was not seen at NTNP sites. Veterans were highly satisfied with Teleneurology care.

Novel RT-ddPCR assays for measuring the levels of subgenomic and genomic SARS-CoV-2 transcripts.

The replication of SARS-CoV-2 and other coronaviruses depends on transcription of negative-sense RNA intermediates that serve as the templates for the synthesis of positive-sense genomic RNA (gRNA) and multiple different subgenomic mRNAs (sgRNAs) encompassing fragments arising from discontinuous transcription. Recent studies have aimed to characterize the expression of subgenomic SARS-CoV-2 transcripts in order to investigate their clinical significance. Here, we describe a novel panel of reverse transcription droplet digital PCR (RT-ddPCR) assays designed to specifically quantify multiple different subgenomic SARS-CoV-2 transcripts and distinguish them from transcripts that do not arise from discontinuous transcription at each locus. These assays can be applied to samples from SARS-CoV-2 infected patients to better understand the regulation of SARS-CoV-2 transcription and how different sgRNAs may contribute to viral pathogenesis and clinical disease severity.

Provider Communication and Telepresence Enhance Veteran Satisfaction With Telestroke Consultations.

BACKGROUND AND PURPOSE: Telestroke has been demonstrated to be a cost-effective means to expand access to care and improve outcomes in stroke; however, information on patient perceptions of this system of care delivery are limited. This study seeks to examine patient feedback of a national telestroke system within the Veterans Health Administration. METHODS: Patients who received a telestroke consultation were eligible for a phone interview 2 weeks later, including questions about technology quality, telepresence, and telestroke provider communication. Satisfaction scores ranged from 1 to 7 (higher=more satisfied) and for analyses were dichotomized as 6 to 7 indicating high satisfaction versus <6. Patient variables including stroke severity (measured by the National Institutes of Health Stroke Scale) were obtained from study records. Generalized estimating equation models were used to determine what factors were associated with patient satisfaction. RESULTS: Over 18 months, 186 interviews were completed, and 142 (76%) reported high satisfaction with telestroke. Patients with more severe stroke were less likely to recall the consultation. Factors significantly associated with patient satisfaction were higher ratings of the technology (P<0.0001), telepresence (P<0.0001), provider communication ratings (P<0.0001), and overall Veterans Affairs satisfaction (P=0.02). In the multivariate model, telepresence (odds ratio, 3.10 [95% CI, 1.81-5.31]) and provider ratings (odds ratio, 2.37 [95% CI, 1.20-4.68]) were independently associated with satisfaction. Veterans who were satisfied were more likely to recommend the technology (P<0.0001). CONCLUSIONS: Provider qualities, including telepresence and provider ratings, were associated with overall Veteran satisfaction with the telestroke consultation. Technology quality may be necessary but not sufficient to impact patient experience. Training providers to improve telepresence could improve patient experience with telestroke consultation.

An exploration of patient-provider dynamics and childbirth experiences in rural and urban Peru: a qualitative study.

BACKGROUND: Between 2006 and 2013, Peru implemented national programs which drastically decreased rates of maternal and neonatal mortality. However, since 2013, maternal and neonatal mortality in Peru have increased. Additionally, discrimination, abuse, and violence against women persists globally and impacts birthing experiences and mental health. This qualitative study sought to better understand the attitudes and beliefs regarding childbirth among women and providers in Southern Peru. This study also explores how these beliefs influence utilization of skilled care, patient-provider dynamics, and childbirth experiences and identifies factors that impact providers' provision of care. METHODS: Thirty semi-structured interviews were conducted with 15 participants from rural Colca Canyon and 15 participants from urban Arequipa between April and May 2018. In each region, 10 women who had experienced recent births and five providers were interviewed. Provider participants predominantly identified as female and were mostly midwives. All interviews were conducted, transcribed, and coded in Spanish. A framework analysis was followed, and data were charted into two separate thematic frameworks using contextual and evaluative categories of conceptualization of childbirth. RESULTS: All recent births discussed were facility-based births. Four domains emerged: women's current birth experiences, provision of childbirth care, beliefs about childbirth among women and providers, and future health-seeking behavior. Findings suggest that women's feelings of helplessness and frustration were exacerbated by their unmet desire for respectful maternity care and patient advocacy or companionship. Providers attributed strain to perceived patient characteristics and insufficient support, including resources and staff. CONCLUSIONS: Our findings suggest current childbirth experiences placed strain on the patient-provider dynamic and influenced women's attitudes and beliefs about future experiences. Currently, the technical quality of safe childbirth is the main driver of skilled birth attendance and facility-based births for women regardless of negative experiences. However, lack of respectful maternity care has been shown to have major long-term implications for women and subsequently, their children. This is one of the first studies to describe the nuances of patient-provider relationships and women's childbirth experiences in rural and urban Peru.

Building cohesion in distributed telemedicine teams: findings from the Department of Veterans Affairs National Telestroke Program.

BACKGROUND: As telemedicine adoption increases, so does the importance of building cohesion among physicians in telemedicine teams. For example, in acute telestroke services, stroke specialists provide rapid remote stroke assessment and treatment to patients at hospitals without stroke specialty care. In the National Telestroke Program (NTSP) of the U.S. Department of Veterans Affairs, a virtual (distributed) hub of stroke specialists throughout the country provides 24/7 consultations nationwide. We examined how these specialists adapted to distributed teamwork, and we identified cohesion-related factors in program development and support. METHODS: We studied the virtual hub of stroke specialists employed by the NTSP. Semi-structured, confidential interviews with stroke specialists in the virtual hub were recorded and transcribed. We explored the extent to which these specialists had developed a sense of shared identity and team cohesion, and we identified factors in this development. Using a qualitative approach with constant comparison methods, two researchers coded each interview transcript independently using a shared codebook. We used matrix displays to identify themes, with special attention to team cohesion, communication, trust, and satisfaction. RESULTS: Of 13 specialists with at least 8 months of NTSP practice, 12 completed interviews; 7 had previously practiced in telestroke programs in other healthcare systems. Interviewees reported high levels of trust and team cohesion, sometimes even more with their virtual colleagues than with co-located colleagues. Factors facilitating perceived team cohesion included a weekly case conference call, a sense of transparency in discussing challenges, engagement in NTSP development tasks, and support from the NTSP leadership. Although lack of in-person contact was associated with lower cohesion, annual in-person NTSP meetings helped mitigate this issue. Despite technical challenges in establishing a new telehealth system within existing national infrastructure, providers reported high levels of satisfaction with the NTSP. CONCLUSION: A virtual telestroke hub can provide a sense of team cohesion among stroke specialists at a level comparable with a standard co-located practice. Engaging in transparent discussion of challenging cases, reviewing new clinical evidence, and contributing to program improvements may promote cohesion in distributed telemedicine teams.

Mfd protects against oxidative stress in Bacillus subtilis independently of its canonical function in DNA repair.

BACKGROUND: Previous reports showed that mutagenesis in nutrient-limiting conditions is dependent on Mfd in Bacillus subtilis. Mfd initiates one type of transcription-coupled repair (TCR); this type of repair is known to target bulky lesions, like those associated with UV exposure. Interestingly, the roles of Mfd in repair of oxidative-promoted DNA damage and regulation of transcription differ. Here, we used a genetic approach to test whether Mfd protected B. subtilis from exposure to two different oxidants. RESULTS: Wild-type cells survived tert-butyl hydroperoxide (t-BHP) exposure significantly better than Mfd-deficient cells. This protective effect was independent of UvrA, a component of the canonical TCR/nucleotide excision repair (NER) pathway. Further, our results suggest that Mfd and MutY, a DNA glycosylase that processes 8-oxoG DNA mismatches, work together to protect cells from lesions generated by oxidative damage. We also tested the role of Mfd in mutagenesis in starved cells exposed to t-BHP. In conditions of oxidative stress, Mfd and MutY may work together in the formation of mutations. Unexpectedly, Mfd increased survival when cells were exposed to the protein oxidant diamide. Under this type of oxidative stress, cells survival was not affected by MutY or UvrA. CONCLUSIONS: These results are significant because they show that Mfd mediates error-prone repair of DNA and protects cells against oxidation of proteins by affecting gene expression; Mfd deficiency resulted in increased gene expression of the OhrR repressor which controls the cellular response to organic peroxide exposure. These observations point to Mfd functioning beyond a DNA repair factor in cells experiencing oxidative stress.

Role of SHP2 phosphatase in KIT-induced transformation: identification of SHP2 as a druggable target in diseases involving oncogenic KIT.

Intracellular mechanism(s) that contribute to promiscuous signaling via oncogenic KIT in systemic mastocytosis and acute myelogenous leukemia are poorly understood. We show that SHP2 phosphatase is essential for oncogenic KIT-induced growth and survival in vitro and myeloproliferative disease (MPD) in vivo. Genetic disruption of SHP2 or treatment of oncogene-bearing cells with a novel SHP2 inhibitor alone or in combination with the PI3K inhibitor corrects MPD by disrupting a protein complex involving p85α, SHP2, and Gab2. Importantly, a single tyrosine at position 719 in oncogenic KIT is sufficient to develop MPD by recruiting p85α, SHP2, and Gab2 complex to oncogenic KIT. Our results demonstrate that SHP2 phosphatase is a druggable target that cooperates with lipid kinases in inducing MPD.

Patient acceptance of teleneurology across neurologic conditions.

INTRODUCTION: Patient acceptability with outpatient teleneurology has been reported within specific conditions, but less is known about acceptability across neurologic conditions. The study objective was to compare the acceptability of teleneurology between patients with various neurological conditions and determine what other factors influence acceptability. METHODS: This was a prospective study of Veterans who completed new outpatient teleneurology visits with the Department of Veterans Affairs National Teleneurology Program. Visits were conducted via video to home or video to the outpatient clinic. Patient acceptability was assessed via telephone interview two weeks post-visit. Acceptability was a summed score (3-21) of three 7-point Likert questions (higher = more acceptable). Clinical diagnosis categories were based on the neurologists' ICD10 diagnosis code. Acceptability score was modeled using a censored Tobit model controlling for demographics, type of tele-visit, medical comorbidity, and ICD10 category. RESULTS: In FY 2021, 277 of 637 (43.5%) patients completed an interview with analyzable acceptability data. Of these 277, 70 (25.3%) had codes indicating headache, 46 (16.6%) movement disorder, 45 (16.2%) general symptoms, and 116 (41.9%) for all other categories. Mean patient acceptability was 18.3 (SD 3.2). There was no significant difference in scores between these groups. The only factor independently related to acceptability was medical comorbidity, with higher comorbidity associated with higher acceptability scores. DISCUSSION: Patients find their outpatient teleneurology experience highly acceptable independent of neurologic condition. Those with more comorbidity report higher acceptability. Use of teleneurology may be useful and acceptable across many outpatient neurologic conditions including for more medically complex patients.

Neurodevelopmental outcomes of umbilical cord blood transplantation in metachromatic leukodystrophy.

Metachromatic leukodystrophy (MLD) is an inherited demyelinating disease that causes progressive neurologic deterioration, leading to severe motor disability, developmental regression, seizures, blindness, deafness, and death. The disease presents as a late-infantile, juvenile, or adult form. Hematopoietic stem cell transplantation has been shown to slow disease progression. The purpose of this longitudinal study was to evaluate long-term treatment outcomes after unrelated donor umbilical cord blood (UCB) transplantation in pediatric patients according to disease burden and age at onset (ie, late-infantile versus juvenile). Engraftment, survival, treatment-related toxicity, graft-versus-host disease, neurophysiologic measures, and neurodevelopmental function were assessed. To evaluate whether signal intensity abnormalities on magnetic resonance imaging (ie, modified Loes scores) predict post-transplant cognitive and gross motor development, a general linear mixed model was fit to the data. Twenty-seven patients underwent transplantation after myeloablative chemotherapy; 24 patients engrafted after the initial transplantation. Seven patients died of infection, regimen-related toxicity, or disease progression. Twenty patients (6 with late-infantile onset and 14 with juvenile onset) were followed for a median of 5.1 years (range, 2.4 to 14.7). We found that patients with motor function symptoms at the time of transplant did not improve after transplantation. Brainstem auditory evoked responses, visual evoked potentials, electroencephalogram, and/or peripheral nerve conduction velocities stabilized or improved in juvenile patients but continued to worsen in most patients with the late-infantile presentation. Pretransplant modified Loes scores were highly correlated with developmental outcomes and predictive of cognitive and motor function. Children who were asymptomatic at the time of transplantation benefited most from the procedure. Children with juvenile onset and minimal symptoms showed stabilization or deterioration of motor skills but maintained cognitive skills. Overall, children with juvenile onset had better outcomes than those with late-infantile onset. As in other leukodystrophies, early intervention correlated with optimal outcomes. We conclude that UCB transplantation benefits children with presymptomatic late-infantile MLD or minimally symptomatic juvenile MLD.

The PI3K pathway drives the maturation of mast cells via microphthalmia transcription factor.

Mast cell maturation is poorly understood. We show that enhanced PI3K activation results in accelerated maturation of mast cells by inducing the expression of microphthalmia transcription factor (Mitf). Conversely, loss of PI3K activation reduces the maturation of mast cells by inhibiting the activation of AKT, leading to reduced Mitf but enhanced Gata-2 expression and accumulation of Gr1(+)Mac1(+) myeloid cells as opposed to mast cells. Consistently, overexpression of Mitf accelerates the maturation of mast cells, whereas Gata-2 overexpression mimics the loss of the PI3K phenotype. Expressing the full-length or the src homology 3- or BCR homology domain-deleted or shorter splice variant of the p85α regulatory subunit of PI3K or activated AKT or Mitf in p85α-deficient cells restores the maturation but not growth. Although deficiency of both SHIP and p85α rescues the maturation of SHIP(-/-) and p85α(-/-) mast cells and expression of Mitf; in vivo, mast cells are rescued in some, but not all tissues, due in part to defective KIT signaling, which is dependent on an intact src homology 3 and BCR homology domain of p85α. Thus, p85α-induced maturation, and growth and survival signals, in mast cells can be uncoupled.

The VA National TeleNeurology Program implementation: a mixed-methods evaluation guided by RE-AIM framework.

Introduction: The Veteran Affairs (VA) Office of Rural Health (ORH) funded the Veterans Health Administration (VHA) National TeleNeurology Program (NTNP) as an Enterprise-Wide Initiative (EWI). NTNP is an innovative healthcare delivery model designed to fill the patient access gap for outpatient neurological care especially for Veterans residing in rural communities. The specific aim was to apply the RE-AIM framework in a pragmatic evaluation of NTNP services. Materials and methods: We conducted a prospective implementation evaluation. Guided by the pragmatic application of the RE-AIM framework, we conceptualized a mixed-methods evaluation for key metrics: (1) reach into the Veteran patient population assessed as total NTNP new patient consult volume and total NTNP clinical encounters (new and return); (2) effectiveness through configurational analysis of conditions leading to high Veteran satisfaction and referring providers perceived effectiveness; (3) adoption and implementation by VA sites through site staff and NTNP interviews; (4) implementation success through perceived management, implementation barriers, facilitators, and adaptations and through rapid qualitative analysis of multiple stakeholders' assessments; and (5) maintenance of NTNP through monitoring quarterly TeleNeurology consultation volume. Results: NTNP was successfully implemented in 13 VA Medical Centers over 2 years. The total NTNP new patient consult volume in fiscal year 2021 (FY21) was 836 (58% rurally residing); this increased to 1,706 in fiscal year 2022 (FY22) (55% rurally residing). Total (new and follow-up) NTNP clinical encounters were 1,306 in FY21 and 3,730 in FY22. Overall, the sites reported positive experiences with program implementation and perceived that the program was serving Veterans with little access to neurological care. Veterans also reported high satisfaction with the NTNP program. We identified the patient level of perceived excellent teleneurologist-patient communications, reduced need to drive to get care, and that NTNP provided care that the Veteran otherwise could not access as key factors related to high Veteran satisfaction. Conclusions: The VA NTNP demonstrated substantial reach, adoption, effectiveness, implementation success, and maintenance over the first 2 years of the program. The NTNP was highly acceptable to both the clinical providers making the referrals and the Veterans receiving the referred video care. The pragmatic application of the RE-AIM framework to guide implementation evaluations is appropriate, comprehensive, and recommended for future applications.

Impact of Telestroke Implementation on Emergency Department Transfer Rate.

BACKGROUND AND OBJECTIVES: Telestroke networks are associated with improved outcomes from acute ischemic stroke (AIS) and facilitate greater access to care, particularly in underserved regions. These networks also have the potential to influence patient disposition through avoiding unnecessary interhospital transfers. This study examines the effect of implementation of the VA National Telestroke Program (NTSP) on interhospital transfer among Veterans. METHODS: We analyzed patients with AIS presenting to the emergency departments of 21 VA hospitals before and after telestroke implementation. Transfer rates were determined through review of administrative data and chart review and patient and facility-level characteristics were collected to identify predictors of transfer. Comparisons were made using t test, Wilcoxon rank sum, and χ 2 analysis. Multivariable logistic regression with sensitivity analysis was conducted to assess the influence of telestroke implementation on transfer rates. RESULTS: We analyzed 3,488 stroke encounters (1,056 pre-NTSP and 2,432 post-NTSP). Following implementation, we observed an absolute 14.4% decrease in transfers across all levels of stroke center designation. Younger age, higher stroke severity, and shorter duration from symptom onset were associated with transfer. At the facility level, hospitals with lower annual stroke volume were more likely to transfer; 1 hospital saw an increase in transfer rates following implementation. After adjusting for patient and facility characteristics, the implementation of VA NTSP resulted in a nearly 60% reduction in odds of transfer (odds ratio 0.39 [0.19, 0.77]). DISCUSSION: In addition to improving treatment in acute stroke, telestroke networks have the potential to positively affect the efficiency of interhospital networks through disposition optimization and the avoidance of unnecessary transfers.

Novel assays to investigate the mechanisms of latent infection with HIV-2.

Although there have been great advancements in the field of HIV treatment and prevention, there is no cure. There are two types of HIV: HIV-1 and HIV-2. In addition to genetic differences between the two types of HIV, HIV-2 infection causes a slower disease progression, and the rate of new HIV-2 infections has dramatically decreased since 2003. Like HIV-1, HIV-2 is capable of establishing latent infection in CD4+ T cells, thereby allowing the virus to evade viral cytopathic effects and detection by the immune system. The mechanisms underlying HIV latency are not fully understood, rendering this a significant barrier to development of a cure. Using RT-ddPCR, we previously demonstrated that latent infection with HIV-1 may be due to blocks to HIV transcriptional elongation, distal transcription/polyadenylation, and multiple splicing. In this study, we describe the development of seven highly-specific RT-ddPCR assays for HIV-2 that can be applied to the study of HIV-2 infections and latency. We designed and validated seven assays targeting different HIV-2 RNA regions along the genome that can be used to measure the degree of progression through different blocks to HIV-2 transcription and splicing. Given that HIV-2 is vastly understudied relative to HIV-1 and that it can be considered a model of a less virulent infection, application of these assays to studies of HIV-2 latency may inform new therapies for HIV-2, HIV-1, and other retroviruses.

Non-B DNA-Forming Motifs Promote Mfd-Dependent Stationary-Phase Mutagenesis in Bacillus subtilis.

Transcription-induced mutagenic mechanisms limit genetic changes to times when expression happens and to coding DNA. It has been hypothesized that intrinsic sequences that have the potential to form alternate DNA structures, such as non-B DNA structures, influence these mechanisms. Non-B DNA structures are promoted by transcription and induce genome instability in eukaryotic cells, but their impact in bacterial genomes is less known. Here, we investigated if G4 DNA- and hairpin-forming motifs influence stationary-phase mutagenesis in Bacillus subtilis. We developed a system to measure the influence of non-B DNA on B. subtilis stationary-phase mutagenesis by deleting the wild-type argF at its chromosomal position and introducing IPTG-inducible argF alleles differing in their ability to form hairpin and G4 DNA structures into an ectopic locus. Using this system, we found that sequences predicted to form non-B DNA structures promoted mutagenesis in B. subtilis stationary-phase cells; such a response did not occur in growing conditions. We also found that the transcription-coupled repair factor Mfd promoted mutagenesis at these predicted structures. In summary, we showed that non-B DNA-forming motifs promote genetic instability, particularly in coding regions in stressed cells; therefore, non-B DNA structures may have a spatial and temporal mutagenic effect in bacteria. This study provides insights into mechanisms that prevent or promote mutagenesis and advances our understanding of processes underlying bacterial evolution.

Valuation of Life With Disability: An International Comparison Study in Vietnam, Peru, and Haiti.

The authors measured perceived quality of life for 4 disabilities among 450 adults in 3 resource-limited countries, measuring mean utilities using time trade-off, and surveying participants on 35 sociocultural characteristics to compare utilities for disabilities by country and examine associated sociocultural characteristics. Mean utilities were >0 for mild and moderate, but <0 for severe and profound. Utilities differed across countries (P = .007, .000, .017, .000 for mild, moderate, severe, profound, respectively). Vietnamese utilities correlated with residence (P = .03, moderate), education (P = .03, severe), and number of children (P = .03, moderate). Peruvian utilities correlated with education (P = .05, mild; P = .05, severe), experience with disability (P = .001, mild), gender (P = .04, moderate; P = .03, profound), number of hospitalizations (P = .04, severe). In Haiti, the only correlate was rejection (P = .02, moderate). Culture-specific variables differentially shape perceptions of disability in developing countries, thereby affecting cost-effectiveness calculations. Given substantially negative perceptions, reducing major disability would improve cost-effectiveness of health-policy decisions more than reducing mortality.

Abemaciclib as initial therapy for advanced breast cancer: MONARCH 3 updated results in prognostic subgroups.

In MONARCH 3, continuous dosing of abemaciclib with an aromatase inhibitor (AI) conferred significant clinical benefit to postmenopausal women with HR+, HER2- advanced breast cancer. We report data for clinically prognostic subgroups: liver metastases, progesterone receptor status, tumor grade, bone-only disease, ECOG performance status, and treatment-free interval (TFI) from an additional 12-month follow-up (after final progression-free survival [PFS] readout). In the intent-to-treat population, after median follow-up of approximately 39 months, the updated PFS was 28.2 versus 14.8 months (hazard ratio [HR], 0.525; 95% confidence interval, 0.415-0.665) in abemaciclib versus placebo arms, respectively. Time to chemotherapy (HR, 0.513), time to second disease progression (HR, 0.637), and duration of response (HR, 0.466) were also statistically significantly prolonged with the addition of abemaciclib to AI. Treatment benefit was observed across all subgroups, as evidenced by objective response rate change from the addition of abemaciclib to AI, with the largest effects observed in patients with liver metastases, progesterone receptor-negative tumors, high-grade tumors, or TFI < 36 months. Extended follow-up in the MONARCH 3 trial further confirmed that the addition of abemaciclib to AI conferred significant treatment benefit to all subgroups, including those with poorer prognosis.

Mfd Affects Global Transcription and the Physiology of Stressed Bacillus subtilis Cells.

For several decades, Mfd has been studied as the bacterial transcription-coupled repair factor. However, recent observations indicate that this factor influences cell functions beyond DNA repair. Our lab recently described a role for Mfd in disulfide stress that was independent of its function in nucleotide excision repair and base excision repair. Because reports showed that Mfd influenced transcription of single genes, we investigated the global differences in transcription in wild-type and mfd mutant growth-limited cells in the presence and absence of diamide. Surprisingly, we found 1,997 genes differentially expressed in Mfd- cells in the absence of diamide. Using gene knockouts, we investigated the effect of genetic interactions between Mfd and the genes in its regulon on the response to disulfide stress. Interestingly, we found that Mfd interactions were complex and identified additive, epistatic, and suppressor effects in the response to disulfide stress. Pathway enrichment analysis of our RNASeq assay indicated that major biological functions, including translation, endospore formation, pyrimidine metabolism, and motility, were affected by the loss of Mfd. Further, our RNASeq findings correlated with phenotypic changes in growth in minimal media, motility, and sensitivity to antibiotics that target the cell envelope, transcription, and DNA replication. Our results suggest that Mfd has profound effects on the modulation of the transcriptome and on bacterial physiology, particularly in cells experiencing nutritional and oxidative stress.

Transcription-associated mutation in Bacillus subtilis cells under stress.

Adaptive (stationary phase) mutagenesis is a phenomenon by which nondividing cells acquire beneficial mutations as a response to stress. Although the generation of adaptive mutations is essentially stochastic, genetic factors are involved in this phenomenon. We examined how defects in a transcriptional factor, previously reported to alter the acquisition of adaptive mutations, affected mutation levels in a gene under selection. The acquisition of mutations was directly correlated to the level of transcription of a defective leuC allele placed under selection. To further examine the correlation between transcription and adaptive mutation, we placed a point-mutated allele, leuC427, under the control of an inducible promoter and assayed the level of reversion to leucine prototrophy under conditions of leucine starvation. Our results demonstrate that the level of Leu(+) reversions increased significantly in parallel with the induced increase in transcription levels. This mutagenic response was not observed under conditions of exponential growth. Since transcription is a ubiquitous biological process, transcription-associated mutagenesis may influence evolutionary processes in all organisms.