Sarecycline inhibits protein translation in Cutibacterium acnes 70S ribosome using a two-site mechanism.

Acne vulgaris is a chronic disfiguring skin disease affecting ∼1 billion people worldwide, often having persistent negative effects on physical and mental health. The Gram-positive anaerobe, Cutibacterium acnes is implicated in acne pathogenesis and is, therefore, a main target for antibiotic-based acne therapy. We determined a 2.8-Šresolution structure of the 70S ribosome of Cutibacterium acnes by cryogenic electron microscopy and discovered that sarecycline, a narrow-spectrum antibiotic against Cutibacterium acnes, may inhibit two active sites of this bacterium's ribosome in contrast to the one site detected previously on the model ribosome of Thermus thermophilus. Apart from the canonical binding site at the mRNA decoding center, the second binding site for sarecycline exists at the nascent peptide exit tunnel, reminiscent of the macrolides class of antibiotics. The structure also revealed Cutibacterium acnes-specific features of the ribosomal RNA and proteins. Unlike the ribosome of the Gram-negative bacterium Escherichia coli, Cutibacterium acnes ribosome has two additional proteins, bS22 and bL37, which are also present in the ribosomes of Mycobacterium smegmatis and Mycobacterium tuberculosis. We show that bS22 and bL37 have antimicrobial properties and may be involved in maintaining the healthy homeostasis of the human skin microbiome.

Herbal and Non-Herbal Dietary Supplements for Psychiatric Indications: Considerations in Liver Transplantation.

PURPOSE OF REVIEW: Traditional, complementary, and integrative medicine (TCIM) modalities are widely employed. However, TCIM, specifically herbal and non-herbal dietary supplements, can pose challenges in the context of organ transplantation. In this review, we discuss common supplements used for psychiatric purposes and highlight important considerations for candidates and recipients of liver transplants. RECENT FINDINGS: Ashwagandha, kava kava, green tea extract, skullcap, turmeric, and valerian have known idiosyncratic hepatotoxic potential and may complicate the liver transplantation course. Multiple supplements reportedly carry a lower risk of hepatotoxicity, though evidence for widespread use in those at risk for or with hepatic impairment is limited. Psychiatrists caring for candidates and recipients of liver transplants must recognize that patients may find supplements helpful in alleviating psychiatric symptoms, despite an overall limited evidence base. Evaluating benefit versus risk ratios and reviewing drug-drug interactions is essential to promote transplant candidacy and mitigate the possibility of native or graft liver dysfunction.

Direct tracking of reverse-transcriptase speed and template sensitivity: implications for sequencing and analysis of long RNA molecules.

Although reverse-transcriptase (RT) enzymes are critical reagents for research and biotechnology, their mechanical properties are not well understood. In particular, we know little about their relative speed and response to structural obstacles in the template. Commercial retroviral RTs stop at many positions along mixed sequence templates, resulting in truncated cDNA products that complicate downstream analysis. By contrast, group II intron-encoded RTs appear to copy long RNAs with high processivity and minimal stops. However, their speed, consistency and pausing behavior have not been explored. Here, we analyze RT velocity as the enzyme moves through heterogeneous sequences and structures that are embedded within a long noncoding RNA transcript. We observe that heterogeneities in the template are highly disruptive to primer extension by retroviral RTs. However, sequence composition and template structure have negligible effects on behavior of group II intron RTs, such as MarathonRT (MRT). Indeed, MRT copies long RNAs in a single pass, and displays synchronized primer extension at a constant speed of 25 nt/sec. In addition, it passes through stable RNA structural motifs without perturbation of velocity. Taken together, the results demonstrate that consistent, robust translocative behavior is a hallmark of group II intron-encoded RTs, some of which operate at high velocity.

Global Microbiota-Dependent Histone Acetylation Patterns Are Irreversible and Independent of Short Chain Fatty Acids.

BACKGROUND AND AIMS: Although germ-free mice are an indispensable tool in studying the gut microbiome and its effects on host physiology, they are phenotypically different than their conventional counterparts. While antibiotic-mediated microbiota depletion in conventional mice leads to physiologic alterations that often mimic the germ-free state, the degree to which the effects of microbial colonization on the host are reversible is unclear. The gut microbiota produce abundant short chain fatty acids (SCFAs), and previous studies have demonstrated a link between microbial-derived SCFAs and global hepatic histone acetylation in germ-free mice. APPROACH AND RESULTS: We demonstrate that global hepatic histone acetylation states measured by mass spectrometry remained largely unchanged despite loss of luminal and portal vein SCFAs after antibiotic-mediated microbiota depletion. In contrast to stable hepatic histone acetylation states, we see robust hepatic transcriptomic alterations after microbiota depletion. Additionally, neither dietary supplementation with supraphysiologic levels of SCFA nor the induction of hepatocyte proliferation in the absence of microbiota-derived SCFAs led to alterations in global hepatic histone acetylation. CONCLUSIONS: These results suggest that microbiota-dependent landscaping of the hepatic epigenome through global histone acetylation is static in nature, while the hepatic transcriptome is responsive to alterations in the gut microbiota.

Disordered eating and eating disorders among women seeking fertility treatment: A systematic review.

The purpose of this systematic review is to evaluate the prevalence of disordered eating and eating disorders among women seeking fertility treatment.Observational studies were searched in Ovid MEDLINE, Web of Science, Embase, and PsycInfo. Studies published prior to September 2020 when the search was conducted were considered. Inclusion criteria included (1) original and empirical research, (2) published in a peer-reviewed journal, and (3) reported on disordered eating among women seeking fertility treatment in the sample or reported on prevalence of eating disorders among women seeking fertility treatment in the sample. Independent screening of abstracts was conducted by two authors (LH and AH). Ten studies met the inclusion criteria. Sample size, study location, measures, and results for each study in this review were reported.Among women pursuing fertility treatment, rates of current eating disorders ranged from 0.5 to 16.7%, while past eating disorder prevalence rates ranged from 1.4 to 27.5%. Current anorexia nervosa or bulimia nervosa was reported by up to 2% and 10.3% of women, respectively, while history of anorexia nervosa or bulimia nervosa was reported by up to 8.5% and 3.3% of women, respectively. Binge eating disorder or other eating disorders were reported by up to 18.5% and 9.1% of women, respectively. Disordered eating pathology was endorsed by 1.6 to 48% of women seeking fertility treatment. Endorsement of pathological eating attitudes was generally higher among women seeking fertility treatment with current or past eating disorders as compared to community samples, with the exception of dietary restraint. Rates of current and past eating disorders are higher among women seeking fertility treatment than in the general population. Providers treating women with infertility should be cognizant of these prevalence rates and consider screening for eating pathology in their patients as this may contribute to their likelihood of successful conception and/or subsequent pregnancy outcomes.

A Brief Psychological Intervention for Chronic Pain in Primary Care: A Pilot Randomized Controlled Trial.

OBJECTIVE: Although evidence-based psychological interventions improve chronic pain, many patients do not engage in behavioral health services. Offering a brief intervention in a medical setting may provide benefits to patients with chronic pain. The purpose of this study was to examine preliminary outcomes of a brief psychological intervention for chronic pain delivered in primary care. DESIGN: Pilot randomized controlled trial. SETTING: Primary care clinic. SUBJECTS: Sixty participants with chronic pain were randomized to a 5-session psychological intervention or treatment-as-usual control group. METHODS: Participants completed pre- and post-intervention measures assessing pain severity, pain interference, pain catastrophizing, depression, and anxiety. RESULTS: Most participants (76.7%) randomized to the intervention completed all sessions. Compared to the control group, those in the intervention had decreases in pain severity (P = .048), pain catastrophizing (P = .04), and depression (P = .01) from pre- to post-intervention. Within the intervention group, there was a significant improvement in pain interference scores (P = 0.02). Within the intervention group, effect sizes were medium to large for changes in pain severity, pain interference, pain catastrophizing, and depression scores. There were no significant changes in anxiety scores. CONCLUSION: Results suggest that delivery of a brief psychological intervention for chronic pain in primary care appears to offer improvements in pain severity, pain interference, pain catastrophizing, and depression. Findings suggest that shorter-term psychological interventions may offer similar benefits as longer-term ones. Furthermore, offering a brief intervention in primary care may increase access and engagement in behavioral pain management services. Future research should examine this through a fully-powered trial with longer-term outcomes.

Prevalence and risk factors of burnout in community pharmacists.

OBJECTIVES: This study aimed to identify the prevalence and risk factors for occupational burnout in community pharmacists. METHODS: Community pharmacists were solicited through a professional network e-mail Listserv to complete an anonymous, electronic survey on burnout. The survey included the Maslach Burnout Inventory-Human Services Survey (MBI-HSS) and a work-factors-based questionnaire. The MBI-HSS assessed burnout on the basis of feelings of emotional exhaustion, depersonalization, and reduced personal accomplishment. The additional questionnaire was used to evaluate risk factors for burnout by collecting information on pharmacist demographics, position characteristics, and pharmacy store characteristics. Logistic regression was performed to identify the risk factors associated with burnout. RESULTS: A total of 412 community pharmacists responded to the survey (7.4% response rate), of whom 411 were included in the final analysis. Overall, 308 (74.9%) of responding community pharmacists experienced burnout in at least 1 of the 3 subscales of the MBI-HSS. Most of the pharmacists experienced burnout owing to emotional exhaustion (68.9%), followed by depersonalization (50.4%) and reduced personal accomplishment (30.7%). The significant risk factors for burnout included shorter years of experience, practicing primarily in a chain pharmacy, and a lack of resources for burnout or resiliency. CONCLUSION: There is a high degree of burnout in community pharmacists (74.9%). Future research is warranted to examine optimal strategies to prevent burnout and promote resiliency in the profession.

Assessment and Optimization of the Empiric Treatment of Urinary Tract Infections in an Academic Emergency Department Observation Unit.

BACKGROUND: Poor adherence to evidence-based guidelines and overuse of broad-spectrum antibiotics has been noted in the emergency department (ED). There is limited evidence on guideline-congruent empiric therapy for urinary tract infections (UTIs) and uropathogen susceptibilities in the ED observation unit (EDOU). OBJECTIVE: The primary objective was to evaluate the prescribing patterns for the empiric treatment of UTI in the EDOU. Secondary objectives were to analyze uropathogen susceptibilities in the EDOU and implement an algorithm for the empiric treatment of UTI. METHODS: This study retrospectively reviewed adult patients who received empiric UTI treatment in the EDOU from January 1, 2018 to April 1, 2018. Eligible patients were categorized as having either uncomplicated or complicated cystitis, or pyelonephritis based on their clinical diagnosis. Antimicrobial therapy was evaluated in accordance with national practice guidelines, institutional guidelines, and local antimicrobial susceptibility patterns. RESULTS: Patients with uncomplicated or complicated cystitis (n = 115) were provided guideline-congruent empiric treatment in 87% of cases. Patients with pyelonephritis (n = 35) were provided guideline-congruent empiric treatment in 57% of cases. Susceptibility patterns of uropathogens isolated from this patient sample differed slightly from the institutional antibiogram, notably depicting a lower Escherichia coli susceptibility rate. Fluoroquinolones were prescribed for a longer than recommended duration in 18 patients (60%). CONCLUSIONS: The majority of patients in this study were provided guideline-congruent empiric therapy. Nevertheless, there are opportunities to optimize empiric UTI treatment and improve antibiotic stewardship in the EDOU.

Fluorescence quenching by metal centered porphyrins and poryphyrin enzymes.

Fluorescence spectroscopy and microscopy have been used extensively to monitor biomolecules, especially reactive oxygen species (ROS) and, more recently, reactive sulfide (RSS) species. Nearly all fluorophores are either excited by or emit light between 450 and 550 nm, which is similar to the absorbance of heme proteins and metal-centered porphyrins. Here we examined the effects of catalase (Cat), reduced and oxidized hemoglobin (Hb and metHb), albumin (alb), manganese (III) tetrakis (4-benzoic acid) porphyrin chloride (MnTBAP), iron protoporphyrin IX (hemin), and copper protoporphyrin IX (CuPPIX) on the fluorescence properties of fluorescein. We also examined the effects of catalase and MnTBAP on fluorophores for ROS (dichlorofluorescein, DCF), polysulfides (3',6'-di(O-thiosalicyl)fluorescein, SSP4), and H2S (7-azido-4-methylcoumarin, AzMC) previously activated by H2O2, a mixed polysulfide (H2Sn, n = 1-7) and H2S, respectively. All except albumin concentration dependently inhibited fluorophore fluorescence and absorbed light between 450 and 550 nm, suggesting that the inhibitory effect was physical not catalytic. Catalase inhibition of fluorescein fluorescence was unaffected by sodium azide, dithiothreitol, diamide, tris(2-carboxyethyl)phosphine (TCEP), or iodoacetate, supporting a physical inhibitory mechanism. Catalase and TBAP augmented, then inhibited DCF fluorescence, but only inhibited SSP4 and AzMC fluorescence indicative of a substrate-specific catalytic oxidation of DCF and nonspecific fluorescence inhibition of all three fluorophores. These results suggest caution must be exercised when using any fluorescent tracers in the vicinity of metal-centered porphyrins.

A case of mistaken identity: are reactive oxygen species actually reactive sulfide species?

Stepwise one-electron reduction of oxygen to water produces reactive oxygen species (ROS) that are chemically and biochemically similar to reactive sulfide species (RSS) derived from one-electron oxidations of hydrogen sulfide to elemental sulfur. Both ROS and RSS are endogenously generated and signal via protein thiols. Given the similarities between ROS and RSS, we wondered whether extant methods for measuring the former would also detect the latter. Here, we compared ROS to RSS sensitivity of five common ROS methods: redox-sensitive green fluorescent protein (roGFP), 2', 7'-dihydrodichlorofluorescein, MitoSox Red, Amplex Red, and amperometric electrodes. All methods detected RSS and were as, or more, sensitive to RSS than to ROS. roGFP, arguably the "gold standard" for ROS measurement, was more than 200-fold more sensitive to the mixed polysulfide H2Sn(n = 1-8) than to H2O2 These findings suggest that RSS may be far more prevalent in intracellular signaling than previously appreciated and that the contribution of ROS may be overestimated. This conclusion is further supported by the observation that estimated daily sulfur metabolism and ROS production are approximately equal and the fact that both RSS and antioxidant mechanisms have been present since the origin of life, nearly 4 billion years ago, long before the rise in environmental oxygen 600 million years ago. Although ROS are assumed to be the most biologically relevant oxidants, our results question this paradigm. We also anticipate our findings will direct attention toward development of novel and clinically relevant anti-(RSS)-oxidants.

Estradiol Elicits Proapoptotic and Antiproliferative Effects in Human Trophoblast Cells.

During the first trimester of pregnancy, appropriate regulation of estradiol (E2) is essential for normal placental development. Previous studies demonstrate that premature elevation in E2 concentrations can lead to abnormal placentation, but have not fully elaborated the mechanism of this effect in the first-trimester trophoblast. Our aim was to determine whether E2 elicits trophoblast cell death or inhibits proliferation. The first-trimester human cytotrophoblast cell line HTR-8/SVneo was cultured in phenol red-free medium containing charcoal-stripped serum and treated with 17beta-E2 at concentrations between 0 and 100 nM. TUNEL and invasion assays indicated that E2 significantly increased cell death and reduced cell invasion at 10 nM, and nuclear Ki67 expression revealed that it decreased cell proliferation at 1 nM. A similar effect on cell death was observed in first-trimester placental explants. The E2 antagonist fulvestrant blocked all effects of E2. Immunohistochemistry showed that protein expression of proapoptotic caspases 3, 8, and 9 increased at E2 concentrations of 25 nM and greater, whereas expression of antiapoptotic BCL2-alpha decreased at E2 concentrations of 10 nM and greater. Additionally, treatments with estrogen receptor (ER) alpha-specific and ERbeta-specific agonists at concentrations between 0 and 1000 nM indicated that only ERalpha mediates E2's effects, although immunohistochemistry and Western immunoblotting showed that HTR-8/SVneo cells and placental explants express both ERalpha and ERbeta. Taken together, these findings reveal the interplay between elevated serum E2 and apoptosis in the first trimester of pregnancy. These factors could be associated with pregnancy complications including infertility and uteroplacental insufficiency.

Factors Associated With Interest in Engaging in Psychological Interventions for Pain Management.

OBJECTIVE: Engagement in evidence-based psychological interventions for pain management is low. Identifying characteristics associated with interest in interventions can inform approaches to increase uptake and engagement. The purpose of this study was to examine factors associated with interest in psychological interventions among persons with chronic noncancer pain receiving prescription opioids. METHODS: Participants with chronic noncancer pain and a new 30 to 90 day opioid prescription were recruited from 2 health systems. Participants (N=845) completed measures regarding pain, opioid use, psychiatric symptoms, emotional support, and interest in psychological interventions for pain management. RESULTS: There were 245 (29.0%) participants who reported a high interest in psychological interventions for pain management. In bivariate analyses, variables associated with interest included younger age, female sex, greater pain severity, greater pain interference, greater number of pain sites, lower emotional support, depression, anxiety, and post-traumatic stress disorder ( P <0.05). In a multivariate model, greater pain severity (odds ratio [OR]=1.17; CI: 1.04-1.32), depression (OR=2.10; CI: 1.39-3.16), post-traumatic stress disorder (OR=1.85; CI: 1.19-2.95), and lower emotional support (OR=0.69; CI: 0.5-0.97) remained statistically significant. DISCUSSION: The rate of interest in psychological interventions for pain management was low, which may indicate that patients initiating opioid treatment of chronic noncancer pain have low interest in psychological interventions. Greater pain severity and psychiatric distress were related to interest, and patients with these characteristics may especially benefit from psychological interventions. Providers may want to refer to psychological interventions before or when opioids are initiated. Additional work is needed to determine whether this would reduce long-term opioid use.

Psychotherapy Disruption Before and After the Transition to Virtual Mental Health Care Induced by the COVID-19 Pandemic.

OBJECTIVE: This study aimed to examine population-level disruption in psychotherapy before and after the rapid shift to virtual mental health care induced by the onset of the COVID-19 pandemic in the United States. METHODS: This retrospective study used electronic health record and insurance claims data from three U.S. health systems. The sample included 110,089 patients with mental health conditions who were members of the health systems' affiliated health plans and attended at least two psychotherapy visits from June 14, 2019, through December 15, 2020. Data were subdivided into two 9-month periods (before vs. after COVID-19 onset, defined in this study as March 14, 2020). Psychotherapy visits were measured via health records and categorized as in person or virtual. Disruption was defined as a gap of >45 days between visits. RESULTS: Visits in the preonset period were almost exclusively in person (97%), whereas over half of visits in the postonset period were virtual (52%). Approximately 35% of psychotherapy visits were followed by a disruption in the preonset period, compared with 18% in the postonset period. Disruption continued to be less common (adjusted OR=0.45) during the postonset period after adjustment for visit, mental health, and sociodemographic factors. The magnitude of the difference in disruption between periods was homogeneous across sociodemographic characteristics but heterogeneous across psychiatric diagnoses. CONCLUSIONS: This study found fewer population-level disruptions in psychotherapy receipt after rapid transition to virtual mental health care following COVID-19 onset. These data support the continued availability of virtual psychotherapy.

Making RNA: Using T7 RNA polymerase to produce high yields of RNA from DNA templates.

RNA is playing an ever-growing role in molecular biology and biomedicine due to the many ways it influences gene expression and its increasing use in modern therapeutics. Hence, production of RNA molecules in large quantity and high purity has become essential for advancing basic scientific research and for developing next-generation therapeutics. T7 RNA polymerase (RNAP) is a DNA-dependent RNA polymerase of bacteriophage origin and it is the most widely-utilized tool enzyme for producing RNA. Here we describe a set of robust methods for in vitro transcribing RNA molecules from DNA templates using T7 RNAP, along with a set of subsequent RNA purification schemes. In the first part of this chapter, we provide the general method for T7 RNAP-based in vitro transcription and technical notes for troubleshooting failed or inefficient transcription. We also provide modified protocols for preparing specialized RNA transcripts. In the second part, we provide two purification methods using either gel-based denaturing purification or size exclusion column-based non-denaturing purification for isolating high-purity RNA products from transcription reaction mixtures and preparing them for downstream applications. This chapter is designed to provide researchers with versatile ways to efficiently generate RNA molecules of interest and a troubleshooting guide should they encounter problems while working with in vitro transcription using T7 RNAP.

Systematic detection of tertiary structural modules in large RNAs and RNP interfaces by Tb-seq.

Compact RNA structural motifs control many aspects of gene expression, but we lack methods for finding these structures in the vast expanse of multi-kilobase RNAs. To adopt specific 3-D shapes, many RNA modules must compress their RNA backbones together, bringing negatively charged phosphates into close proximity. This is often accomplished by recruiting multivalent cations (usually Mg2+), which stabilize these sites and neutralize regions of local negative charge. Coordinated lanthanide ions, such as terbium (III) (Tb3+), can also be recruited to these sites, where they induce efficient RNA cleavage, thereby revealing compact RNA 3-D modules. Until now, Tb3+ cleavage sites were monitored via low-throughput biochemical methods only applicable to small RNAs. Here we present Tb-seq, a high-throughput sequencing method for detecting compact tertiary structures in large RNAs. Tb-seq detects sharp backbone turns found in RNA tertiary structures and RNP interfaces, providing a way to scan transcriptomes for stable structural modules and potential riboregulatory motifs.