Associations among drug acquisition and use behaviors, psychosocial attributes, and opioid-involved overdoses.

AIMS: This study sought to develop and assess an exploratory model of how demographic and psychosocial attributes, and drug use or acquisition behaviors interact to affect opioid-involved overdoses. DESIGN: We conducted exploratory and confirmatory factor analysis (EFA/CFA) to identify a factor structure for ten drug acquisition and use behaviors. We then evaluated alternative structural equation models incorporating the identified factors, adding demographic and psychosocial attributes as predictors of past-year opioid overdose. SETTING AND PARTICIPANTS: We used interview data collected for two studies recruiting opioid-misusing participants receiving services from a community-based syringe services program. The first investigated current attitudes toward drug-checking (N = 150). The second was an RCT assessing a telehealth versus in-person medical appointment for opioid use disorder treatment referral (N = 270). MEASUREMENTS: Demographics included gender, age, race/ethnicity, education, and socioeconomic status. Psychosocial measures were homelessness, psychological distress, and trauma. Self-reported drug-related risk behaviors included using alone, having a new supplier, using opioids with benzodiazepines/alcohol, and preferring fentanyl. Past-year opioid-involved overdoses were dichotomized into experiencing none or any. FINDINGS: The EFA/CFA revealed a two-factor structure with one factor reflecting drug acquisition and the second drug use behaviors. The selected model (CFI = .984, TLI = .981, RMSEA = .024) accounted for 13.1% of overdose probability variance. A latent variable representing psychosocial attributes was indirectly associated with an increase in past-year overdose probability (ß = .234, p = .001), as mediated by the EFA/CFA identified latent variables: drug acquisition (ß = .683, p < .001) and drug use (ß = .567, p = .001). Drug use behaviors (ß = .287, p = .04) but not drug acquisition (ß = .105, p = .461) also had a significant, positive direct effect on past-year overdose. No demographic attributes were significant direct or indirect overdose predictors. CONCLUSIONS: Psychosocial attributes, particularly homelessness, increase the probability of an overdose through associations with risky drug acquisition and drug-using behaviors. Further research is needed to replicate these findings with populations at high-risk of an opioid-related overdose to assess generalizability and refine the metrics used to assess psychosocial characteristics.

Current attitudes toward drug checking services and a comparison of expected with actual drugs present in street drug samples collected from opioid users.

BACKGROUND: The opioid epidemic continues to be associated with high numbers of fatalities in the USA and other countries, driven mainly by the inclusion of potent synthetic opioids in street drugs. Drug checking by means of various technologies is being increasingly implemented as a harm reduction strategy to inform users about constituent drugs in their street samples. We assessed how valued drug checking services (DCS) would be for opioid street drug users given the ubiquity of fentanyl and related analogs in the drug supply, the information they would most value from drug checking, and compared expected versus actual constituent drugs in collected samples. METHODS: A convenience sample of opioid street drug users (N = 118) was recruited from two syringe service exchange programs in Chicago between 2021 and 2022. We administered brief surveys asking about overdose history, whether fentanyl was their preferred opioid, and interest in DCS. We also collected drug samples and asked participants what drug(s) they expected were in the sample. Provided samples were analyzed using LC-MS technology and the results compared to their expected drugs. RESULTS: Participants reported an average of 4.4 lifetime overdoses (SD = 4.8, range = 0-20) and 1.1 (SD = 1.8, range = 0-10) past-year overdoses. A majority (92.1%) believed they had recently used drugs containing fentanyl whether intentionally or unintentionally. Opinions about the desirability of fentanyl were mixed with 56.1% indicating they did not and 38.0% indicating they did prefer fentanyl over other opioids, mainly heroin. Attitudes toward DCS indicated a general but not uniform receptiveness with a majority indicating interest in DCS though sizeable minorities believed DCS was "too much trouble" (25.2%) or there was "no point" in testing (35.4%). Participants were especially inaccurate identifying common cutting agents and potentiating drugs such as diphenhydramine in their samples (sensitivity = .17). CONCLUSIONS: Results affirmed street drug users remain interested in using DCS to monitor their drugs and such services should be more widely available. Advanced checking technologies that provide information on the relative quantities and the different drugs present in a given sample available at point-of-care, would be most valuable but remain challenging to implement.

A Latent Class Analysis of Chronic Health Conditions Among HIV-Positive Transgender Women of Color.

Research on the health of transgender people has focused on the risk for and health consequences of HIV and other sexually transmitted infections with little known about the prevalence of a broader range of medical conditions experienced by transgender people. This study used latent class (LC) analysis to examine a range of chronic medical conditions among 223 HIV-positive transgender women of color receiving primary care and psychosocial services in Chicago. The best-fitting model had 2 classes: low and moderate/high multimorbidity with 26% of participants classified in the moderate/high multimorbidity LC. Age group (i.e., under 35 vs 35 and older; AOR 13.8, p < 0.001), ever having AIDS (AOR 4.0, p < 0.05) and psychological distress (AOR 5.1, p < 0.05) were associated with increased probability of moderate/high multimorbidity class membership. The results suggest focusing on HIV-related care or hormonal treatment and potential cardiovascular issues could result in sub-optimal treatment for a population dis-engaged from primary care but which has a broad spectrum of largely untreated medical conditions.

RESUMEN: La investigación sobre la salud de las personas transgénero se ha centrado en el riesgo y las consecuencias del VIH y otras infecciones de transmisión sexual, y se sabe poco acerca de la prevalencia de una gama más amplia de condiciones médicas experimentadas por las personas transgénero. Este estudio utilizó un análisis de clase latente (LC) para examinar una gama de condiciones médicas crónicas entre 223 mujeres transgénero VIH positivas que reciben atención primaria y servicios psicosociales en Chicago. El modelo que mejor se ajustó tuvo 2 clases: multimorbilidad baja y moderada/alta, con 26% de los participantes clasificados en la LC de multimorbilidad moderada/alta. Grupo de edad (es decir, menores de 35 contra 35 y más; AOR = 13.8, p < 0.001), con SIDA (AOR = 4.0, p < 0.05) y angustia psicológica (AOR = 5.1, p < 0.05) fueron asociado con una mayor probabilidad de membresía de clase de multimorbilidad moderada/alta. Los resultados sugieren que centrarse en la atención relacionada con el VIH o en el tratamiento hormonal y los posibles problemas cardiovasculares podrían resultar en un tratamiento subóptimo para una población que participa muy poco en la atención primaria, pero que tiene un amplio espectro de condiciones médicas en gran parte no tratadas.

Syringe service program-based telemedicine linkage to opioid use disorder treatment: protocol for the STAMINA randomized control trial.

BACKGROUND: A key strategy for mitigating the current opioid epidemic is expanded access to medications for treating opioid use disorder (MOUD). However, interventions developed to expand MOUD access have limited ability to engage opioid users at higher levels of overdose risk, such as those who inject opioids. This paper describes the study protocol for testing STAMINA (Syringe Service Telemedicine Access for Medication-assisted Intervention through NAvigation), an intervention that engages high-risk opioid users at community-based syringe service programs (SSP) and quickly links them to MOUD using a telemedicine platform. METHODS: This randomized control trial will be conducted at three SSP sites in Chicago. All participants will complete an initial assessment with a provider from a Federally Qualified Health Center who can prescribe or refer MOUD services as appropriate. The control arm will receive standard referral to treatment and the intervention arm will receive immediate telemedicine linkage to the provider and (depending on the type of MOUD prescribed) provided transportation to pick up their induction prescription (for buprenorphine or naltrexone) or attend their intake appointment (for methadone). We aim to recruit a total of 273 participants over two years to provide enough power to detect a difference in our primary outcome of MOUD treatment linkage. Secondary outcomes include treatment engagement, treatment retention, and non-MOUD opioid use. Data will be collected using structured interviews and saliva drug tests delivered at baseline, three months, and six months. Fixed and mixed effects generalized linear regression analyses and survival analysis will be conducted to compare the probabilities of a successful treatment linkage between the two arms, days retained in treatment, and post-baseline opioid and other drug use. DISCUSSION: If successful, STAMINA's telemedicine approach will significantly reduce the amount of time between SSP clients' initial indication of interest in the medication and treatment initiation. Facilitating this process will likely lead to stronger additional treatment- and recovery-oriented outcomes. This study is also timely given the need for more rigorous testing of telemedicine interventions in light of temporary regulatory changes that have occurred during the COVID-19 pandemic. TRIAL REGISTRATION: ClinicalTrials.gov (Clinical Trials ID: NCT04575324 and Protocol Number: 1138-0420). Registered 29 September 2020. The study protocol is also registered on the Open Science Framework (DOI 10.17605/OSF.IO/4853 M).

Streamlined circular proximity ligation assay provides high stringency and compatibility with low-affinity antibodies.

Proximity ligation assay (PLA) is a powerful tool for quantitative detection of protein biomarkers in biological fluids and tissues. Here, we present the circular proximity ligation assay (c-PLA), a highly specific protein detection method that outperforms traditional PLA in stringency, ease of use, and compatibility with low-affinity reagents. In c-PLA, two proximity probes bind to an analyte, providing a scaffolding that positions two free oligonucleotides such that they can be ligated into a circular DNA molecule. This assay format stabilizes antigen proximity probe complexes and enhances stringency by reducing the probability of random background ligation events. Circle formation also increases selectivity, since the uncircularized DNA can be removed enzymatically. We compare this method with traditional PLA on several biomarkers and show that the higher stringency for c-PLA improves reproducibility and enhances sensitivity in both buffer and human plasma. The limit of detection ranges from femtomolar to nanomolar concentrations for both methods. Kinetic analyses using surface plasmon resonance (SPR) and biolayer interferometry (BLI) reveal that the variation in limit of detection is due to the variation in antibody affinity and that c-PLA outperforms traditional PLA for low-affinity antibodies. The lower background signal can be used to increase proximity probe concentration while maintaining a high signal-to-noise ratio, thereby enabling the use of low-affinity reagents in a homogeneous assay format. We anticipate that the advantages of c-PLA will be useful in a variety of clinical protein detection applications where high-affinity reagents are lacking.

Patterns of Exposure to Socio-structural Stressors and HIV Care Engagement Among Transgender Women of Color.

Transgender women are disproportionately affected by HIV and experiences of social adversity that may interfere with engagement in care and viral suppression. We used latent class analysis to examine patterns of social adversity and their impact on HIV care continuum outcomes in an urban sample of transgender women of color. Participants (n = 224) were median age 29 and 86% non-Hispanic Black. Lack of resources, unemployment, and housing instability were reported by over 50%, and 41% reported history of incarceration. Latent class analysis identified 2 distinct classes representing higher and lower levels of social adversity. In latent class regression, membership in the higher social adversity class was associated with statistically significantly lower odds of viral suppression and HIV care engagement in univariate analysis; when adjusted for age, race, and recruitment site the association remained statistically significant for viral suppression (aOR 0.38, 95% CI 0.18-0.79; chi-square = 6.681, d.f. = 1, p = 0.010), though not for HIV care engagement. Our findings highlight the impact of socio-structural barriers on engagement in the HIV care continuum among transgender women.

RESUMEN: Las mujeres transgénero son desproporcionadamente afectadas por el VIH y las experiencias de adversidad social que pueden interferir con la participación en la atención medica y la supresión viral. Utilizamos un análisis de clase latente para examinar los patrones de adversidad social y su impacto en los resultados continuos de la atención medica del VIH en una muestra urbana de mujeres transgénero de color. Los participantes (n = 224) tenían una mediana de edad de 29 años y 86% negros no hispanos. La falta de recursos, el desempleo y la inestabilidad de la vivienda fueron reportados en más del 50%, y el 41% reportó antecedentes de encarcelamiento. El análisis de clase latente identificó 2 clases distintas que representan niveles más altos y más bajos de adversidad social. En la regresión de clase latente, la pertenencia a la clase de mayor adversidad social se asoció con probabilidades estadísticamente significante más bajas de supresión viral y participación en la atención medica del VIH en el análisis univariante; cuando se ajustó por edad, raza y sitio de reclutamiento, la asociación siguió siendo estadísticamente significativa para la supresión viral (aOR 0.38, IC 95% 0.18­0.79; chi-cuadrado = 6.681, df = 1, p = 0.010), aunque no para la participación en la atención medica del VIH. Nuestros hallazgos destacan el impacto de las barreras socioestructurales en la participación en el continuo de atención medica del VIH entre las mujeres transgénero.

Expanding biological applications using cell-free metabolic engineering: An overview.

Expanding the concept of cell-free biology, implemented both with purified components and crude extracts, is continuing to deepen our appreciation of biological fundamentals while enlarging the range of applications. We are no longer intimidated by the complexity of crude extracts and complicated reaction systems with hundreds of active components, and, instead, coordinately activate and inactivate metabolic processes to focus and expand the capabilities of natural biological processes. This, in turn, dramatically increases the range of benefits offered by new products, both natural and supernatural, that were previously infeasible and/or unimaginable. This overview of cell-free metabolic engineering provides a broad range of examples and insights to guide and motivate continued research that will further expand fundamental understanding and beneficial applications. However, this survey also reveals how far we are from fully unlocking the potential offered by natural and engineered biological components and systems. This is an exciting conclusion, but metabolic engineering by itself is not sufficient. Going forward, innovative metabolic engineering must be intimately combined with creative process engineering to fully realize potential contributions toward a sustainable global civilization.

System analysis and improved [FeFe] hydrogenase O2 tolerance suggest feasibility for photosynthetic H2 production.

Photosynthetic H2 production has been a compelling but elusive objective. Here we describe how coordinated bioreactor, metabolic pathway, and protein engineering now suggest feasibility for the sustainable, solar-powered production of a storable fuel to complement our expanding photovoltaic and wind based capacities. The need to contain and harvest the gaseous products provides decisive solar bioreactor design advantages by limiting O2 exposure to prolific, but O2-sensitive H2 producing enzymes-[FeFe] hydrogenases. CO2 supply and cell growth can also be limited so that most of the photosynthetic reduction capacity is directed toward H2 production. Yet, natural [FeFe] hydrogenases are still too O2 sensitive for technology implementation. We report the discovery of new variants and a new O2 tolerance mechanism that significantly reduce the sensitivity to O2 exposure without lowering H2 production rates or losing electrons to O2 reduction. Testing the improved hydrogenases with a biologically derived, light-dependent electron source provides evidence that this game changing technology has the potential for sustainable large-scale fuel production.

A Study of the Longitudinal Patterns of Stimulant and Amyl Nitrite Use and Sexual Behavior Pre- and Post-HIV Seroconversion Among MSM.

The use of stimulant drugs alone or in combination with amyl nitrites (stimulant/nitrites) has been associated with higher rates of risky sexual behavior and predictive of HIV infection among men who have sex with men. However, the temporal pattern of stimulant/nitrite use pre- and post-seroconversion has not been well established. This study assessed changes in stimulant/nitrite use and risky sexual behavior among seroconverting MSM over time. Data were collected in the Baltimore-Washington, DC; Pittsburgh; Chicago; and Los Angeles sites of the Multicenter AIDS Cohort Study (MACS), a longitudinal study of the natural history of HIV infection among MSM. We used propensity scores to select 1044 MSM from 7087 MACS participants composed of 348 seroconverting, 348 seronegative, and 348 seroprevalent participants matched on demographics, recruitment cohort, and study visits. We centered up to four-years of semi-annual data around the seroconversion visit of the seroconverting case within each matched group of participants. Mixed effects regressions estimated the effects of serostatus, recruitment cohort, and time on self-reported stimulant/nitrite use, numbers of male intercourse partners, and numbers of unprotected receptive anal intercourse (URAI) partners. Covariates included demographics, binge drinking, and marijuana use. Seroconverters had the highest odds of stimulant/inhaled nitrite use (AOR 10.3, CI 4.8-22.0), incident rates of intercourse (IRR 1.6, CI 1.3-2.1), and URAI partners (IRR 5.1, CI 3.5-7.3). All participants decreased drug use and sexual risk behavior over time. However, the decreases were largest for seroconverters who nevertheless maintained the highest rates of stimulant/nitrite use and sexual risk. Cohort-related effects were associated with sharp reductions in stimulant/nitrite use and URAI in the early 1990s that rebounded considerably within the first decade of the 2000s. Although all participants decreased risky sexual behavior and stimulant and/or nitrite use over time, seroconverters had the largest decreases. There was no evidence for abrupt or substantial increases in drug use or risky sex post-seroconversion. However, there was substantial variation at the individual level, with the factors underlying this variation not well understood and worth further study. Moreover, stimulant/nitrite use and risky sexual behavior appear to have been strongly influenced by contextual historical and socio-cultural effects. The manner in which contextual factors influence individual behavior is also not well understood and also warrants further study.

Assessing sequence plasticity of a virus-like nanoparticle by evolution toward a versatile scaffold for vaccines and drug delivery.

Virus-like particles (VLPs) have been extensively explored as nanoparticle vehicles for many applications in biotechnology (e.g., vaccines, drug delivery, imaging agents, biocatalysts). However, amino acid sequence plasticity relative to subunit expression and nanoparticle assembly has not been explored. Whereas the hepatitis B core protein (HBc) VLP appears to be the most promising model for fundamental and applied studies; particle instability, antigen fusion limitations, and intrinsic immunogenicity have limited its development. Here, we apply Escherichia coli-based cell-free protein synthesis (CFPS) to rapidly produce and screen HBc protein variants that still self-assemble into VLPs. To improve nanoparticle stability, artificial covalent disulfide bridges were introduced throughout the VLP. Negative charges on the HBc VLP surface were then reduced to improve surface conjugation. However, removal of surface negative charges caused low subunit solubility and poor VLP assembly. Solubility and assembly as well as surface conjugation were greatly improved by transplanting a rare spike region onto the common shell structure. The newly stabilized and extensively modified HBc VLP had almost no immunogenicity in mice, demonstrating great promise for medical applications. This study introduces a general paradigm for functional improvement of complex protein assemblies such as VLPs. This is the first study, to our knowledge, to systematically explore the sequence plasticity of viral capsids as an approach to defining structure function relationships for viral capsid proteins. Our observations on the unexpected importance of the HBc spike tip charged state may also suggest new mechanistic routes toward viral therapeutics that block capsid assembly.

Cysteine as a ligand platform in the biosynthesis of the FeFe hydrogenase H cluster.

Hydrogenases catalyze the redox interconversion of protons and H2, an important reaction for a number of metabolic processes and for solar fuel production. In FeFe hydrogenases, catalysis occurs at the H cluster, a metallocofactor comprising a [4Fe-4S]H subcluster coupled to a [2Fe]H subcluster bound by CO, CN(-), and azadithiolate ligands. The [2Fe]H subcluster is assembled by the maturases HydE, HydF, and HydG. HydG is a member of the radical S-adenosyl-L-methionine family of enzymes that transforms Fe and L-tyrosine into an [Fe(CO)2(CN)] synthon that is incorporated into the H cluster. Although it is thought that the site of synthon formation in HydG is the "dangler" Fe of a [5Fe] cluster, many mechanistic aspects of this chemistry remain unresolved including the full ligand set of the synthon, how the dangler Fe initially binds to HydG, and how the synthon is released at the end of the reaction. To address these questions, we herein show that L-cysteine (Cys) binds the auxiliary [4Fe-4S] cluster of HydG and further chelates the dangler Fe. We also demonstrate that a [4Fe-4S]aux[CN] species is generated during HydG catalysis, a process that entails the loss of Cys and the [Fe(CO)2(CN)] fragment; on this basis, we suggest that Cys likely completes the coordination sphere of the synthon. Thus, through spectroscopic analysis of HydG before and after the synthon is formed, we conclude that Cys serves as the ligand platform on which the synthon is built and plays a role in both Fe(2+) binding and synthon release.

X-ray crystallographic and EPR spectroscopic analysis of HydG, a maturase in [FeFe]-hydrogenase H-cluster assembly.

Hydrogenases use complex metal cofactors to catalyze the reversible formation of hydrogen. In [FeFe]-hydrogenases, the H-cluster cofactor includes a diiron subcluster containing azadithiolate, three CO, and two CN(-) ligands. During the assembly of the H cluster, the radical S-adenosyl methionine (SAM) enzyme HydG lyses the substrate tyrosine to yield the diatomic ligands. These diatomic products form an enzyme-bound Fe(CO)x(CN)y synthon that serves as a precursor for eventual H-cluster assembly. To further elucidate the mechanism of this complex reaction, we report the crystal structure and EPR analysis of HydG. At one end of the HydG (ßα)8 triosephosphate isomerase (TIM) barrel, a canonical [4Fe-4S] cluster binds SAM in close proximity to the proposed tyrosine binding site. At the opposite end of the active-site cavity, the structure reveals the auxiliary Fe-S cluster in two states: one monomer contains a [4Fe-5S] cluster, and the other monomer contains a [5Fe-5S] cluster consisting of a [4Fe-4S] cubane bridged by a µ2-sulfide ion to a mononuclear Fe(2+) center. This fifth iron is held in place by a single highly conserved protein-derived ligand: histidine 265. EPR analysis confirms the presence of the [5Fe-5S] cluster, which on incubation with cyanide, undergoes loss of the labile iron to yield a [4Fe-4S] cluster. We hypothesize that the labile iron of the [5Fe-5S] cluster is the site of Fe(CO)x(CN)y synthon formation and that the limited bonding between this iron and HydG may facilitate transfer of the intact synthon to its cognate acceptor for subsequent H-cluster assembly.

Characterization of [FeFe] Hydrogenase O2 Sensitivity Using a New, Physiological Approach.

[FeFe] hydrogenases catalyze rapid H2 production but are highly O2-sensitive. Developing O2-tolerant enzymes is needed for sustainable H2 production technologies, but the lack of a quantitative and predictive assay for O2 tolerance has impeded progress. We describe a new approach to provide quantitative assessment of O2 sensitivity by using an assay employing ferredoxin NADP+ reductase (FNR) to transfer electrons from NADPH to hydrogenase via ferredoxins (Fd). Hydrogenase inactivation is measured during H2 production in an O2-containing environment. An alternative assay uses dithionite (DTH) to provide reduced Fd. This second assay measures the remaining hydrogenase activity in periodic samples taken from the NADPH-driven reaction solutions. The second assay validates the more convenient NADPH-driven assay, which better mimics physiological conditions. During development of the NADPH-driven assay and while characterizing the Clostridium pasteurianum (Cp) [FeFe] hydrogenase, CpI, we detected significant rates of direct electron loss from reduced Fd to O2 However, this loss does not interfere with measurement of first order hydrogenase inactivation, providing rate constants insensitive to initial hydrogenase concentration. We show increased activity and O2 tolerance for a protein fusion between Cp ferredoxin (CpFd) and CpI mediated by a 15-amino acid linker but not for a longer linker. We suggest that this precise, solution phase assay for [FeFe] hydrogenase O2 sensitivity and the insights we provide constitute an important advance toward the discovery of the O2-tolerant [FeFe] hydrogenases required for photosynthetic, biological H2 production.

Production and stabilization of the trimeric influenza hemagglutinin stem domain for potentially broadly protective influenza vaccines.

The rapid dissemination of the 2009 pandemic H1N1 influenza virus emphasizes the need for universal influenza vaccines that would broadly protect against multiple mutated strains. Recent efforts have focused on the highly conserved hemagglutinin (HA) stem domain, which must undergo a significant conformational change for effective viral infection. Although the production of isolated domains of multimeric ectodomain proteins has proven difficult, we report a method to rapidly produce the properly folded HA stem domain protein from influenza virus A/California/05/2009 (H1N1) by using Escherichia coli-based cell-free protein synthesis and a simple refolding protocol. The T4 bacteriophage fibritin foldon placed at the C terminus of the HA stem domain induces trimer formation. Placing emphasis on newly exposed protein surfaces, several hydrophobic residues were mutated, two polypeptide segments were deleted, and the number of disulfide bonds in each monomer was reduced from four to two. High pH and Brij 35 detergent emerged as the most beneficial factors for improving the refolding yield. To stabilize the trimer of the HA stem-foldon fusion, new intermolecular disulfide bonds were finally introduced between foldon monomers and between stem domain monomers. The correct immunogenic conformation of the stabilized HA stem domain trimer was confirmed by using antibodies CR6261, C179, and FI6 that block influenza infection by binding to the HA stem domain trimer. These results suggest great promise for a broadly protective vaccine and also demonstrate a unique approach for producing individual domains of complex multimeric proteins.

High-Throughput Screening of Catalytic H2 Production.

Hydrogenases, ferredoxins, and ferredoxin-NADP+ reductases (FNR) are redox proteins that mediate electron metabolism in vivo, and are also potential components for biological H2 production technologies. A high-throughput H2 production assay device (H2 PAD) is presented that enables simultaneous evaluation of 96 individual H2 production reactions to identify components that improve performance. Using a CCD camera and image analysis software, H2 PAD senses the chemo-optical response of Pd/WO3 thin films to the H2 produced. H2 PAD-enabled discovery of hydrogenase and FNR mutants that enhance biological H2 production is reported. From a library of 10 080 randomly mutated Clostridium pasteurianum [FeFe] hydrogenases, we found a mutant with nearly 3-fold higher H2 production specific activity. From a library of 400 semi-randomly mutated Oryza sativa FNR, the top hit enabled a 60 % increase in NADPH-driven H2 production rates. H2 PAD can also facilitate elucidation of fundamental biochemical mechanisms within these systems.

The Radical SAM Enzyme HydG Requires Cysteine and a Dangler Iron for Generating an Organometallic Precursor to the [FeFe]-Hydrogenase H-Cluster.

Three maturase enzymes-HydE, HydF, and HydG-synthesize and insert the organometallic component of the [FeFe]-hydrogenase active site (the H-cluster). HydG generates the first organometallic intermediates in this process, ultimately producing an [Fe(CO)2(CN)] complex. A limitation in understanding the mechanism by which this complex forms has been uncertainty regarding the precise metallocluster composition of HydG that comprises active enzyme. We herein show that the HydG auxiliary cluster must bind both l-cysteine and a dangler Fe in order to generate the [Fe(CO)2(CN)] product. These findings support a mechanistic framework in which a [(Cys)Fe(CO)2(CN)](-) species is a key intermediate in H-cluster maturation.

A Multi-Group Latent Class Analysis of Chronic Medical Conditions Among Men Who Have Sex with Men.

Until recently, research on the health of gay and other men who have sex with men (MSM) has focused on risk for and the health consequences of HIV and other sexually transmitted infections. A multigroup latent class analysis examined a range of lifetime chronic medical conditions (CMCs) among MSM. Covariates included sociodemographics, substance use, psychological distress, and HIV serostatus. A two-class model best fit the medical condition data: a low probabilities class for most CMCs and a moderate to high probabilities (MHP) class. HIV serostatus was associated with increased within-class probabilities for some CMCs, particularly gastrointestinal and skin disorders. Only increasing age and use of erectile dysfunction drugs were directly associated with increased odds of being in the MHP class whereas methamphetamine use, identifying as gay, and lower alcohol use were indirectly associated. Implications of the findings for future research and the health care needs of MSM are discussed.

Biosynthesis of the [FeFe] Hydrogenase H Cluster: A Central Role for the Radical SAM Enzyme HydG.

Hydrogenase enzymes catalyze the rapid and reversible interconversion of H2 with protons and electrons. The active site of the [FeFe] hydrogenase is the H cluster, which consists of a [4Fe-4S]H subcluster linked to an organometallic [2Fe]H subcluster. Understanding the biosynthesis and catalytic mechanism of this structurally unusual active site will aid in the development of synthetic and biological hydrogenase catalysts for applications in solar fuel generation. The [2Fe]H subcluster is synthesized and inserted by three maturase enzymes-HydE, HydF, and HydG-in a complex process that involves inorganic, organometallic, and organic radical chemistry. HydG is a member of the radical S-adenosyl-l-methionine (SAM) family of enzymes and is thought to play a prominent role in [2Fe]H subcluster biosynthesis by converting inorganic Fe(2+), l-cysteine (Cys), and l-tyrosine (Tyr) into an organometallic [(Cys)Fe(CO)2(CN)](-) intermediate that is eventually incorporated into the [2Fe]H subcluster. In this Forum Article, the mechanism of [2Fe]H subcluster biosynthesis is discussed with a focus on how this key [(Cys)Fe(CO)2(CN)](-) species is formed. Particular attention is given to the initial metallocluster composition of HydG, the modes of substrate binding (Fe(2+), Cys, Tyr, and SAM), the mechanism of SAM-mediated Tyr cleavage to CO and CN(-), and the identification of the final organometallic products of the reaction.

Cell-free co-production of an orthogonal transfer RNA activates efficient site-specific non-natural amino acid incorporation.

We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9-1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p-azido-l-phenylalanine (pAzF) or p-propargyloxy-l-phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50-88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA(opt)) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.

A vaccine directed to B cells and produced by cell-free protein synthesis generates potent antilymphoma immunity.

Clinical studies of idiotype (Id) vaccination in patients with lymphoma have established a correlation between the induced anti-Id antibody responses and favorable clinical outcomes. To streamline the production of an Id vaccine, we engineered a small diabody (Db) molecule containing both a B-cell-targeting moiety (anti-CD19) and a lymphoma Id. This molecule (αCD19-Id) was designed to penetrate lymph nodes and bind to noncognate B cells to form an antigen presentation array. Indeed, the αCD19-Id molecule accumulated on B cells in vivo after s.c. administration. These noncognate B cells, decorated with the diabody, could then stimulate the more rare Id-specific B cells. Peptide epitopes present in the diabody linker augmented the response by activating CD4(+) helper T cells. Consequently, the αCD19-Id molecule induced a robust Id-specific antibody response and protected animals from tumor challenge. Such diabodies are produced in a cell-free protein expression system within hours of amplification of the specific Ig genes from the B-cell tumor. This customized product can now be available to vaccinate patients before they receive other, potentially immunosuppressive, therapies.