Development of 2nd generation aminomethyl spectinomycins that overcome native efflux in Mycobacterium abscessus.

Mycobacterium abscessus (Mab), a nontuberculous mycobacterial (NTM) species, is an emerging pathogen with high intrinsic drug resistance. Current standard-of-care therapy results in poor outcomes, demonstrating the urgent need to develop effective antimycobacterial regimens. Through synthetic modification of spectinomycin (SPC), we have identified a distinct structural subclass of N-ethylene linked aminomethyl SPCs (eAmSPCs) that are up to 64-fold more potent against Mab over the parent SPC. Mechanism of action and crystallography studies demonstrate that the eAmSPCs display a mode of ribosomal inhibition consistent with SPC. However, they exert their increased antimicrobial activity through enhanced accumulation, largely by circumventing efflux mechanisms. The N-ethylene linkage within this series plays a critical role in avoiding TetV-mediated efflux, as lead eAmSPC 2593 displays a mere fourfold susceptibility improvement against Mab ΔtetV, in contrast to the 64-fold increase for SPC. Even a minor shortening of the linkage by a single carbon, akin to 1st generation AmSPC 1950, results in a substantial increase in MICs and a 16-fold rise in susceptibility against Mab ΔtetV. These shifts suggest that longer linkages might modify the kinetics of drug expulsion by TetV, ultimately shifting the equilibrium towards heightened intracellular concentrations and enhanced antimicrobial efficacy. Furthermore, lead eAmSPCs were also shown to synergize with various classes of anti-Mab antibiotics and retain activity against clinical isolates and other mycobacterial strains. Encouraging pharmacokinetic profiles coupled with robust efficacy in Mab murine infection models suggest that eAmSPCs hold the potential to be developed into treatments for Mab and other NTM infections.

Discovery of L-threonine transaldolases for enhanced biosynthesis of beta-hydroxylated amino acids.

Beta-hydroxy non-standard amino acids (ß-OH-nsAAs) have utility as small molecule drugs, precursors for beta-lactone antibiotics, and building blocks for polypeptides. While the L-threonine transaldolase (TTA), ObiH, is a promising enzyme for ß-OH-nsAA biosynthesis, little is known about other natural TTA sequences. We ascertained the specificity of the TTA enzyme class more comprehensively by characterizing 12 candidate TTA gene products across a wide range (20-80%) of sequence identities. We found that addition of a solubility tag substantially enhanced the soluble protein expression level within this difficult-to-express enzyme family. Using an optimized coupled enzyme assay, we identified six TTAs, including one with less than 30% sequence identity to ObiH that exhibits broader substrate scope, two-fold higher L-Threonine (L-Thr) affinity, and five-fold faster initial reaction rates under conditions tested. We harnessed these TTAs for first-time bioproduction of ß-OH-nsAAs with handles for bio-orthogonal conjugation from supplemented precursors during aerobic fermentation of engineered Escherichia coli, where we observed that higher affinity of the TTA for L-Thr increased titer. Overall, our work reveals an unexpectedly high level of sequence diversity and broad substrate specificity in an enzyme family whose members play key roles in the biosynthesis of therapeutic natural products that could benefit from chemical diversification.

EFdA efficiently suppresses HIV replication in the male genital tract and prevents penile HIV acquisition.

Sexually transmitted HIV infections in heterosexual men are acquired through the penis. Low adherence to condom usage and the fact that 40% of circumcised men are not protected indicate the need for additional prevention strategies. Here, we describe a new approach to evaluate the prevention of penile HIV transmission. We demonstrated that the entire male genital tract (MGT) of bone marrow/liver/thymus (BLT) humanized mice is repopulated with human T and myeloid cells. The majority of the human T cells in the MGT express CD4 and CCR5. Direct penile exposure to HIV leads to systemic infection including all tissues of the MGT. HIV replication throughout the MGT was reduced 100-1,000-fold by treatment with 4'-ethynyl-2-fluoro-2'-deoxyadenosine (EFdA), resulting in the restoration of CD4+ T cell levels. Importantly, systemic preexposure prophylaxis with EFdA effectively protects from penile HIV acquisition. IMPORTANCE Over 84.2 million people have been infected by the human immunodeficiency virus type 1 (HIV-1) during the past 40 years, most through sexual transmission. Men comprise approximately half of the HIV-infected population worldwide. Sexually transmitted HIV infections in exclusively heterosexual men are acquired through the penis. However, direct evaluation of HIV infection throughout the human male genital tract (MGT) is not possible. Here, we developed a new in vivo model that permits, for the first time, the detail analysis of HIV infection. Using BLT humanized mice, we showed that productive HIV infection occurs throughout the entire MGT and induces a dramatic reduction in human CD4 T cells compromising immune responses in this organ. Antiretroviral treatment with novel drug EFdA suppresses HIV replication in all tissues of the MGT, restores normal levels of CD4 T cells and is highly efficient at preventing penile transmission.

Investigating Intraindividual Variability of Psychological Needs Satisfaction and Relations With Subsequent Physical Activity.

Heeding recent calls to capture dynamic variability of physical activity (PA) motivation within a self-determination theory framework, this study examined the extent to which psychological needs satisfaction in PA predicted subsequent PA, disaggregating within-person and between-persons data. University students (N = 89) wore an ActiGraph GT3X accelerometer for 6 days and reported basic psychological needs satisfaction daily. Multilevel models examined whether competence, autonomy, and relatedness for the previous day's PA (>2,020 counts per minute) predicted the following day's minutes of PA (>2,020 counts per minute), controlling for previous-day PA. Participants who, on average, reported greater feelings of autonomy and competence tended to engage in more minutes of PA the following day. When participants reported feeling greater relatedness than what was typical for them, they tended to engage in more PA the following day. Psychological needs vary day to day, but how and to what extent they predict PA depends on the specific need.

Combinatorial gene inactivation of aldehyde dehydrogenases mitigates aldehyde oxidation catalyzed by E. coli resting cells.

Aldehydes are attractive chemical targets both as end products in the flavors and fragrances industry and as synthetic intermediates due to their propensity for C-C bond formation. Here, we identify and address unexpected oxidation of a model collection of aromatic aldehydes, including many that originate from biomass degradation. When diverse aldehydes are supplemented to E. coli cells grown under aerobic conditions, as expected they are either reduced by the wild-type MG1655 strain or stabilized by a strain engineered for reduced aromatic aldehyde reduction (the E. coli RARE strain). Surprisingly, when these same aldehydes are supplemented to resting cell preparations of either E. coli strain, under many conditions we observe substantial oxidation. By performing combinatorial inactivation of six candidate aldehyde dehydrogenase genes in the E. coli genome using multiplexed automatable genome engineering (MAGE), we demonstrate that this oxidation can be substantially slowed, with greater than 50% retention of 6 out of 8 aldehydes when assayed 4 h after their addition. Given that our newly engineered strain exhibits reduced oxidation and reduction of aromatic aldehydes, we dubbed it the E. coli ROAR strain. We applied the new strain to resting cell biocatalysis for two kinds of reactions - the reduction of 2-furoic acid to furfural and the condensation of 3-hydroxybenzaldehyde and glycine to form a non-standard ß-hydroxy-α-amino acid. In each case, we observed substantial improvements in product titer 20 h after reaction initiation (9-fold and 10-fold, respectively). Moving forward, the use of this strain to generate resting cells should allow aldehyde product isolation, further enzymatic conversion, or chemical reactivity under cellular contexts that better accommodate aldehyde toxicity.

Population-based correlates of COVID-19 infection: An analysis from the DFW COVID-19 prevalence study.

BACKGROUND: COVID-19 has resulted in over 1 million deaths in the U.S. as of June 2022, with continued surges after vaccine availability. Information on related attitudes and behaviors are needed to inform public health strategies. We aimed to estimate the prevalence of COVID-19, risk factors of infection, and related attitudes and behaviors in a racially, ethnically, and socioeconomically diverse urban population. METHODS: The DFW COVID-19 Prevalence Study Protocol 1 was conducted from July 2020 to March 2021 on a randomly selected sample of adults aged 18-89 years, living in Dallas or Tarrant Counties, Texas. Participants were asked to complete a 15-minute questionnaire and COVID-19 PCR and antibody testing. COVID-19 prevalence estimates were calculated with survey-weighted data. RESULTS: Of 2969 adults who completed the questionnaire (7.4% weighted response), 1772 (53.9% weighted) completed COVID-19 testing. Overall, 11.5% of adults had evidence of COVID-19 infection, with a higher prevalence among Hispanic and non-Hispanic Black persons, essential workers, those in low-income neighborhoods, and those with lower education attainment compared to their counterparts. We observed differences in attitudes and behaviors by race and ethnicity, with non-Hispanic White persons being less likely to believe in the importance of mask wearing, and racial and ethnic minorities more likely to attend social gatherings. CONCLUSION: Over 10% of an urban population was infected with COVID-19 early during the pandemic. Differences in attitudes and behaviors likely contribute to sociodemographic disparities in COVID-19 prevalence.

Targeting Helicobacter pylori for antibacterial drug discovery with novel therapeutics.

Helicobacter pylori is an important human pathogen with increasing antimicrobial resistance to standard-of-care antibiotics. Treatment generally includes a combination of classical broad-spectrum antibiotics and a proton-pump inhibitor, which often leads to perturbation of the gut microbiome and the potential for the development of antibiotic resistance. In this review, we examine reports, primarily from the past decade, on the discovery of new anti-H. pylori therapeutics, including approaches to develop narrow-spectrum and mechanistically unique antibiotics to treat these infections in their gastric niche. Compound series that target urease, respiratory complex I, and menaquinone biosynthesis are discussed in this context, along with bivalent antibiotic approaches that suppress resistance development. With increases in the understanding of the unique physiology of H. pylori and technological advances in the field of antibacterial drug discovery, there is a clear promise that novel therapeutics can be developed to effectively treat H. pylori infections.

Solvent Effects and Mechanistic Studies for Re2O7-Catalyzed Allylative Annulation Reactions.

The sequence of allylic alcohol transposition, carbonyl group trapping, oxocarbenium ion formation, and nucleophilic addition results in the formation of a ring while serving as a fragment-coupling and stereocenter-generating reaction. Successful applications of these processes require a balancing of the kinetics of numerous productive and unproductive steps. This work describes the manner in which solvent changes can be used to expand the scope and change the stereochemical outcomes of these processes. Mechanistic studies provide greater insight into the nuances of the transformations and the reactive species that are generated.

Dietary ecology of Alaskan polar bears (Ursus maritimus) through time and in response to Arctic climate change.

Arctic climate change poses serious threats to polar bears (Ursus maritimus) as reduced sea ice makes seal prey inaccessible and marine ecosystems undergo bottom-up reorganization. Polar bears' elongated skulls and reduced molar dentition, as compared to their sister species the grizzly bear (Ursus arctos), are adaptations associated with hunting seals on sea ice and a soft, lipid-rich diet of blubber and meat. With significant declines in sea ice, it is unclear if and how polar bears may be altering their diets. Clarifying polar bear dietary responses to changing climates, both today and in the past, is critical to proper conservation and management of this apex predator. This is particularly important when a dietary strategy may be maladaptive. Here, we test the hypothesis that hard-food consumption (i.e., less preferred foods including bone), inferred from dental microwear texture analysis, increased with Arctic warming. We find that polar bears demonstrate a conserved absence of hard-object feeding in Alaska through time (including approximately 1000 years ago), until the 21st century, consistent with a highly conserved and specialized diet of soft blubber and flesh. Notably, our results also suggest that some 21st-century polar bears may be consuming harder foods (e.g., increased carcass utilization, terrestrial foods including garbage), despite having skulls and metabolisms poorly suited for such a diet. Prior to the 21st century, only polar bears with larger mandibles demonstrated increased hard-object feeding, though to a much lower degree than closely related grizzly bears which regularly consume mechanically challenging foods. Polar bears, being morphologically specialized, have biomechanical constraints which may limit their ability to consume mechanically challenging diets, with dietary shifts occurring only under the most extreme scenarios. Collectively, the highly specialized diets and cranial morphology of polar bears may severely limit their ability to adapt to a warming Arctic.

Minimal impact of selective susceptibility reporting on the use of piperacillin-tazobactam for Escherichia coli and Klebsiella bacteremia.

Selective cascade reporting of antibiotic susceptibilities did not have a significant impact on de-escalation from piperacillin-tazobactam (PT), duration of PT use, length of stay, or rates of acute kidney injury and Clostridioides difficile infection in patients with positive monomicrobial blood cultures with either Escherichia coli or Klebsiella spp.

Clinical Remission of Treatment-Resistant Depression, Polysubstance Abuse, and Antidepressant Discontinuation Syndrome Through Engagement of Lifestyle Interventions.

BACKGROUND: This case illustrates the relationship between gut, hormonal, and brain function in that dietary changes, mindfulness interventions, and detoxification led to resolution of disabling psychiatric symptoms and protracted psychotropic medication withdrawal symptoms. SUMMARY: A 50-year-old partnered, unemployed, Caucasian female with a history of major depressive disorder, multiple suicide attempts, extensive trauma and abuse, and substance abuse presented for outpatient management. The patient reported limited benefit from over two decades of conventional treatment with psychotropic medications. She presented with depression and symptoms of protracted withdrawal after self-discontinuation of multiple psychiatric medications and was prescribed a dietary, detoxification, and supplementation regimen by the primary author. Additional lifestyle interventions implemented included daily meditation, dry-skin brushing, and coffee enemas. CONCLUSION: This case exemplifies dramatic clinical remission after cessation of medication treatment and engagement of lifestyle interventions, which include dietary change, meditation, and detoxification. As such, when limited results are achieved by psychotropic medication, tapering combined with dietary interventions as the first-line therapy should be considered. This case is also evidence of the role of lifestyle interventions in treating protracted withdrawal symptoms associated with discontinuing psychotropic medications.

Standardizing the Use of Albumin in Large Volume Paracentesis.

BACKGROUND: Albumin after large volume paracentesis (LVP) reduces paracentesis-induced circulatory dysfunction (PICD). The most efficacious dose of albumin for LVP is unclear. OBJECTIVE: To evaluate the impact of implementing a standardized LVP order set on albumin utilization and patient outcomes. METHODS: This is a retrospective review of patients with ascites due to cirrhosis who received a therapeutic paracentesis at a large, academic institution. Primary outcome was amount of albumin used prior to and after order set implementation. Albumin doses were standardized in the order set to 25 g (5-6 L removed), 50 g (7-10 L), and 75 g (>10 L). Patient outcomes were rates of hyponatremia, renal impairment, and hypotension. RESULTS: There were 100 patients included in each arm of the final analysis. Patients prior to order set implementation received a higher amount of albumin per liter removed compared to those post-implementation (8.3 g/L vs 6.5 g/L, respectively; P < .01). There were no significant differences between groups in absolute changes in serum sodium (0 mEq/L vs -1 mEq/L, P = .64), serum creatinine (0.06 mg/dL vs 0.05 mg/dL, P = .94), or systolic blood pressure (-4 mm Hg vs -3 mm Hg, P = .96). There were no differences between groups in rates of hyponatremia (1.6% vs 6.6%, P = .21), renal impairment (11.3% vs 11.5%, P = .97), or hypotension (17.4% vs 17.6%, P = .97). CONCLUSIONS: Use of an order set to guide albumin dosing based on amount of ascitic fluid removed during LVP resulted in a significant reduction in the amount of albumin given with no difference in adverse effects.

GCC Consolidated Feedback to ICH on the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline.

The 13th GCC Closed Forum for Bioanalysis was held in New Orleans, Louisiana, USA on April 5th, 2019. This GCC meeting was organized to discuss the contents of the 2019 ICH M10 Bioanalytical Method Validation Draft Guideline published in February 2019 and consolidate the feedback of the GCC members. In attendance were 63 senior-level participants from eight countries representing 44 bioanalytical CRO companies/sites. This event represented a unique opportunity for CRO bioanalytical experts to share their opinions and concerns regarding the ICH M10 Bioanalytical Method Validation Draft Guideline and to build unified comments to be provided to the ICH.

Assessment of the Biotransformation of Low-Turnover Drugs in the HµREL Human Hepatocyte Coculture Model.

Metabolic profiles of four drugs possessing diverse metabolic pathways (timolol, meloxicam, linezolid, and XK469) were compared following incubations in both suspended cryopreserved human hepatocytes and the HµREL hepatocyte coculture model. In general, minimal metabolism was observed following 4-hour incubations in both suspended hepatocytes and the HµREL model, whereas incubations conducted up to 7 days in the HµREL coculture model resulted in more robust metabolic turnover. In the case of timolol, in vivo human data suggest that 22% of the dose is transformed via multistep oxidative opening of the morpholine moiety. Only the first-step oxidation was detected in suspended hepatocytes, whereas the relevant downstream metabolites were produced in the HµREL model. For meloxicam, both the hydroxymethyl and subsequent carboxylic acid metabolites were abundant following incubation in the HµREL model, while only a trace amount of the hydroxymethyl metabolite was observed in suspension. Similar to timolol, linezolid generated substantially higher levels of morpholine ring-opened carboxylic acid metabolites in the HµREL model. Finally, while the major aldehyde oxidase-mediated mono-oxidative metabolite of XK469 was minimally produced in hepatocyte suspension, the HµREL model robustly produced this metabolite, consistent with a pathway reported to account for 54% of the total urinary excretion in human. In addition, low-level taurine and glycine conjugates were identified in the HµREL model. In summary, continuous metabolite production was observed for up to 7 days of incubation in the HµREL model, covering cytochrome P450, aldehyde oxidase, and numerous conjugative pathways, while predominant metabolites correlated with relevant metabolites reported in human in vivo studies.

Complement 3 Receptor Expression in Individuals with Type 2 Diabetes.

BACKGROUND: According to the World Health Organization, as of 2014 9% of the world's adult population is affected by diabetes. Uncontrolled diabetes is a pro-inflammatory process that increases generation of reactive oxygen species (ROS). METHODS: The production of ROS leads to a chronic increase in oxidative stress which results in an increased susceptibility to infections. Individuals with type 2 diabetes mellitus (T2DM) are highly susceptible to Mycobacterium tuberculosis (M. tb) infection. Previous research has demonstrated that glutathione (GSH) plays an important role in the control of M. tb infection. Recent studies have demonstrated that phagocytosis of M.tb is diminished in patients with T2DM. Phagocytosis in macrophages is thought to be mediated in part by complement protein 3b (C3b)-complement protein receptor 3b (C3R) interactions. Since C3b production is not diminished in patients with T2DM we propose that C3R production is reduced and is the cause for impaired macrophage phagocytosis as well as IL-12 and IFN-γ signaling. CONCLUSION: This study utilizes a quantitative PCR (qPCR), demonstrating decreased transcription of C3R mRNA in patients with T2DM as compared to non-diabetics.

Low-Turnover Drug Molecules: A Current Challenge for Drug Metabolism Scientists.

In vitro assays using liver subcellular fractions or suspended hepatocytes for characterizing the metabolism of drug candidates play an integral role in the optimization strategy employed by medicinal chemists. However, conventional in vitro assays have limitations in their ability to predict clearance and generate metabolites for low-turnover (slowly metabolized) drug molecules. Due to a rapid loss in the activity of the drug-metabolizing enzymes, in vitro incubations are typically performed for a maximum of 1 hour with liver microsomes to 4 hours with suspended hepatocytes. Such incubations are insufficient to generate a robust metabolic response for compounds that are slowly metabolized. Thus, the challenge of accurately estimating low human clearance with confidence has emerged to be among the top challenges that drug metabolism scientists are confronted with today. In response, investigators have evaluated novel methodologies to extend incubation times and more sufficiently measure metabolism of low-turnover drugs. These methods include plated human hepatocytes in monoculture, and a novel in vitro methodology using a relay of sequential incubations with suspended cryopreserved hepatocytes. In addition, more complex in vitro cellular models, such as HepatoPac (Hepregen, Medford, MA), a micropatterned hepatocyte-fibroblast coculture system, and the HµREL (Beverley Hills, CA) hepatic coculture system, have been developed and characterized that demonstrate prolonged enzyme activity. In this review, the advantages and disadvantages of each of these in vitro methodologies as it relates to the prediction of clearance and metabolite identification will be described in an effort to provide drug metabolism scientists with the most up-to-date experimental options for dealing with the complex issue of low-turnover drug candidates.

Synthesis of heterocyclic triads by Pd-catalyzed cross-couplings and evaluation of their cell-specific toxicity profile.

Two complementary approaches for the preparation of linked 5-membered heterocycles were developed. The Pd-catalyzed Suzuki-Miyaura cross-coupling with halogenated furan, thiophene, and selenophene led to higher overall yields, but C,H-bond activation was a more efficient strategy for the coupling at C(2) of oxazoles. Potency and selectivity of the final hydroxymethyl products in renal (A498), lung (NCI-H226), kidney (CAKI-1), and breast (MDA-MB-468, MCF7) carcinoma cell lines were determined.

Condensation reactions of guanidines with bis-electrophiles: Formation of highly nitrogenous heterocycles.

2-Amino-1,4-dihydropyrimidines were reacted with bis-electrophiles to produce novel fused bi-pyrimidine, pyrimido-aminotriazine, and pyrimido-sulfonamide scaffolds. In addition, a quinazoline library was constructed using a guanidine Atwal-Biginelli reaction with 1-(quinazolin-2-yl)guanidines. The product heterocycles have novel constitutions with high nitrogen atom counts and represent valuable additions to screening libraries for the discovery of new modulators of biological targets.