Akt Kinase Activation Mechanisms Revealed Using Protein Semisynthesis.

Akt is a critical protein kinase that drives cancer proliferation, modulates metabolism, and is activated by C-terminal phosphorylation. The current structural model for Akt activation by C-terminal phosphorylation has centered on intramolecular interactions between the C-terminal tail and the N lobe of the kinase domain. Here, we employ expressed protein ligation to produce site-specifically phosphorylated forms of purified Akt1 that are well suited for mechanistic analysis. Using biochemical, crystallographic, and cellular approaches, we determine that pSer473-Akt activation is driven by an intramolecular interaction between the C-tail and the pleckstrin homology (PH)-kinase domain linker that relieves PH domain-mediated Akt1 autoinhibition. Moreover, dual phosphorylation at Ser477/Thr479 activates Akt1 through a different allosteric mechanism via an apparent activation loop interaction that reduces autoinhibition by the PH domain and weakens PIP3 affinity. These results provide a new framework for understanding how Akt is controlled in cell signaling and suggest distinct functions for differentially modified Akt forms.

Spatial atlas of the mouse central nervous system at molecular resolution.

Spatially charting molecular cell types at single-cell resolution across the 3D volume is critical for illustrating the molecular basis of brain anatomy and functions. Single-cell RNA sequencing has profiled molecular cell types in the mouse brain1,2, but cannot capture their spatial organization. Here we used an in situ sequencing method, STARmap PLUS3,4, to profile 1,022 genes in 3D at a voxel size of 194 × 194 × 345 nm3, mapping 1.09 million high-quality cells across the adult mouse brain and spinal cord. We developed computational pipelines to segment, cluster and annotate 230 molecular cell types by single-cell gene expression and 106 molecular tissue regions by spatial niche gene expression. Joint analysis of molecular cell types and molecular tissue regions enabled a systematic molecular spatial cell-type nomenclature and identification of tissue architectures that were undefined in established brain anatomy. To create a transcriptome-wide spatial atlas, we integrated STARmap PLUS measurements with a published single-cell RNA-sequencing atlas1, imputing single-cell expression profiles of 11,844 genes. Finally, we delineated viral tropisms of a brain-wide transgene delivery tool, AAV-PHP.eB5,6. Together, this annotated dataset provides a single-cell resource that integrates the molecular spatial atlas, brain anatomy and the accessibility to genetic manipulation of the mammalian central nervous system.

Irreversible synthesis of an ultrastrong two-dimensional polymeric material.

Polymers that extend covalently in two dimensions have attracted recent attention1,2 as a means of combining the mechanical strength and in-plane energy conduction of conventional two-dimensional (2D) materials3,4 with the low densities, synthetic processability and organic composition of their one-dimensional counterparts. Efforts so far have proven successful in forms that do not allow full realization of these properties, such as polymerization at flat interfaces5,6 or fixation of monomers in immobilized lattices7-9. Another frequently employed synthetic approach is to introduce microscopic reversibility, at the cost of bond stability, to achieve 2D crystals after extensive error correction10,11. Here we demonstrate a homogenous 2D irreversible polycondensation that results in a covalently bonded 2D polymeric material that is chemically stable and highly processable. Further processing yields highly oriented, free-standing films that have a 2D elastic modulus and yield strength of 12.7 ± 3.8 gigapascals and 488 ± 57 megapascals, respectively. This synthetic route provides opportunities for 2D materials in applications ranging from composite structures to barrier coating materials.

Carbon isotope fractionation by an ancestral rubisco suggests that biological proxies for CO2 through geologic time should be reevaluated.

The history of Earth's carbon cycle reflects trends in atmospheric composition convolved with the evolution of photosynthesis. Fortunately, key parts of the carbon cycle have been recorded in the carbon isotope ratios of sedimentary rocks. The dominant model used to interpret this record as a proxy for ancient atmospheric CO2 is based on carbon isotope fractionations of modern photoautotrophs, and longstanding questions remain about how their evolution might have impacted the record. Therefore, we measured both biomass (εp) and enzymatic (εRubisco) carbon isotope fractionations of a cyanobacterial strain (Synechococcus elongatus PCC 7942) solely expressing a putative ancestral Form 1B rubisco dating to ≫1 Ga. This strain, nicknamed ANC, grows in ambient pCO2 and displays larger εp values than WT, despite having a much smaller εRubisco (17.23 ± 0.61‰ vs. 25.18 ± 0.31‰, respectively). Surprisingly, ANC εp exceeded ANC εRubisco in all conditions tested, contradicting prevailing models of cyanobacterial carbon isotope fractionation. Such models can be rectified by introducing additional isotopic fractionation associated with powered inorganic carbon uptake mechanisms present in Cyanobacteria, but this amendment hinders the ability to accurately estimate historical pCO2 from geological data. Understanding the evolution of rubisco and the CO2 concentrating mechanism is therefore critical for interpreting the carbon isotope record, and fluctuations in the record may reflect the evolving efficiency of carbon fixing metabolisms in addition to changes in atmospheric CO2.

HIV-1 Incidence, Adherence, and Drug Resistance in Individuals Taking Daily Emtricitabine/Tenofovir Disoproxil Fumarate for HIV-1 Pre-Exposure Prophylaxis: Pooled Analysis From 72 Global Studies.

BACKGROUND: Oral pre-exposure prophylaxis (PrEP) with emtricitabine/tenofovir disoproxil fumarate (F/TDF) has high efficacy against HIV-1 acquisition. Seventy-two prospective studies of daily oral F/TDF PrEP were conducted to evaluate HIV-1 incidence, drug resistance, adherence, and bone and renal safety in diverse settings. METHODS: HIV-1 incidence was calculated from incident HIV-1 diagnoses after PrEP initiation and within 60 days of discontinuation. Tenofovir concentration in dried blood spots (DBS), drug resistance, and bone/renal safety indicators were evaluated in a subset of studies. RESULTS: Among 17,274 participants, there were 101 cases with new HIV-1 diagnosis (0.77 per 100 person-years; 95% CI 0.63-0.94). In 78 cases with resistance data, 18 (23%) had M184I or V, one (1.3%) had K65R, and three (3.8%) had both mutations. In 54 cases with tenofovir concentration data from DBS, 45 (83.3%), 2 (3.7%), 6 (11.1%), and 1 (1.9%) had average adherence of <2, 2-3, 4-6, and ≥7 doses/week, respectively, and the corresponding incidence was 3.9 (95% CI 2.9-5.3), 0.24 (0.060-0.95), 0.27 (0.12-0.60), and 0.054 (0.008-0.38) per 100 person-years. Adherence was low in younger participants, Hispanic/Latinx and Black participants, cisgender women, and transgender women. Bone and renal adverse event incidence rates were 0.69 and 11.8 per 100 person-years, respectively, consistent with previous reports. CONCLUSIONS: Leveraging the largest pooled analysis of global PrEP studies to date, we demonstrate that F/TDF is safe and highly effective, even with less than daily dosing, in diverse clinical settings, geographies, populations, and routes of HIV-1 exposure.

Colloidal robotics.

Robots have components that work together to accomplish a task. Colloids are particles, usually less than 100 µm, that are small enough that they do not settle out of solution. Colloidal robots are particles capable of functions such as sensing, computation, communication, locomotion and energy management that are all controlled by the particle itself. Their design and synthesis is an emerging area of interdisciplinary research drawing from materials science, colloid science, self-assembly, robophysics and control theory. Many colloidal robot systems approach synthetic versions of biological cells in autonomy and may find ultimate utility in bringing these specialized functions to previously inaccessible locations. This Perspective examines the emerging literature and highlights certain design principles and strategies towards the realization of colloidal robots.

KardiaMobile 6L versus 12-lead ECG: Effects on clinic utilization time.

INTRODUCTION: We aimed to study whether KardiaMobile 6L 30-second capture technology could shorten ECG collection time compared to standard 12L ECG without compromising data usability. METHODS: A single-center, non-randomized trial was performed on patients presenting for follow-up visits to the electrophysiology (EP) clinic. Providers in the KardiaMobile 6L group were allowed to request a standard 12L if the 6L was deemed insufficient for clinical care. Room utilization times, defined as the time from medical assistant room entry to exit, were compared for each group. RESULTS: There were 100 patients in the study, with 50 in each arm. Average room utilization time for the 12L group and 6L groups were 10.33 ± 2.2 and 7.27 ± 1.93 min, respectively (p < .001). In 8 (16%) visits for the 6L group, an additional 12L was requested. CONCLUSION: For EP follow-up visits, clinic utilization time was significantly reduced with the KardiaMobile 6L compared to the 12L ECG with infrequent need for an additional 12L.

Occupation, Retirement Age, and 20-Year Cognitive Decline: The Atherosclerosis Risk in Communities Neurocognitive Study.

INTRODUCTION: We examined the association of both midlife occupation and age at retirement with cognitive decline in the Atherosclerosis Risk in Communities (ARIC) biracial community-based cohort. METHODS: Current or most recent occupation at ARIC baseline (1987-89; ages 45-64y) was categorized based on 1980 US census major occupation groups and tertiles of the Nam-Powers-Boyd occupational status score (n=14,090). Retirement status via annual follow-up questionnaires administered ascertained in 1999-2007 was classified as occurring before or after age 70 (n=7,503). Generalized estimating equation models were used to examine associations of occupation and age at retirement with trajectories of global cognitive factor scores, assessed from visit 2 (1990-92) to visit 5 (2011-2013). Models were a priori stratified by race and sex and adjusted for demographics and comorbidities. RESULTS: Low occupational status and blue-collar occupations were associated with low baseline cognitive scores in all race-sex strata. Low occupational status and homemaker status were associated with faster decline in White women but slower decline in Black women compared to high occupational status. Retirement before age 70 was associated with slower cognitive decline in White men and women and in Black men. Results did not change substantially after accounting for attrition. CONCLUSION: Low occupational status was associated with cognitive decline in women but not in men. Earlier retirement was associated with a slower cognitive decline in White participants and in Black men. Further research should explore reasons for the observed associations and race-sex differences.

Implementation of an intravenous sotalol initiation protocol: Implications for feasibility, safety, and length of stay.

INTRODUCTION: Oral sotalol initiation requires a multiple-day, inpatient admission to monitor for QT prolongation during loading. A 1-day intravenous (IV) sotalol loading protocol was approved by the United States Food and Drug Administration in March 2020, but limited data on clinical use and administration currently exists. This study describes implementation of an IV sotalol protocol within an integrated health system, provides initial efficacy and safety outcomes, and examines length of stay (LOS) compared with oral sotalol initiation. METHODS: IV sotalol was administered according to a prespecified initiation protocol to adult patients with refractory atrial or ventricular arrhythmias. Baseline characteristics, safety and feasibility outcomes, and LOS were compared with patients receiving oral sotalol over a similar time period. RESULTS: From January 2021 to June 2022, a total of 29 patients (average age 66.0 ± 8.6 years, 27.6% women) underwent IV sotalol load and 20 patients (average age 60.4 ± 13.9 years, 65.0% women) underwent oral sotalol load. The load was successfully completed in 22/29 (75.9%) patients receiving IV sotalol and 20/20 (100%) of patients receiving oral sotalol, although 7/20 of the oral sotalol patients (35.0%) required dose reduction. Adverse events interrupting IV sotalol infusion included bradycardia (seven patients, 24.1%) and QT prolongation (three patients, 10.3%). No patients receiving IV or oral sotalol developed sustained ventricular arrhythmias before discharge. LOS for patients completing IV load was 2.6 days shorter (mean 1.0 vs. 3.6, p < .001) compared with LOS with oral load. CONCLUSION: IV sotalol loading has a safety profile that is similar to oral sotalol. It significantly shortens hospital LOS, potentially leading to large cost savings.

Randomized Controlled Trial of Automated Directly Observed Therapy for Measurement and Support of PrEP Adherence Among Young Men Who have Sex with Men.

Measurement of adherence to oral pre-exposure prophylaxis (PrEP) in real-time has been challenging. We developed DOT Diary, a smartphone application that combines automated directly observed therapy with a PrEP adherence visualization toolkit, and tested its ability to measure PrEP adherence and to increase adherence among a diverse cohort of young men who have sex with men (MSM). We enrolled 100 MSM in San Francisco and Atlanta and randomly assigned them 2:1 to DOT Diary versus standard of care. Concordance between DOT Diary measurement and drug levels in dried blood spots was substantial, with 91.0% and 85.3% concordance between DOT Diary and emtricitabine-triphosphate and tenofovir-diphosphate, respectively. There was no significant difference in the proportion of participants with detectable PrEP drug levels at 24 weeks between study arms. These results suggest DOT Diary is substantially better than self-reported measures of adherence, but additional interventions are needed to improve PrEP adherence over time.

RESUMEN: La medición de la adherencia a la profilaxis oral previa a la exposición (PrEP) en tiempo real ha constituido un desafío. Hemos desarrollado DOT Diary, una aplicación para teléfonos inteligentes que combina la terapia automatizada observada de forma directa con un kit de herramientas para visualizar la adherencia a la PrEP, y testeamos su capacidad para medir la adherencia a la PrEP, así como para aumentar la adherencia entre una cohorte variada de hombres jóvenes que tienen sexo con hombres (HSH). Reclutamos a 100 HSH en San Francisco y Atlanta y los asignamos aleatoriamente 2:1 a DOT Diary con respecto a la asistencia estándar. La concordancia entre la medición del DOT Diary y los niveles de fármacos en gotas de sangre seca fue sustancial, con un 91,0% y un 85,3% de concordancia entre el uso del DOT Diary y el de emtricitabina-trifosfato y tenofovir-difosfato, respectivamente. No hubo diferencias significativas en la proporción de participantes con niveles detectables del fármaco de la PrEP a las 24 semanas entre los brazos del estudio. Estos resultados sugieren que DOT Diary es sustancialmente superior a las medidas de adherencia que se notifican de forma personal, aunque hacen falta intervenciones adicionales para mejorar la adherencia a la PrEP a largo plazo.

Discretized hexagonal boron nitride quantum emitters and their chemical interconversion.

Quantum emitters in two-dimensional hexagonal boron nitride (hBN) are of significant interest because of their unique photophysical properties, such as single-photon emission at room temperature, and promising applications in quantum computing and communications. The photoemission from hBN defects covers a wide range of emission energies but identifying and modulating the properties of specific emitters remain challenging due to uncontrolled formation of hBN defects. In this study, more than 2000 spectra are collected consisting of single, isolated zero-phonon lines (ZPLs) between 1.59 and 2.25 eV from diverse sample types. Most of ZPLs are organized into seven discretized emission energies. All emitters exhibit a range of lifetimes from 1 to 6 ns, and phonon sidebands offset by the dominant lattice phonon in hBN near 1370 cm-1. Two chemical processing schemes are developed based on water and boric acid etching that generate or preferentially interconvert specific emitters, respectively. The identification and chemical interconversion of these discretized emitters should significantly advance the understanding of solid-state chemistry and photophysics of hBN quantum emission.

Off-axis imaging with off-axis parabolic mirrors.

Off-axis parabolic mirrors are commonly used to focus beams of light propagating along their optical axis. However, certain applications require the focusing of beams displaced from the optical axis. As this regime is less commonly encountered, we clarify certain unintuitive aspects of the imaging. These considerations have direct applications in implementing non-collinear optical geometries using a single parabolic mirror.

A latent trajectory analysis of young sexual and gender minorities' adherence to three rectal microbicide placebo formulations (MTN-035; a randomized crossover trial).

BACKGROUND: Rectal microbicides (RM) are biomedical HIV prevention products that aim to prevent or reduce the transmission of HIV and other sexually transmitted infections (STIs). RM modalities may be beneficial for populations who have complex lifestyles, difficulties adhering to pre-exposure prophylaxis (PrEP) regimens, and/or have limited access to care. MTN-035 (DESIRE; Developing and Evaluating Short-Acting Innovations for Rectal Use), a randomized crossover trial, aimed to evaluate the safety and acceptability of, and adherence to, three placebo RM modalities (douche, insert, and suppository) prior to receptive anal intercourse. METHODS: We conducted latent trajectory analysis to identify clusters of individuals who shared similar trajectories in acceptability and adherence for each product (douche, insert, and suppository) over time. We analyzed weekly short messaging service (SMS) use reports for each modality as reported by enrolled sexual and gender minority (SGM) participants. RESULTS: Two trajectories for each product were identified: a "protocol compliant" trajectory (i.e., at least one product use occasion per week) and "high use" trajectory (i.e., more than three product use occasions per week). Participants with high use were more likely to lack access to PrEP and have higher intentions to utilize RM modalities compared to those who were protocol compliant. CONCLUSIONS: This study highlighted high adherence to RM modalities among SGM. As research into viable HIV prevention modalities continues to evolve, tailored intervention strategies are needed to support the uptake of and adherence to alternative prevention modalities that are behaviorally congruent with targeted users. TRIAL REGISTRATION: NCT03671239 (14/09/2018).

Sexual behavior and medication adherence in men who have sex with men participating in a pre-exposure prophylaxis study of combinations of Maraviroc, Tenofovir Disoproxil Fumarate and/or Emtricitabine (HPTN 069/ACTG 5305).

HPTN 069/ACTG 5305 was designed to evaluate potential new PrEP regimens that included maraviroc, tenofovir disoproxil fumarate, and/or emtricitabine. The current analyses assessed antiretroviral (ARV) plasma concentrations in relation to sexual behavior in 224 cisgender men who have sex with men and 2 transgender women at risk for HIV. Poisson generalized estimating equations (GEE) regression were used to test for associations between self-reported sexual behavior, sociodemographic, behavioral variables, and study drug levels The median (IQR) age was 30 [25, 37] years old; 48.2% had completed college; 27.4% were Black and 21.7% Latino. At weeks 24 and 48, one third of participants reported condomless anal sex (CAS) in the prior month with more than one partner. CAS was associated with daily ARV drug use (χ2 = 12.64, p = 0.002). Older individuals and those with greater education were more likely to ingest ARV drugs daily (χ2 = 9.36, p = 0.009 and χ2 = 8.63, p = 0.013, respectively), while neither race nor ethnicity was associated with daily ARV drug use. Participants who reported recent condomless anal sex and/or advanced education had higher rates of daily ARV drug use. These data support the need for ongoing adherence counseling in clinical trials of new PrEP modalities.

Autoperforation of two-dimensional materials to generate colloidal state machines capable of locomotion.

A central ambition of the robotics field has been to increasingly miniaturize such systems, with perhaps the ultimate achievement being the synthetic microbe or cell sized machine. To this end, we have introduced and demonstrated prototypes of what we call colloidal state machines (CSMs) as particulate devices capable of integrating sensing, memory, and energy harvesting as well as other functions onto a single particle. One technique that we have introduced for creating CSMs based on 2D materials such as graphene or monolayer MoS2 is "autoperforation", where the nanometer-scale film is fractured around a designed strain field to produce structured particles upon liftoff. While CSMs have been demonstrated with functions such as memory, sensing, and energy harvesting, the property of locomotion has not yet been demonstrated. In this work, we introduce an inversion moulding technique compatible with autoperforation that allows for the patterning of an external catalytic surface that enables locomotion in an accompanying fuel bath. Optimal processing conditions for electroplating a catalytic Pt layer to one side of an autoperforated CSM are elucidated. The self-driven propulsion of the resulting Janus CSM in H2O2 is studied, including the average velocity, as a function of fluid surface tension and H2O2 concentration in the bath. Since machines have to encode for a specific task, this work summarizes efforts to create a microfluidic testbed that allows for CSM designs to be evaluated for the ultimate purpose of navigation through complex fluidic networks, such as the human circulatory system. We introduce two CSM designs that mimic aspects of human immunity to solve search and recruitment tasks in such environments. These results advance CSM design concepts closer to promising applications in medicine and other areas.

DOT Diary: Developing a Novel Mobile App Using Artificial Intelligence and an Electronic Sexual Diary to Measure and Support PrEP Adherence Among Young Men Who Have Sex with Men.

Young men who have sex with men (YMSM) are highly vulnerable to HIV. While pre-exposure prophylaxis (PrEP) has demonstrated effectiveness, adherence has been low among YMSM and difficult to measure accurately. In collaboration with a healthcare company, we configured an automated directly-observed therapy (aDOT) platform for monitoring and supporting PrEP use. Based on interest expressed in focus groups among 54 YMSM, we combined aDOT with an electronic sexual diary to provide feedback on level of protection during sex and to motivate app use. In an 8-week optimization pilot with 20 YMSM in San Francisco and Atlanta, the app was found to be highly acceptable, with median System Usability Scale scores in the "excellent" range (80/100). App use was high, with median PrEP adherence of 91% based on aDOT-confirmed dosing. Most (84%) participants reported the app helped with taking PrEP. These promising findings support further evaluation of DOT Diary in future effectiveness studies.

Cabotegravir Is Not Associated With Weight Gain in Human Immunodeficiency Virus-uninfected Individuals in HPTN 077.

Studies in human immunodeficiency virus (HIV)-infected individuals suggest excess weight gain with integrase inhibitor-based antiretroviral therapy. The HIV Prevention Trials Network Study 077 evaluated changes in weight and fasting metabolic parameters in HIV-uninfected individuals randomized to cabotegravir or a placebo. No differences between arms were found for change in weight or fasting metabolic parameters overall or for subgroups.

Immobilization and Function of nIR-Fluorescent Carbon Nanotube Sensors on Paper Substrates for Fluidic Manipulation.

Nanoparticle-based optical sensors are capable of highly sensitive and selective chemical interactions and can form the basis of molecular recognition for various classes of analytes. However, their incorporation into standardized in vitro assays has been limited by their incompatibility with packaging or form factors necessary for specific applications. Here, we have developed a technique for immobilizing nIR-fluorescent single-walled carbon nanotube (SWCNT) sensors on seven different types of paper substrates including nitrocellulose, nylon, poly(vinylidene fluoride), and cellulose. Sensors remain functional upon immobilization and exhibit nIR fluorescence in nonaqueous solvent systems. We then extend this system to the Corona Phase Molecular Recognition (CoPhMoRe) approach of synthetic molecular recognition by screening ssDNA-wrapped SWCNTs with different sequences against a panel of fat-soluble vitamins in canola oil, identifying a sensor which responds to ß-carotene with a dissociation constant of 2.2 µM. Moreover, we pattern hydrophobic regions onto nitrocellulose using the wax printing method and form one-dimensional sensor barcodes for rapid multiplexing. Using a sensor array of select ssDNA wrappings, we are able to distinguish between Cu(II), Cd(II), Hg(II), and Pb(II) at a concentration of 100 µM. Finally, we demonstrate that immobilized sensors remain fluorescent and responsive for nearly 60 days when stability is addressed. This work represents a significant step toward the deployment of fluorescent nanoparticle sensors for point-of-use applications.

Evaluation of the Risk of Laboratory Microbial Contamination during Routine Testing in Automated Clinical Chemistry and Microbiology Laboratories.

BACKGROUND: Every clinical specimen is potentially infectious, but data regarding risk for contamination of the laboratory environment during routine testing are scarce. We assessed contamination during routine sample analysis in automated clinical chemistry and microbiology laboratories. METHODS: A fluorescent marker was applied to specimen container exteriors to assess the impact of gross contamination. Nonpathogenic MS2 virus was added to remnant blood, urine, and ESwab matrices as a biomarker of cross-contamination. Samples were processed and analyzed using Roche Cobas 8100 and ISE, c502, e602, and c702 modules (blood) and BD Kiestra total laboratory automation (blood, urine, ESwabs) over 3 experiments. Fluorescence transfer to laboratory surfaces and personnel was visualized using ultraviolet light. Surfaces were swabbed and assessed for MS2 cross-contamination by RT-PCR. Adherence to standard precautions by laboratory staff was assessed by observation. RESULTS: Fluorescence was observed on 49 of 165 (30%) laboratory surfaces and personnel and 21 of 93 (23%) total laboratory automation instruments. Fluorescence transferred most frequently to gloves (31/40), computer accessories (9/18), and specimen loading racks (12/12). None of 123 areas swabbed were positive for MS2. Improper personal protective equipment use occurred at a rate of 0.36 and 0.15 events per staff per hour in the chemistry and microbiology laboratories, respectively. Hand-washing compliance was observed for 61 of 132 (46%) staff members evaluated. CONCLUSIONS: Analysis of grossly contaminated specimens on automated chemistry and microbiology equipment elicits a low likelihood of instrument contamination. However, handling contaminated specimen containers can result in contamination of environmental laboratory surfaces, representing a source of risk that is heightened by low adherence to appropriate personal protective equipment.