Patterns of mean age at drug use initiation by race and ethnicity, 2004-2019.

INTRODUCTION: Adolescent drug use can result in clinically significant psychiatric outcomes later in life mitigated by targeted prevention strategies. While mean age of drug initiation has increased over time, there is little research of mean age of drug initiation among adolescents by race/ethnicity. METHODS: The study used the National Survey on Drug Use and Health data (2004-2019). Sample included individuals aged 12 to 21 years. Year-by-year drug use initiation (i.e., first-time use within the past year) trends examined for each drug by race/ethnicity using jointpoint regression. RESULTS: Sample included 95,022 initiates for any of 18 drugs. Year-by-year mean initiation age significantly increased for alcohol (except Non-Hispanic [NH] White, 2004-2012), tobacco cigarettes (except NH American Indian/Alaska Native [AI/AN]), cigars, marijuana (except NH Asian or Pacific Islander, NH Multiracial), cocaine (except NH Black). Significant increase in mean initiation age found for heroin (Hispanic/Latinx only), hallucinogens (NH White, NH Black only), LSD (NH White only), methamphetamines (NH White only), smokeless tobacco (NH White, NH Black only), inhalants (only NH White, NH AI/AN; NH Multiracial, 2004-2011), sedatives (NH White, Hispanic/Latinx only), stimulants (NH White, Hispanic/Latinx only), and ecstasy (NH White, NH Black, Hispanic/Latinx only). Significant decrease in mean initiation age found for alcohol (only NH White, 2013-2019), smokeless tobacco (only Hispanic/Latinx, 2015-2019; NH AI/AN, 2012-2019), and inhalants (only NH Multiracial, 2012-2019). CONCLUSION: Mean initiation age differed widely by race/ethnicity. Mean initiation age in most racial/ethnic groups increased for several drugs including alcohol, marijuana, and tobacco products and decreased for some drugs such as inhalants. These findings could help inform groups to target for future prevention strategies.

Investigating Secondary Alcohol Outcomes in a Contingency Management Intervention among American Indian and Alaska Native Adults.

OBJECTIVE: The aim of the study is to investigate clinically meaningful, secondary outcomes in a randomized trial of a culturally adapted contingency management (CM) intervention for alcohol use in 3 diverse American Indian and Alaska Native communities. METHODS: Three American Indian and Alaska Native communities located in the Northern Plains, Alaska, and the Inland Northwest were partnering sites. A total of 158 individuals were randomized to either a 12-week CM intervention or a noncontingent (NC) control group. The CM group received reinforcers for providing alcohol-negative ethyl glucuronide (EtG < 150 ng/mL) urine samples, while the NC group received reinforcers unconditionally. Outcomes included EtG as a continuous measure (range, 0-2,000 ng/mL), EtG > 499 ng/mL (a measure of higher levels of recent alcohol use), longest duration of abstinence, and time-to-first alcohol-positive EtG during the trial. Generalized estimating equations along with Cox proportional hazard and negative binomial regressions were used. RESULTS: Participants randomized to the CM group had lower mean EtG levels (-241.9 ng/mL; 95% confidence interval [CI], -379.0 to -104.8 ng/mL) and 45.7% lower odds (95% CI, 0.31 to 0.95) of providing an EtG sample indicating higher levels of alcohol use during the intervention. Longest duration of abstinence was 43% longer for the CM group than the NC group (95% CI, 1.0 to 1.9). Risk of time-to-first drink during treatment did not differ significantly. CONCLUSIONS: These secondary outcome analyses provide evidence that CM is associated with reductions in alcohol use and longer durations of abstinence (as assessed by EtG), both clinically meaningful endpoints and analyses that differ from the primary study outcome.

Cannabis for Healing in a Native Community Clinic: Development and Results from an Informatics Research Tool.

This paper describes how the Puyallup Tribe created a clinic specializing in cannabis-based treatments and partnered with a university research team to assess the impacts of cannabis on patient outcomes. Clinic leaders and research team co-developed an informatics research tool that included survey questions about patient demographics, cannabis use, and measures of pain, depression, anxiety, other substance use, and trauma. Over the first 2.5 years of operations, 69 patients completed a survey. Participants were an average age of 50 years old (SD = 16.7), female (77.6%) and American Indian/Alaska Native (61.5%) with more than 12 years of education (66.7%). Over 77% of the participants used either cannabidiol-dominant (CBD) alone or both CBD and Tetrahydrocannabinol-dominant (THC) products, nearly 23% used neither CBD nor THC products. Most came to the clinic for a pain relief appointment (70.3%). Compared to the general population, participants experienced more pain-related comorbidities, such as anxiety, fatigue, sleep, and pain, and fewer physical functioning capabilities. Over half reported symptoms consistent with depressive or post-traumatic stress disorder. The informatics research tool was successfully integrated into a unique Tribally owned medical clinic.

Factors Associated with Child Removal Among American Indian and Alaska Native People in an Alcohol Intervention Study.

This study was a secondary data analysis of factors associated with alcohol-related child removal among American Indian/Alaska Native (AI/AN) adults enrolled in a clinical trial of an alcohol intervention. Among 326 parent participants, 40% reported ever having a child removed from their care in part because of the parent's alcohol use, defined here as alcohol-related child removal. Seventy-five percent of parents reported at least one separation during their own childhood (M = 1.3, SD = 1.0). In a multivariable analysis, alcohol-related child removal was associated with parental boarding school attendance. No relationship was found between alcohol-related child removal and alcohol intervention outcomes. Results may provide evidence of multigenerational child removal impacts of boarding schools on AI/AN adults receiving an alcohol use disorder intervention. Assessment of parental history of child removal by practitioners, strategies to prevent alcohol-related separation and to support reunification should be integrated into addiction treatment in AI/AN communities.

Drug-Drug Interaction Between Orally Administered Hydrocodone-Acetaminophen and Inhalation of Cannabis Smoke: A Case Report.

Objective: To determine if a 2-day protocol measuring pharmacokinetic and pharmacodynamic characteristics can demonstrate drug-drug interactions when smoked cannabis is added to orally administered hydrocodone/acetaminophen combination products. Case Summary: A 51-year-old non-Hispanic white male with chronic pain diagnoses participated in a 2-day pilot protocol. The participant attended two 7-hour in-lab days where he received 10 blood draws each day and completed self-administered pain and anxiety surveys. For both days, the participant took his prescribed dose of hydrocodone/acetaminophen (1/2 tablet of 7.5 mg/325 mg combination product) with the addition of 1 smoked pre-rolled marijuana cigarette (labeled as 0.5 g; 22.17% Δ9-tetrahydrocannabinol; 0.12% cannabidiol) on Day 2. Blood specimens were analyzed using mass spectrometry to quantify the difference of plasma hydrocodone levels between Day 1 and Day 2. Results: Compared to Day 1, lower levels of pain and anxiety were reported during Day 2 with the addition of cannabis to oral hydrocodone/acetaminophen. Day 2 pharmacokinetic analysis also revealed more rapid absorption and overall lower levels of hydrocodone in plasma. Discussion: Lower hydrocodone plasma levels in Day 2 may indicate cannabis's effect on metabolism and reduce the risk of opioid toxicity. The quicker absorption rate of hydrocodone could explain lower pain and anxiety scores reported on the second day. Conclusion and Relevance: A 2-day protocol was able to capture differences across time in pharmacokinetic and pharmacodynamic measurements. Larger studies can be designed to better characterize the potential drug-drug interaction of cannabis and opioids.

Variations in national availability of waivered buprenorphine prescribers by racial and ethnic composition of zip codes.

BACKGROUND: Opioid overdose remains a public health crisis in diverse communities. Between 2019 and 2020, there was an almost 40% increase in drug fatalities primarily due to opioid analogues of both stimulants and opioids. Medications for opioid use disorder (MOUD; e.g., buprenorphine) are effective, evidence-based treatments that can be delivered in office-based primary care settings. We investigated disparities in the proportion of national prescribers who have obtained a waiver issued to prescribe MOUD by demographic characteristics. METHODS: Data for the secondary data analyses were obtained from the Drug Enforcement Administration that maintains data on waivered MOUD prescribers across the US. Proportion of waivered prescribers were examined by ZIP code, race and ethnicity composition, socioeconomic status, insurance, and urban-rural designation using generalized linear mixed effects models. RESULTS: Compared with predominantly Non-Hispanic White ZIP codes, other racially and ethnically diverse areas had a higher proportion of waivered buprenorphine prescribers. Differences in prescriber availability between predominant racial group was dependent on rurality based on the interaction found in our fitted model. In metropolitan areas, we found that predominantly Non-Hispanic White ZIP codes had a lower rate of waivered prescribers compared to predominantly Black/African American ZIP codes. CONCLUSIONS: Our findings suggest that among AI/AN and Black/African American neighborhoods, availability of waivered prescribers may not be a primary barrier. However, availability of waivered prescribers and prescribing might potentially be an obstacle for Hispanic/Latinx and rural communities. Additional research to determine factors related to improving MOUD availability among diverse communities therefore remains vital to advancing health equity.

Contingency management for alcohol use disorder reduces cannabis use among American Indian and Alaska Native adults.

OBJECTIVE: Determine whether a culturally tailored contingency management (CM) intervention targeting alcohol abstinence resulted in secondary effects on cannabis use among American Indian and Alaska Native (AI/AN) adults. METHODS: The research team conducted this secondary data analysis to examine cannabis abstinence using data from a randomized control trial of CM for alcohol use among three AI/AN-serving organizations. One hundred and fifty-eight adults met the randomization criteria (i.e., submission of 50% or more urine samples and one alcohol-positive urine test during a 4-week, pre-randomization, observation period). For 12 weeks after randomization, participants received incentives for submitting a urine test negative for ethyl glucuronide (EtG < 150 ng/mL, CM group) or incentives for submitting a urine sample regardless of abstinence (Non-contingent [NC] Control group). Generalized linear mixed effects models assessed group differences in cannabis abstinence during the intervention, verified by urine tetrahydrocannabinol negative tests (11-nor-delta-9-tetrahydrocannabinol-9-carboxylic acid <50 ng/mL). RESULTS: At baseline, 42.2% (n = 35) of participants in the NC group and 40.0% (n = 30) of those in the CM group had a cannabis positive urine test. An overall intervention by time interaction was detected for a cannabis negative urine test (χ2 = 13.40, p = 0.001). Compared to the NC group, the CM group had 3.92 (95% CI:1.23-12.46) times higher odds of having a cannabis negative urine test during the intervention period and 5.13 (95% CI:1.57-16.76) times higher odds of having a negative cannabis test at the end of intervention period. CONCLUSION: CM addressing alcohol misuse may be an effective strategy for decreasing cannabis use among AI/AN adults. TRIAL REGISTRATION: ClinicalTrials.gov number, Identifier: NCT02174315.

Onset of alcohol use disorder among alcohol initiates by race/ethnicity.

Prevalence of alcohol use disorders (AUD) varies across racial/ethnic groups. It remains unclear whether rapid transition from first-time alcohol use to developing AUD varies by race and ethnicity. In this study, we investigate racial/ethnic differences in AUD onset among first-time alcohol drinkers and identify specific predictors of AUD onset by racial/ethnic group. The study population was non-institutionalized US residents aged 12 and older. Within four nationally representative probability samples (n ∼70,000/year) drawn from the 2015-2018 National Surveys on Drug Use and Health, we identified 9,381 individuals who initiated alcohol use within 1-12 months prior to the survey. The probability of AUD after initiation was estimated for the entire sample, followed by racial/ethnic group stratification. Bivariate and multivariable logistic regression models were used to identify predictors of AUD onset among alcohol initiates. The overall incidence estimate of AUD among alcohol initiates was 3.7% (95% CI = 3.0%, 4.6%). There was no significant variation in the incidence of AUD between racial/ethnic groups. Drug use, drug use disorders, and major depressive episode were significant predictors of AUD onset among all alcohol initiates. However, these predictors were not significant among non-Hispanic/Latinx Black individuals. Drug use and drug use disorders were strong predictors of AUD onset among alcohol initiates, except among non-Hispanic/Latinx Black individuals. These findings strengthen the importance of focusing on the co-use of alcohol and other drugs and the need to further investigate the risk profile differences between racial/ethnic groups.

Effect of Incentives for Alcohol Abstinence in Partnership With 3 American Indian and Alaska Native Communities: A Randomized Clinical Trial.

Importance: Many American Indian and Alaska Native communities are disproportionately affected by problems with alcohol use and seek culturally appropriate and effective interventions for individuals with alcohol use disorders. Objective: To determine whether a culturally tailored contingency management intervention, in which incentives were offered for biologically verified alcohol abstinence, resulted in increased abstinence among American Indian and Alaska Native adults. This study hypothesized that adults assigned to receive a contingency management intervention would have higher levels of alcohol abstinence than those assigned to the control condition. Design, Setting, and Participants: This multisite randomized clinical trial, the Helping Our Native Ongoing Recovery (HONOR) study, included a 1-month observation period before randomization and a 3-month intervention period. The study was conducted at 3 American Indian and Alaska Native health care organizations located in Alaska, the Pacific Northwest, and the Northern Plains from October 10, 2014, to September 2, 2019. Recruitment occurred between October 10, 2014, and February 20, 2019. Eligible participants were American Indian or Alaska Native adults who had 1 or more days of high alcohol-use episodes within the last 30 days and a current diagnosis of alcohol dependence. Data were analyzed from February 1 to April 29, 2020. Interventions: Participants received treatment as usual and were randomized to either the contingency management group, in which individuals received 12 weeks of incentives for submitting a urine sample indicating alcohol abstinence, or the control group, in which individuals received 12 weeks of incentives for submitting a urine sample without the requirement of alcohol abstinence. Regression models fit with generalized estimating equations were used to assess differences in abstinence during the intervention period. Main Outcomes and Measures: Alcohol-negative ethyl glucuronide (EtG) urine test result (defined as EtG<150 ng/mL). Results: Among 1003 adults screened for eligibility, 400 individuals met the initial criteria. Of those, 158 individuals (39.5%; mean [SD] age, 42.1 [11.4] years; 83 men [52.5%]) met the criteria for randomization, which required submission of 4 or more urine samples and 1 alcohol-positive urine test result during the observation period before randomization. A total of 75 participants (47.5%) were randomized to the contingency management group, and 83 participants (52.5%) were randomized to the control group. At 16 weeks, the number who submitted an alcohol-negative urine sample was 19 (59.4%) in the intervention group vs 18 (38.3%) in the control group. Participants randomized to the contingency management group had a higher likelihood of submitting an alcohol-negative urine sample (averaged over time) compared with those randomized to the control group (odds ratio, 1.70; 95% CI, 1.05-2.76; P = .03). Conclusions and Relevance: The study's findings indicate that contingency management may be an effective strategy for increasing alcohol abstinence and a tool that can be used by American Indian and Alaska Native communities for the treatment of individuals with alcohol use disorders. Trial Registration: ClinicalTrials.gov Identifier: NCT02174315.

Assessing the Predictive Validity of the Stages of Change Readiness and Treatment Eagerness Scale (SOCRATES) in Alaska Native and American Indian People.

OBJECTIVES: The objective of this study was to examine the predictive validity of the Stages of Change Readiness and Treatment Eagerness Scale (SOCRATES) among Alaska Native and American Indian (ANAI) people with an alcohol use disorder. METHODS: The sample was 170 ANAI adults with an alcohol use disorder living in Anchorage, Alaska who were part of a larger alcohol intervention study. The primary outcome of this study was alcohol use as measured by mean urinary ethyl glucuronide (EtG). EtG urine tests were collected at baseline and then up to twice a week for four weeks. We conducted bivariate linear regression analyses to evaluate associations between mean EtG value and each of the three SOCRATES subscales (Recognition, Ambivalence, and Taking Steps) and other covariates such as demographic characteristics, alcohol use history, and chemical dependency service utilization. We then performed multivariable linear regression modeling to examine these associations after adjusting for covariates. RESULTS: After adjusting for covariates, mean EtG values were negatively associated with the Taking Steps (P = 0.017) and Recognition (P = 0.005) subscales of the SOCRATES among ANAI people living in Alaska. We did not find an association between mean EtG values and the Ambivalence subscale (P = 0.129) of the SOCRATES after adjusting for covariates. CONCLUSIONS: Higher scores on the Taking Steps and Recognition subscales of the SOCRATES at baseline among ANAI people predicted lower mean EtG values. This study has important implications for communities and clinicians who need tools to assist ANAI clients in initiating behavior changes related to alcohol use.

A Chemical Strategy for Protease Substrate Profiling.

The dipeptidyl peptidases (DPPs) regulate hormones, cytokines, and neuropeptides by cleaving dipeptides after proline from their amino termini. Due to technical challenges, many DPP substrates remain unknown. Here, we introduce a simple method, termed CHOPS (chemical enrichment of protease substrates), for the discovery of protease substrates. CHOPS exploits a 2-pyridinecarboxaldehyde (2PCA)-biotin probe, which selectively biotinylates protein N-termini except those with proline in the second position. CHOPS can, in theory, discover substrates for any protease, but is particularly well suited to discover canonical DPP substrates, as cleaved but not intact DPP substrates can be identified by gel electrophoresis or mass spectrometry. Using CHOPS, we show that DPP8 and DPP9, enzymes that control the Nlrp1 inflammasome through an unknown mechanism, do not directly cleave Nlrp1. We further show that DPP9 robustly cleaves short peptides but not full-length proteins. More generally, this work delineates a practical technology for identifying protease substrates, which we anticipate will complement available "N-terminomic" approaches.

Total Chemical Synthesis of Human Thyroid-Stimulating Hormone (hTSH) ß-Subunit: Application of Arginine-tagged Acetamidomethyl (AcmR) Protecting Groups.

The ß-subunit of human thyroid stimulating hormone (hTSH) has been synthesized as a single glycoform bearing a chitobiose disaccharide at the native glycosylation site. Key to the successful completion of this synthesis was the introduction of an arginine-tagged acetamidomethyl group, which served to greatly facilitate handling of a glycopeptide fragment with poor aqueous solubility. This general solution to the challenge of working with intractable peptides is expected to find wide use in protein synthesis.

Life history factors, personality and the social clustering of sexual experience in adolescents.

Adolescent sexual behaviour may show clustering in neighbourhoods, schools and friendship networks. This study aims to assess how experience with sexual intercourse clusters across the social world of adolescents and whether predictors implicated by life history theory or personality traits can account for its between-individual variation and social patterning. Using data on 2877 adolescents from the Avon Longitudinal Study of Parents and Children, we ran logistic multiple classification models to assess the clustering of sexual experience by approximately 17.5 years in schools, neighbourhoods and friendship networks. We examined how much clustering at particular levels could be accounted for by life history predictors and Big Five personality factors. Sexual experience exhibited substantial clustering in friendship networks, while clustering at the level of schools and neighbourhoods was minimal, suggesting a limited role for socio-ecological influences at those levels. While life history predictors did account for some variation in sexual experience, they did not explain clustering in friendship networks. Personality, especially extraversion, explained about a quarter of friends' similarity. After accounting for life history factors and personality, substantial unexplained similarity among friends remained, which may reflect a tendency to associate with similar individuals or the social transmission of behavioural norms.

[Injuries to blood vessels near the heart caused by central venous catheters]. / Verletzungen herznaher Gefäße durch zentralvenöse Katheter.

Injuries to blood vessels near the heart can quickly become life-threatening and include arterial injuries during central venous puncture, which can lead to hemorrhagic shock. We report 6 patients in whom injury to the subclavian artery and vein led to life-threatening complications. Central venous catheters are associated with a multitude of risks, such as venous thrombosis, air embolism, systemic or local infections, paresthesia, hemothorax, pneumothorax, and cervical hematoma, which are not always immediately discernible. The subclavian catheter is at a somewhat lower risk of catheter-associated sepsis and symptomatic venous thrombosis than approaches via the internal jugular and femoral veins. Indeed, access via the subclavian vein carries a substantial risk of pneumo- and hemothorax. Damage to the subclavian vein or artery can also occur during deliberate and inadvertent punctures and result in life-threatening complications. Therefore, careful consideration of the access route is required in relation to the patient and the clinical situation, to keep the incidence of complications as low as possible. For catheterization of the subclavian vein, puncture of the axillary vein in the infraclavicular fossa is a good alternative, because ultrasound imaging of the target vessel is easier than in the subclavian vein and the puncture can be performed much further from the lung.

[Anesthesia for medical students : A brief guide to practical anesthesia in adults with a web-based video illustration]. / Anästhesie für Medizinstudierende : Kurzanleitung zur praktischen Anästhesie bei Erwachsenen mit webbasierter Videoillustrierung.

In Germany, Austria and Switzerland, anesthesiologists are the second largest group of physicians in hospitals, but this does not correspond to the amount of anesthesiology teaching that medical students receive in medical schools. Accordingly, the chances of medical students recognizing anesthesiology as a promising personal professional career are smaller than in other disciplines with large teaching components. Subsequent difficulties to recruit anesthesiology residents are likely, although many reasons support anesthesiology as a professional career.Traditional strategies to teach medical students in anesthesiology in medical school consist of airway management or cardiopulmonary resuscitation attempts in manikins. Anesthesiology is a complex interaction consisting of anatomy, physiology, pharmacology, clinical evaluation, experience, knowledge, and manual skills. While some medical schools offer teaching in high fidelity simulators, clinical teaching in the operating room is often limited. When medical students opt for a clinical rotation in anesthesiology, there is a chance to demonstrate the fascinating world of anesthesiology, but this chance has to be utilized carefully by anesthesiologists, as young talents have to be discovered, supported, and challenged.We have put together a short guide for medical students for a clinical rotation in anesthesiology in adults in order to generate basic knowledge and interest in anesthesiology as well as a sense of achievement. Basic knowledge about premedication, induction, maintenance and strategies for anesthesia is discussed. Further, the most important anesthesia drugs are discussed and manual skills, such as intravenous cannulation, mask ventilation, intubation, and regional anesthesia are featured with QR-code based video illustrations on a smartphone or personal computer. We did not discuss possible local mannerism and special patient groups (e. g., children, special medical history), local guidelines, or standard operating procedures.Medical students can be inspired by anesthesiology when not simply told about the tools of the trade and strategies, but instead by gaining knowledge and clinical skills that render a sense of achievement, likely during a clinical rotation into anesthesiology. A short theoretical instruction of anesthesiology enables rapid and targeted orientation even before a clinical rotation. Whether this subsequently results in a higher recruiting chance is currently unknown.

Computational, electrochemical, and spectroscopic studies of two mononuclear cobaloximes: the influence of an axial pyridine and solvent on the redox behaviour and evidence for pyridine coordination to cobalt(i) and cobalt(ii) metal centres.

[Co(dmgBF2)2(H2O)2] (where dmgBF2 = difluoroboryldimethylglyoximato) was used to synthesize [Co(dmgBF2)2(H2O)(py)]·0.5(CH3)2CO (where py = pyridine) in acetone. The formulation of complex was confirmed by elemental analysis, high resolution MS, and various spectroscopic techniques. The complex [Co(dmgBF2)2(solv)(py)] (where solv = solvent) was readily formed in situ upon the addition of pyridine to complex . A spectrophotometric titration involving complex and pyridine proved the formation of such a species, with formation constants, log K = 5.5, 5.1, 5.0, 4.4, and 3.1 in 2-butanone, dichloromethane, acetone, 1,2-difluorobenzene/acetone (4 : 1, v/v), and acetonitrile, respectively, at 20 °C. In strongly coordinating solvents, such as acetonitrile, the lower magnitude of K along with cyclic voltammetry, NMR, and UV-visible spectroscopic measurements indicated extensive dissociation of the axial pyridine. In strongly coordinating solvents, [Co(dmgBF2)2(solv)(py)] can only be distinguished from [Co(dmgBF2)2(solv)2] upon addition of an excess of pyridine, however, in weakly coordinating solvents the distinctions were apparent without the need for excess pyridine. The coordination of pyridine to the cobalt(ii) centre diminished the peak current at the Epc value of the Co(I/0) redox couple, which was indicative of the relative position of the reaction equilibrium. Herein we report the first experimental and theoretical (59)Co NMR spectroscopic data for the formation of Co(i) species of reduced cobaloximes in the presence and absence of py (and its derivatives) in CD3CN. From spectroelectrochemical studies, it was found that pyridine coordination to a cobalt(i) metal centre is more favourable than coordination to a cobalt(ii) metal centre as evident by the larger formation constant, log K = 4.6 versus 3.1, respectively, in acetonitrile at 20 °C. The electrosynthesis of hydrogen by complexes and in various solvents demonstrated the dramatic effects of the axial ligand and the solvent on the turnover number of the respective catalyst.

Structural and functional studies of a trans-acyltransferase polyketide assembly line enzyme that catalyzes stereoselective α- and ß-ketoreduction.

While the cis-acyltransferase modular polyketide synthase assembly lines have largely been structurally dissected, enzymes from within the recently discovered trans-acyltransferase polyketide synthase assembly lines are just starting to be observed crystallographically. Here we examine the ketoreductase (KR) from the first polyketide synthase module of the bacillaene nonribosomal peptide synthetase/polyketide synthase at 2.35-Å resolution. This KR naturally reduces both α- and ß-keto groups and is the only KR known to do so during the biosynthesis of a polyketide. The isolated KR not only reduced an N-acetylcysteamine-bound ß-keto substrate to a D-ß-hydroxy product, but also an N-acetylcysteamine-bound α-keto substrate to an L-α-hydroxy product. That the substrates must enter the active site from opposite directions to generate these stereochemistries suggests that the acyl-phosphopantetheine moiety is capable of accessing very different conformations despite being anchored to a serine residue of a docked acyl carrier protein. The features enabling stereocontrolled α-ketoreduction may not be extensive since a KR that naturally reduces a ß-keto group within a cis-acyltransferase polyketide synthase was identified that performs a completely stereoselective reduction of the same α-keto substrate to generate the D-α-hydroxy product. A sequence analysis of trans-acyltransferase KRs reveals that a single residue, rather than a three-residue motif found in cis-acyltransferase KRs, is predictive of the orientation of the resulting ß-hydroxyl group.

A close look at a ketosynthase from a trans-acyltransferase modular polyketide synthase.

The recently discovered trans-acyltransferase modular polyketide synthases catalyze the biosynthesis of a wide range of bioactive natural products in bacteria. Here we report the structure of the second ketosynthase from the bacillaene trans-acyltransferase polyketide synthase. This 1.95 Å resolution structure provides the highest resolution view available of a modular polyketide synthase ketosynthase and reveals a flanking subdomain that is homologous to an ordered linker in cis-acyltransferase modular polyketide synthases. The structure of the cysteine-to-serine mutant of the ketosynthase acylated by its natural substrate provides high-resolution details of how a native polyketide intermediate is bound and helps explain the basis of ketosynthase substrate specificity. The substrate range of the ketosynthase was further investigated by mass spectrometry.

Regulation of respiration and the oxygen diffusion barrier in soybean protect symbiotic nitrogen fixation from chilling-induced inhibition and shoots from premature senescence.

Symbiotic nitrogen fixation is sensitive to dark chilling (7 degrees C-15 degrees C)-induced inhibition in soybean (Glycine max). To characterize the mechanisms that cause the stress-induced loss of nodule function, we examined nodule structure, carbon-nitrogen interactions, and respiration in two soybean genotypes that differ in chilling sensitivity: PAN809 (PAN), which is chilling sensitive, and Highveld Top (HT), which is more chilling resistant. Nodule numbers were unaffected by dark chilling, as was the abundance of the nitrogenase and leghemoglobin proteins. However, dark chilling decreased nodule respiration rates, nitrogenase activities, and NifH and NifK mRNAs and increased nodule starch, sucrose, and glucose in both genotypes. Ureide and fructose contents decreased only in PAN nodules. While the chilling-induced decreases in nodule respiration persisted in PAN even after return to optimal temperatures, respiration started to recover in HT by the end of the chilling period. The area of the intercellular spaces in the nodule cortex and infected zone was greatly decreased in HT after three nights of chilling, an acclimatory response that was absent from PAN. These data show that HT nodules are able to regulate both respiration and the area of the intercellular spaces during chilling and in this way control the oxygen diffusion barrier, which is a key component of the nodule stress response. We conclude that chilling-induced loss of symbiotic nitrogen fixation in PAN is caused by the inhibition of respiration coupled to the failure to regulate the oxygen diffusion barrier effectively. The resultant limitations on nitrogen availability contribute to the greater chilling-induced inhibition of photosynthesis in PAN than in HT.

The role of low soil temperature in the inhibition of growth and PSII function during dark chilling in soybean genotypes of contrasting tolerance.

Dark chilling affects growth and yield of warm-climate crops such as soybean [Glycine max (L.) Merr.]. Several studies have investigated chilling-stress effects on photosynthesis and other aspects of metabolism, but none have compared effects of whole-plant chilling (WPC; shoots and roots) with that of aboveground chilling in legumes. This is important because low root temperatures might induce additional constraints, such as inhibition of N(2) fixation, thereby aggravating chilling-stress symptoms. Effects of dark chilling on PSII, shoot growth, leaf ureide content and photosynthetic capacity were studied in two soybean genotypes, Highveld Top (chilling tolerant) and PAN809 (chilling sensitive), in experiments comparing effects of WPC with that of shoot chilling (SC). Both treatments inhibited shoot growth in PAN809 but not Highveld Top. Also, WPC in PAN809 caused a decrease in leaf ureide content followed by severe chlorosis and alterations in O-J-I-P fluorescence-rise kinetics, distinct from SC. A noteworthy difference was the appearance of a Delta K peak in the O-J-I-P fluorescence rise in response to WPC. These genotypic and treatment differences also reflected in the degree of inhibition of CO(2) assimilation rates. The appearance of a Delta K peak, coupled with growth inhibition, reduced ureide content, chlorosis and lower CO(2) assimilation rates, provides mechanistic information about how WPC might have aggravated chilling-stress symptoms in PAN809. We introduce a model explaining how chilling soil temperatures might trigger N-limitation in sensitive genotypes and how characteristic changes in O-J-I-P fluorescence-rise kinetics are linked to changes in carbon and nitrogen metabolism.