Transcriptional and chromatin-based partitioning mechanisms uncouple protein scaling from cell size.

Biosynthesis scales with cell size such that protein concentrations generally remain constant as cells grow. As an exception, synthesis of the cell-cycle inhibitor Whi5 "sub-scales" with cell size so that its concentration is lower in larger cells to promote cell-cycle entry. Here, we find that transcriptional control uncouples Whi5 synthesis from cell size, and we identify histones as the major class of sub-scaling transcripts besides WHI5 by screening for similar genes. Histone synthesis is thereby matched to genome content rather than cell size. Such sub-scaling proteins are challenged by asymmetric cell division because proteins are typically partitioned in proportion to newborn cell volume. To avoid this fate, Whi5 uses chromatin-binding to partition similar protein amounts to each newborn cell regardless of cell size. Disrupting both Whi5 synthesis and chromatin-based partitioning weakens G1 size control. Thus, specific transcriptional and partitioning mechanisms determine protein sub-scaling to control cell size.

Stakeholder-led design of a mobile HIV clinic model to enhance engagement and retention in HIV care in the Southern United States.

To foster retention of people living with HIV (PLWH) in HIV care in the Southern United States, we aimed to develop a stakeholder-driven mobile HIV clinic (MHC) model. From June 2019 to May 2021 we conducted a mixed-methods study: 50 surveys with out-of-care PLWH and 41 in-depth interviews with PLWH, HIV clinic staff, city officials, AIDS service organizations, and mobile clinics to examine preferences for MHC implementation. Survey data was analyzed descriptively, and interview transcripts were coded thematically. Participants recommended the MHC: (1) have nondescript exterior and HIV services nested in non-HIV care to foster confidentiality, (2) be located along public transportation and have extended hours to promote accessibility, (3) have established protocols addressing security, biosafety, and data safety; (4) provide comprehensive clinical and support services to address retention barriers; and (5) be integrated within the health system, use low-cost, diverse staffing, and establish appointment notification systems. By informing MHC design, these findings add to the toolbox of strategies that can render HIV care more accessible.

"I Get the Flavors and It Makes Me Love Vaping More": How and Why Youth Users Modify Electronic Nicotine Delivery Systems.

INTRODUCTION: Youth in the United States are using electronic nicotine delivery systems (ENDS) at a high rate. Modifications to ENDS by youth can introduce additional health hazards which have not been previously considered. To better understand these risks, we need more information on what these modifications are, the motivations behind them, and the sources of information on modifications. AIMS AND METHODS: Utilizing a trained moderator, in 2020-2021, we conducted one-on-one interviews with 19 youth ENDS users aged 16-17 living in the United States and analyzed their responses using a qualitative description approach. RESULTS: The most prominent modification was to the e-liquid; youth indicated they mixed e-juices to create new flavors and added substances not intended for vaping, including illicit drugs such as cannabis and cocaine. Few youths from our sample were interested in achieving a specific nicotine level to vape, and modifications to the battery, coil and wick were less frequently mentioned. Some of these modifications were motivated by a desire to achieve specific experiences with their device. At other times, modifications were made due to necessity because of limited access to ENDS devices and supplies. YouTube and peers were the main sources of information about modifying. CONCLUSIONS: Youth are making modifications that are both intended and unintended by the manufacturer. Adding illicit drugs and other substances not made for vaping is of particular concern. Understanding how youth modify ENDS and how that changes their use is important to guide regulatory policy intended to reduce harm to youth from ENDS use. IMPLICATIONS: Youth from our study indicated that they make modifications to the ENDS devices, specifically to the e-liquid. These modifications are both intended by the manufacturer, such as changing the e-liquid and replacing coils, and unintended, such as adding substances not meant for vaping. Future policies aimed at reducing youth ENDS use should consider mandating better safeguards against modifications that appeal to youth.

PP2ACdc55 dephosphorylates Pds1 and inhibits spindle elongation in S. cerevisiae.

PP2ACdc55 (the form of protein phosphatase 2A containing Cdc55) regulates cell cycle progression by reversing cyclin-dependent kinase (CDK)- and polo-like kinase (Cdc5)-dependent phosphorylation events. In S. cerevisiae, Cdk1 phosphorylates securin (Pds1), which facilitates Pds1 binding and inhibits separase (Esp1). During anaphase, Esp1 cleaves the cohesin subunit Scc1 and promotes spindle elongation. Here, we show that PP2ACdc55 directly dephosphorylates Pds1 both in vivo and in vitro Pds1 hyperphosphorylation in a cdc55 deletion mutant enhanced the Pds1-Esp1 interaction, which played a positive role in Pds1 nuclear accumulation and in spindle elongation. We also show that nuclear PP2ACdc55 plays a role during replication stress to inhibit spindle elongation. This pathway acted independently of the known Mec1, Swe1 or spindle assembly checkpoint (SAC) checkpoint pathways. We propose a model where Pds1 dephosphorylation by PP2ACdc55 disrupts the Pds1-Esp1 protein interaction and inhibits Pds1 nuclear accumulation, which prevents spindle elongation, a process that is elevated during replication stress.

Understanding and addressing privacy and confidentiality concerns associated with the provision of mobile HIV care: a qualitative study.

Less than half the people with HIV (PLWH) in the United States (US) are retained in HIV care, underscoring the importance of novel reengagement and retention strategies. Mobile HIV clinics (MHCs) are one such strategy, but privacy and confidentiality concerns have limited their use. As part of a larger mixed-methods study in Atlanta, Georgia, from June 2019- July 2020, we conducted 41 qualitative interviews with key stakeholders to explore confidentiality, privacy and stigma concerns and strategies to address them. Interviews were recorded, transcribed and coded thematically. Four key themes emerged: 1) the need to understand MHC acceptance in the context of high HIV stigma in the South, 2) the multidimensionality of confidentiality and stigma concerns (e.g., related to exterior labeling, layout, location attracting unwanted attention), 3) the counter perspective: potential for MHCs to positively reframe HIV and reduce stigma, and 4) strategies to overcome stigma and confidentiality concerns, including co-delivery of non-HIV services, unidirectional flow, and non-HIV exterior labeling. In furthering understanding of the breadth of privacy and confidentiality concerns associated with an MHC and strategies for addressing them, this exploratory study lays a critical foundation for the development of an MHC to reengage and retain PLWH in the US.

Form and function of topologically associating genomic domains in budding yeast.

The genome of metazoan cells is organized into topologically associating domains (TADs) that have similar histone modifications, transcription level, and DNA replication timing. Although similar structures appear to be conserved in fission yeast, computational modeling and analysis of high-throughput chromosome conformation capture (Hi-C) data have been used to argue that the small, highly constrained budding yeast chromosomes could not have these structures. In contrast, herein we analyze Hi-C data for budding yeast and identify 200-kb scale TADs, whose boundaries are enriched for transcriptional activity. Furthermore, these boundaries separate regions of similarly timed replication origins connecting the long-known effect of genomic context on replication timing to genome architecture. To investigate the molecular basis of TAD formation, we performed Hi-C experiments on cells depleted for the Forkhead transcription factors, Fkh1 and Fkh2, previously associated with replication timing. Forkhead factors do not regulate TAD formation, but do promote longer-range genomic interactions and control interactions between origins near the centromere. Thus, our work defines spatial organization within the budding yeast nucleus, demonstrates the conserved role of genome architecture in regulating DNA replication, and identifies a molecular mechanism specifically regulating interactions between pericentric origins.

It's addiction at this Point": A qualitative examination of youth E-cigarette use trajectory in the United States.

E-cigarettes (electronic cigarettes) have been the most used tobacco product among US youth since 2014, reaching a plateau during the COVID-19 pandemic. Youth e-cigarette use is associated with negative health consequences such as impaired cognitive functioning. For many, the COVID-19 pandemic altered social interactions, harm perceptions, and product availability. This changed the frequency and locations in which youth use e-cigarettes. To better understand youth e-cigarette use, we need more information on factors that can alter e-cigarette use, specifically, how the pandemic changed e-cigarette use among youth. In 2020-2021, we conducted online, individual interviews with 19 youth (aged 13-17) e-cigarette users living in the US to explore how COVID-19 impacted their e-cigarette use. Youth described a progression of e-cigarette use from initial experimentation, regular social use, and ultimately to nicotine addiction demonstrated by individual use in isolation. Many youth initiated e-cigarette use due to influences by friends or family members. Youth discussed progression to social use, with social interactions as an important reason for use and an avenue for expanding one's knowledge of e-cigarettes. After a period of time, youth began to recognize that the social interactions mattered less, suggesting to them that they had become addicted. This realization became more apparent during COVID-19, which changed how youth used e-cigarettes, especially around where use was occurring, health concerns, and use behavior and frequency. In our interviews, youth trajectory began with an initiation with family and friends, progressed to social use, and eventually developed to addiction, at which point social use was no longer the primary motivation for e-cigarette use. Understanding the trajectory of e-cigarette use will allow for effective interventions that reduce harm to youth from e-cigarette use.

Cancer detection in clinical practice and using blood-based liquid biopsy: A retrospective audit of over 350 dogs.

BACKGROUND: Guidelines-driven screening protocols for early cancer detection in dogs are lacking, and cancer often is detected at advanced stages. HYPOTHESIS/OBJECTIVES: To examine how cancer typically is detected in dogs and whether the addition of a next-generation sequencing-based "liquid biopsy" test to a wellness visit has the potential to enhance cancer detection. ANIMALS: Client-owned dogs with definitive cancer diagnoses enrolled in a clinical validation study for a novel blood-based multicancer early detection test. METHODS: Retrospective medical record review was performed to establish the history and presenting complaint that ultimately led to a definitive cancer diagnosis. Blood samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer. RESULTS: In an unselected cohort of 359 cancer-diagnosed dogs, 4% of cases were detected during a wellness visit, 8% were detected incidentally, and 88% were detected after the owner reported clinical signs suggestive of cancer. Liquid biopsy detected disease in 54.7% (95% confidence interval [CI], 49.5%-59.8%) of patients, including 32% of dogs with early-stage cancer, 48% of preclinical dogs, and 84% of dogs with advanced-stage disease. CONCLUSIONS/CLINICAL IMPORTANCE: Most cases of cancer were diagnosed after the onset of clinical signs; only 4% of dogs had cancer detected using the current standard of care (i.e., wellness visit). Liquid biopsy has the potential to increase detection of cancer when added to a dog's wellness visit.

Rapid HIV testing in large urban jails.

HIV prevalence is higher in jails than in the community, yet many jails do not conduct HIV testing. Jails in Baltimore, Maryland; Philadelphia, Pennsylvania; and the District of Columbia have implemented innovative rapid HIV testing programs. We have summarized the results of these programs, including the numbers of persons tested, rapid and confirmatory HIV test results, and numbers of persons newly diagnosed with HIV. We have described facilitators and challenges of implementation. These programs confirmed that rapid HIV testing in jails was feasible and identified undiagnosed HIV infection. Challenges included limited space to provide confidential rapid HIV testing and rapid turnover of detainees. Implementation required collaboration between local governments, health agencies, and correctional institutions. These programs serve as models for expanding rapid HIV testing in jails.

Electric field guided assembly of one-dimensional nanostructures for high performance sensors.

Various nanowire or nanotube-based devices have been demonstrated to fulfill the anticipated future demands on sensors. To fabricate such devices, electric field-based methods have demonstrated a great potential to integrate one-dimensional nanostructures into various forms. This review paper discusses theoretical and experimental aspects of the working principles, the assembled structures, and the unique functions associated with electric field-based assembly. The challenges and opportunities of the assembly methods are addressed in conjunction with future directions toward high performance sensors.

Barriers and facilitators to use of a mobile HIV care model to re-engage and retain out-of-care people living with HIV in Atlanta, Georgia.

Novel strategies to re-engage and retain people living with HIV (PLWH) who are out of care are greatly needed. While mobile clinics have been used effectively for HIV testing and linkage, evidence guiding their use in providing HIV care domestically has been limited. To guide the development of a mobile HIV clinic (MHC) model as a strategy to re-engage and retain PLWH who are out of care, we aimed to explore stakeholder perceptions of barriers and facilitators to MHC implementation and use. From June 2019-July 2020, we conducted 41 in-depth interviews with HIV clinic providers, administrators, staff, legal authorities, and community advisory board members, PLWH, AIDS service organizations and city officials in Atlanta, Georgia, and domestic and international mobile health clinics to explore barriers and facilitators to use of MHCs. Interviews were transcribed, coded and thematically analysed. Barriers raised include potential for: breach of confidentiality with resulting heightened stigmatization, fractured continuity of care, safety concerns, staffing challenges, and low community acceptance of MHC presence in their locality. Participants provided suggestions regarding appropriate exterior design, location, timing, and co-delivery of non-HIV services that could facilitate MHC acceptance and address the concerns. In identifying key barriers and facilitators to MHC use, this study informs design and implementation of an MHC as a novel strategy for re-engaging and retaining PLWH who are out of care.

Regulation of mRNA translation as a conserved mechanism of longevity control.

Appropriate regulation of mRNA translation is essential for growth and survival and the pathways that regulate mRNA translation have been highly conserved throughout eukaryotic evolution. Translation is controlled by a complex set of mechanisms acting at multiple levels, ranging from global protein synthesis to individual mRNAs. Recently, several mutations that perturb regulation of mRNA translation have also been found to increase longevity in three model organisms: the buddingyeast Saccharomyces cerevisiae, the nematode Caenorhabditis elegans and the fruit fly Drosophila melanogaster. Many of these translation control factors can be mapped to a single pathway downstream of the nutrient responsive target of rapamycin (TOR) kinase. In this chapter, we will review the data suggesting that mRNA translation is an evolutionarily conserved modifier of longevity and discuss potential mechanisms by which mRNA translation could influence aging and age-associated disease in different species.

The Adder Phenomenon Emerges from Independent Control of Pre- and Post-Start Phases of the Budding Yeast Cell Cycle.

Although it has long been clear that cells actively regulate their size, the molecular mechanisms underlying this regulation have remained poorly understood. In budding yeast, cell size primarily modulates the duration of the cell-division cycle by controlling the G1/S transition known as Start. We have recently shown that the rate of progression through Start increases with cell size, because cell growth dilutes the cell-cycle inhibitor Whi5 in G1. Recent phenomenological studies in yeast and bacteria have shown that these cells add an approximately constant volume during each complete cell cycle, independent of their size at birth. These results seem to be in conflict, as the phenomenological studies suggest that cells measure the amount they grow, rather than their size, and that size control acts over the whole cell cycle, rather than specifically in G1. Here, we propose an integrated model that unifies the adder phenomenology with the molecular mechanism of G1/S cell-size control. We use single-cell microscopy to parameterize a full cell-cycle model based on independent control of pre- and post-Start cell-cycle periods. We find that our model predicts the size-independent amount of cell growth during the full cell cycle. This suggests that the adder phenomenon is an emergent property of the independent regulation of pre- and post-Start cell-cycle periods rather than the consequence of an underlying molecular mechanism measuring a fixed amount of growth.

Sorbitol treatment extends lifespan and induces the osmotic stress response in Caenorhabditis elegans.

The response to osmotic stress is a highly conserved process for adapting to changing environmental conditions. Prior studies have shown that hyperosmolarity by addition of sorbitol to the growth medium is sufficient to increase both chronological and replicative lifespan in the budding yeast, Saccharomyces cerevisiae. Here we report a similar phenomenon in the nematode Caenorhabditis elegans. Addition of sorbitol to the nematode growth medium induces an adaptive osmotic response and increases C. elegans lifespan by about 35%. Lifespan extension from 5% sorbitol behaves similarly to dietary restriction in a variety of genetic backgrounds, increasing lifespan additively with mutation of daf-2(e1370) and independently of daf-16(mu86), sir-2.1(ok434), aak-2(ok524), and hif-1(ia04). Dietary restriction by bacterial deprivation or mutation of eat-2(ad1113) fails to further extend lifespan in the presence of 5% sorbitol. Two mutants with constitutive activation of the osmotic response, osm-5(p813) and osm-7(n1515), were found to be long-lived, and lifespan extension from sorbitol required the glycerol biosynthetic enzymes GPDH-1 and GPDH-2. Taken together, these observations demonstrate that exposure to sorbitol at levels sufficient to induce an adaptive osmotic response extends lifespan in worms and define the osmotic stress response pathway as a longevity pathway conserved between yeast and nematodes.

DOCK8 regulates lymphocyte shape integrity for skin antiviral immunity.

DOCK8 mutations result in an inherited combined immunodeficiency characterized by increased susceptibility to skin and other infections. We show that when DOCK8-deficient T and NK cells migrate through confined spaces, they develop cell shape and nuclear deformation abnormalities that do not impair chemotaxis but contribute to a distinct form of catastrophic cell death we term cytothripsis. Such defects arise during lymphocyte migration in collagen-dense tissues when DOCK8, through CDC42 and p21-activated kinase (PAK), is unavailable to coordinate cytoskeletal structures. Cytothripsis of DOCK8-deficient cells prevents the generation of long-lived skin-resident memory CD8 T cells, which in turn impairs control of herpesvirus skin infections. Our results establish that DOCK8-regulated shape integrity of lymphocytes prevents cytothripsis and promotes antiviral immunity in the skin.

UV-photoconversion of ethosuximide from a longevity-promoting compound to a potent toxin.

The anticonvulsant ethosuximide has been previously shown to increase life span and promote healthspan in the nematode Caenorhabditis elegans at millimolar concentrations. Here we report that following exposure to ultraviolet irradiation at 254 nm, ethosuximide is converted into a compound that displays toxicity toward C. elegans. This effect is specific for ethosuximide, as the structurally related compounds trimethadione and succinimide do not show similar toxicities following UV exposure. Killing by UV-irradiated ethosuximide is not attenuated in chemosensory mutants that are resistant to toxicity associated with high doses of non-irradiated ethosuximide. Non-irradiated ethosuximide extends life span at 15°C or 20°C, but not at 25°C, while irradiated ethosuximide shows similar toxicity at all three temperatures. Dietary restriction by bacterial deprivation does not protect against toxicity from irradiated ethosuximide, while non-irradiated ethosuximide further extends the long life spans of restricted animals. These data support the model that ethosuximide extends life span by a mechanism that is, at least partially, distinct from dietary restriction by bacterial deprivation and demonstrates an unexpected photochemical conversion of ethosuximide into a toxic compound by UV light.

Proteasomal regulation of the hypoxic response modulates aging in C. elegans.

The Caenorhabditis elegans von Hippel-Lindau tumor suppressor homolog VHL-1 is a cullin E3 ubiquitin ligase that negatively regulates the hypoxic response by promoting ubiquitination and degradation of the hypoxic response transcription factor HIF-1. Here, we report that loss of VHL-1 significantly increased life span and enhanced resistance to polyglutamine and beta-amyloid toxicity. Deletion of HIF-1 was epistatic to VHL-1, indicating that HIF-1 acts downstream of VHL-1 to modulate aging and proteotoxicity. VHL-1 and HIF-1 control longevity by a mechanism distinct from both dietary restriction and insulin-like signaling. These findings define VHL-1 and the hypoxic response as an alternative longevity and protein homeostasis pathway.