Probing Nascent RNA with Metabolic Incorporation of Modified Nucleosides.

The discovery of previously unknown functional roles of RNA in biological systems has led to increased interest in revealing novel RNA molecules as therapeutic targets and the development of tools to better understand the role of RNA in cells. RNA metabolic labeling broadens the scope of studying RNA by incorporating of unnatural nucleobases and nucleosides with bioorthogonal handles that can be utilized for chemical modification of newly synthesized cellular RNA. Such labeling of RNA provides access to applications including measurement of the rates of synthesis and decay of RNA, cellular imaging for RNA localization, and selective enrichment of nascent RNA from the total RNA pool. Several unnatural nucleosides and nucleobases have been shown to be incorporated into RNA by endogenous RNA synthesis machinery of the cells. RNA metabolic labeling can also be performed in a cell-specific manner, where only cells expressing an essential enzyme incorporate the unnatural nucleobase into their RNA. Although several discoveries have been enabled by the current RNA metabolic labeling methods, some key challenges still exist: (i) toxicity of unnatural analogues, (ii) lack of RNA-compatible conjugation chemistries, and (iii) background incorporation of modified analogues in cell-specific RNA metabolic labeling. In this Account, we showcase work done in our laboratory to overcome these challenges faced by RNA metabolic labeling.To begin, we discuss the cellular pathways that have been utilized to perform RNA metabolic labeling and study the interaction between nucleosides and nucleoside kinases. Then we discuss the use of vinyl nucleosides for metabolic labeling and demonstrate the low toxicity of 5-vinyluridine (5-VUrd) compared to other widely used nucleosides. Next, we discuss cell-specific RNA metabolic labeling with unnatural nucleobases, which requires the expression of a specific phosphoribosyl transferase (PRT) enzyme for incorporation of the nucleobase into RNA. In the course of this work, we discovered the enzyme uridine monophosphate synthase (UMPS), which is responsible for nonspecific labeling with modified uracil nucleobases. We were able to overcome this background labeling by discovering a mutant uracil PRT (UPRT) that demonstrates highly specific RNA metabolic labeling with 5-vinyluracil (5-VU). Furthermore, we discuss the optimization of inverse-electron-demand Diels-Alder (IEDDA) reactions for performing chemical modification of vinyl nucleosides to achieve covalent conjugation of RNA without transcript degradation. Finally, we highlight our latest endeavor: the development of mutually orthogonal chemical reactions for selective labeling of 5-VUrd and 2-vinyladenosine (2-VAdo), which allows for potential use of multiple vinyl nucleosides for simultaneous investigation of multiple cellular processes involving RNA. We hope that our methods and discoveries encourage scientists studying biological systems to include RNA metabolic labeling in their toolkit for studying RNA and its role in biological systems.

An Optimized Enzyme-Nucleobase Pair Enables In Vivo RNA Metabolic Labeling with Improved Cell-Specificity.

Current transcriptome-wide analyses have identified a growing number of regulatory RNA with expression that is characterized in a cell-type-specific manner. Herein, we describe RNA metabolic labeling with improved cell-specificity utilizing the in vivo expression of an optimized uracil phosphoribosyltransferase (UPRT) enzyme. We demonstrate improved selectivity for metabolic incorporation of a modified nucleobase (5-vinyuracil) into nascent RNA, using a battery of tests. The selective incorporation of vinyl-U residues was demonstrated in 3xUPRT LM2 cells through validation with dot blot, qPCR, LC-MS/MS and microscopy analysis. We also report using this approach in a metastatic human breast cancer mouse model for profiling cell-specific nascent RNA.

Repurposing a DNA-Repair Enzyme for Targeted Protein Degradation.

Herein we present the exploration of the utility of DNA demethylase enzymes for targeted protein degradation. Novel benzylguanine substrates are characterized for their ability to control protein degradation in cells. Our data demonstrate the utility of this approach to degrade fusion proteins in different localizations within living cells.

Lingual Alveolar Soft Part Sarcoma in a 1-Year-Old Infant: Youngest Reported Case With Characteristic ASPSCR1-TFE3 Fusion.

Alveolar soft part sarcoma (ASPS) is an exceptionally rare non-rhabdomyosarcomatous soft tissue sarcoma (NRSTS), characterized by the translocation t(X;17) p(11.2;q25). This translocation results in the chimeric ASPSCR1-TFE3 transcription factor which drives tumorigenesis. Complete surgical resection is crucial in allowing a successful outcome in these cases. Here, we describe an 11-month-old female infant who presented with a well-circumscribed lesion of the tongue, with the clinical and radiologic appearances of an infantile hemangioma. This led to an initial plan for surveillance management. However, the mass continued to enlarge and the lesion was therefore biopsied when the infant was 17 months old. Histology showed plump epithelioid tumor cells, in many places lining pseudoalveolar spaces. Occasional Pas-D inclusions were present in the cytoplasm. Immunostaining showed nuclear positivity for TFE-3. Real-time quantitative polymerase chain reaction testing confirmed the presence of ASPSCR1-TFE3 fusion transcripts, characteristic of the translocation t(X;17) p(11.2;q25) observed in ASPS. This represents the youngest reported ASPS case with a confirmed molecular diagnosis. Complete surgical resection was undertaken and a surveillance imaging schedule implemented. This case highlights the need for regular review of the initial diagnosis and the importance of multidisciplinary discussion and early biopsy where the clinical course does not follow that expected for the putative (nonhistologically confirmed) diagnosis.

Use of labeled tomato lectin for imaging vasculature structures.

Intravascular injections of fluorescent or biotinylated tomato lectin were tested to study labeling of vascular elements in laboratory mice. Injections of Lycopersicon esculentum agglutinin (tomato lectin) (50-100 µg/100 µl) were made intravascularly, through the tail vein, through a cannula implanted in the jugular vein, or directly into the left ventricle of the heart. Tissues cut for thin 10- to 12-µm cryostat sections, or thick 50- to 100-µm vibratome sections, were examined using fluorescence microscopy. Tissue labeled by biotinylated lectin was examined by bright field microscopy or electron microscopy after tissue processing for biotin. Intravascular injections of tomato lectin led to labeling of vascular structures in a variety of tissues, including brain, kidney, liver, intestine, spleen, skin, skeletal and cardiac muscle, and experimental tumors. Analyses of fluorescence in serum indicated the lectin was cleared from circulating blood within 2 min. Capillary labeling was apparent in tissues collected from animals within 1 min of intravascular injections, remained robust for about 1 h, and then declined markedly until difficult to detect 12 h after injection. Light microscopic images suggest the lectin bound to the endothelial cells that form capillaries and endothelial cells that line some larger vessels. Electron microscopic studies confirmed the labeling of luminal surfaces of endothelial cells. Vascular labeling by tomato lectin is compatible with a variety of other morphological labeling techniques, including histochemistry and immunocytochemistry, and thus appears to be a sensitive and useful method to reveal vascular patterns in relationship to other aspects of parenchymal development, structure, and function.

A scoping review of the research literature on eating and body image for transgender and nonbinary adults.

BACKGROUND: Eating disorder treatment approaches and outcome studies have historically centered almost exclusively on cisgender populations. Transgender and nonbinary (TGNB) adults are underrepresented in general and intervention research despite being at increased risk for eating and body image-related problems. AIMS: This scoping review was designed to gather and examine the research with TGNB adults who experience eating and body image related problems, as well as clinical studies on the effectiveness of treatment approaches. METHOD: Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) was used for reporting this review. MEDLINE and PsychInfo were used as electronic databases for searching subject terms. Inclusion criteria for studies required the quantitative measurement or qualitative exploration of body image or eating for TGNB adults. The relevant data were extracted and summarized based on quantitative findings and qualitative themes. RESULTS: After review of over 1258 articles, 59 studies met criteria and data were extracted and summarized. Factors associated with eating disorders and body image problems across studies suggests gender-affirming medical interventions are effective and emphasized treatment for an eating disorder is warranted alongside gender affirming medical care. Body image was associated with eating patterns aimed at meeting gendered ideals of body shape and size. There was variation in guiding theories and absence of consensus in the definition of transgender in the review studies. This likely demonstrates the changing language, social acceptance of TGNB people and identities, diagnostic criteria, and clinical conceptualizations of eating and body image. CONCLUSIONS: Future research should consider the use of theory for guiding inclusion of salient social factors influencing eating patterns, body image, and treatment outcomes. In addition, future research is needed that centers on nonbinary and genderqueer populations, as well as those from minoritized racial and ethnic groups to inform culturally appropriate concerns, needs, and treatment modalities.

Fifty-nine published research studies with transgender adults on eating patterns, body image, and associated risks and comorbidities were collected and summarized. Across studies, gender affirming medical interventions like hormone therapy and surgery were associated with decreases in eating disorder symptoms and improved body image. Studies from interviews with transgender adults found social causes for disordered eating and poor body image including rejection and discrimination. Future studies should use guiding theories for testing the causality and consider rejection and discrimination experienced by transgender adults.

A scoping review of research literature on eating and body image for transgender and nonbinary youth.

BACKGROUND: Transgender and nonbinary (TGNB) adolescents and young adults are underrepresented in the literature on eating disorders and body image-related problems, despite increased mental health disparities and emerging research showing high associations between gender dysphoria, body image, and eating disorders among TGNB youth. AIMS: The scoping review was designed to critically examine the research on TGNB adolescents and young adults who experience eating and body image related problems as well as clinical studies on treatment approaches and effectiveness. METHOD: Preferred Reporting Items for Systematic Reviews and Meta-Analyses extension for Scoping Reviews (PRISMA-ScR) was used for reporting this scoping review. The electronic databases of MEDLINE and PsychInfo were used for searching subject terms. Inclusion criteria for studies required the quantitative measurement or qualitative exploration of body image or eating for transgender minor children, adolescents, or young adult samples (18 to 25 years old) and address differences in eating/body-related problems by age. The relevant data was extracted and narratively summarized. RESULTS: 49 studies were identified, data extracted, and analyzed. Increased prevalence of eating disorders and body image problems were identified for TGNB youth. Body-gender congruence through gender affirming social and medical interventions (e.g., hormone therapy) were noted as significant for alleviating body image problems and facilitating eating disorder treatment. Family and social factors were not well understood in the literature and a need for increased study of TGNB youth from varied racial/ethnic, neurodiverse, and within specific identities (e.g., nonbinary) and families and cultural contexts is still needed. CONCLUSIONS: Future research should consider the use of developmental and family theories for guiding inclusion of salient social factors influencing eating patterns, body image, and treatment outcomes. In addition, more studies are needed with those from minoritized racial and ethnic groups, neurodiversity, and varied gender identities (e.g., nonbinary and gender queer) for identifying important differences.

Seeking gender-affirming medical care: A phenomenological inquiry on skillful coping with transgender and non-binary adults in the United States Midwest.

This study sought to understand how transgender and gender non-binary (TGNB) individuals skillfully cope with healthcare services and to explore how childhood experiences impact expectations, habits, and meaning-making when utilizing healthcare services. Using an interpretive phenomenological approach, we sampled 17, White TGNB adults in the United States, ages 19 to 57, using semi-structed interviews about childhood experiences with healthcare utilization and adult experiences seeking genderaffirming healthcare. Analysis identified one main theme-Anticipate the worst in healthcare and be pleasantly surprised-and three subthemes: i) contrast between positive childhood and negative adulthood experiences in medical care; ii) coping practices for the worst; and iii) finding your unicorn doctor and medical staff for pleasant experiences. Results indicate participants experienced a disruption and acquisition of new coping practices in healthcare settings and the cultivation of a radical imagination for a more liberated medical world for TGNB people. Implications for providers and medical offices for empowering TGNB adults are described.

Associations Between Healthcare Experiences, Mental Health Outcomes, and Substance Use Among Transgender Adults.

INTRODUCTION: Transgender and nonbinary (TGNB) adults face significant barriers to healthcare, including healthcare denials, limited access to clinicians, and mistreatment by healthcare clinicians. While prior studies have explored the consequences of overt discrimination in healthcare, they often overlook the possible impacts of more subtle forms of discrimination. AIM: Is there a relationship between specific healthcare experiences, including both overt and subtle forms of discrimination, and mental health/substance use among TGNB adults? METHODS: This study was a secondary analysis of the 2015 U.S. Transgender Survey (USTS), a cross-sectional survey conducted by the National Center for Transgender Equality (NCTE) that included 27,715 TGNB adults from across the US and several US territories. This study analyzed variables including healthcare experiences, mental health, and substance use outcomes. RESULTS: Doctors refusing to give non-TGNB-related care was associated with 71% increased odds of severe psychological distress and 95% increased odds of suicidal ideation. Further, having to teach doctors about TGNB care and doctors asking invasive questions were associated with all our studied negative mental health outcomes. Doctors asking invasive questions was additionally related to increased odds of heavy alcohol use, marijuana use, and illicit drug use. CONCLUSIONS: The results of this study indicate that negative health care experiences are significantly associated with mental health and substance use for TGNB adults. Specifically, these results emphasize the role of more subtle forms of discrimination, including a lack of clinician knowledge about the care of TGNB patients, asking invasive questions, and treating TGNB patients with respect.

The effects of gender identity change efforts on Black, Latinx, and White transgender and gender nonbinary adults: Implications for ethical clinical practice.

Gender identity change efforts (GICE) and sexual orientation change efforts (SOCE) continue to be practiced by mental health professionals and religious organizations. It is frequently sought out by families who are rejecting loved ones with marginalized sexual orientations and gender identities. This study explored the impact of religious and nonreligious GICE on the mental health of transgender and nonbinary adults through a secondary data analysis of the U.S. Transgender Survey. Results found that both nonreligious and religious GICE were associated with increased odds of suicidal ideation and attempts. Only nonreligious GICE was associated with severe psychological distress. Black individuals showed increased odds of binge alcohol consumption when exposed to nonreligious change efforts, while White individuals showed decreased odds with both nonreligious and religious GICE. This study supports ethical and legal bans on GICE.

Enantioselective decarboxylative alkylation reactions: catalyst development, substrate scope, and mechanistic studies.

α-Quaternary ketones are accessed through novel enantioselective alkylations of allyl and propargyl electrophiles by unstabilized prochiral enolate nucleophiles in the presence of palladium complexes with various phosphinooxazoline (PHOX) ligands. Excellent yields and high enantiomeric excesses are obtained from three classes of enolate precursor: enol carbonates, enol silanes, and racemic ß-ketoesters. Each of these substrate classes functions with nearly identical efficiency in terms of yield and enantioselectivity. Catalyst discovery and development, the optimization of reaction conditions, the exploration of reaction scope, and applications in target-directed synthesis are reported. Experimental observations suggest that these alkylation reactions occur through an unusual inner-sphere mechanism involving binding of the prochiral enolate nucleophile directly to the palladium center.

Investigating ß-Lactam Drug Targets in Mycobacterium tuberculosis Using Chemical Probes.

Tuberculosis (TB), caused by the bacterial pathogen Mycobacterium tuberculosis (Mtb), infects 10 million people a year. An estimated 25% of humans harbor latent TB infections, an asymptomatic form of the disease. In both active and latent infections, Mtb relies on cell wall peptidoglycan for viability. In the current work, we synthesized fluorescent analogues of ß-lactam antibiotics to study two classes of enzymes that maintain Mtb's peptidoglycan: penicillin-binding proteins (PBPs) and l,d-transpeptidases (LDTs). This set of activity-based probes included analogues of three classes of ß-lactams: a monobactam (aztreonam-Cy5), a cephalosporin (cephalexin-Cy5), and a carbapenem (meropenem-Cy5). We used these probes to profile enzyme activity in protein gel-resolved lysates of Mtb. All three out-performed the commercial reagent Bocillin-FL, a penam. Meropenem-Cy5 was used to identify ß-lactam targets by mass spectrometry, including PBPs, LDTs, and the ß-lactamase BlaC. New probes were also used to compare PBP and LDT activity in two metabolic states: dormancy and active replication. We provide the first direct evidence that Mtb dynamically regulates the enzymes responsible for maintaining peptidoglycan in dormancy. Lastly, we profiled drug susceptibility in lysates and found that meropenem inhibits PBPs, LDTs, and BlaC.

Increased incidence of post-operative respiratory failure in patients with pre-operative SARS-CoV-2 infection.

OBJECTIVE: While studies have reported increased post-operative pulmonary complications with SARS-CoV-2 infection, many are limited by use of historical controls or focus on less severe respiratory complications. We characterized the association between pre-operative SARS-CoV-2 infection and post-operative respiratory failure (PORF). DESIGN AND SETTING: This was a single center retrospective cohort study in New York City between March 14-June 14, 2020. PATIENTS: Exclusion criteria were age < 18-years, obstetric procedures, absence of SARS-CoV-2 PCR testing, and pre-operative respiratory failure. A total of 778 patients met criteria, of which 87 had SARS-CoV-2. MEASUREMENTS: The primary outcome, PORF, included inability to extubate for ≥24 h or unplanned re-intubation within 5 days. Multiple exposures were measured including SARS-CoV-2 infection 4 weeks before or 5 days after surgery. Multivariable logistic regression was performed to adjust for pre-operative hypoxemia, oxygen use, and pneumonia as well as tachycardia, gender, Charlson Comorbidity Index (CCI), Surgical Mortality Probability Model (S-MPM) index, and peri-operative blood transfusion. MAIN RESULTS: SARS-CoV patients had higher CCI (P = 0.007) and S-MPM scores (P = 0.02). The incidence of PORF was 16% versus 7% in uninfected comparators (P = 0.001). Amongst infected individuals, 39% exhibited symptoms of COVID-19 and PORF was more common in these patients compared to asymptomatic individuals (26% vs. 9%, P = 0.04). Adjusted analysis revealed increased odds of PORF with infection (OR 2.8, 95% CI 1.2-6.2). This persisted even when adjusting for probable mediators such as pre-operative hypoxemia. Infected patients also demonstrated increased adjusted odds of 30-day mortality (OR 3.5, 95% CI 1.4-9.1). CONCLUSIONS: Detection of SARS-CoV-2 infection within 4 weeks before or 5 days after surgery is associated with increased odds of 5-day PORF and 30-day mortality. This supports delaying elective surgery, but questions remain regarding the applicability of this recommendation for asymptomatic patients needing urgent or semi-urgent procedures such as oncologic surgery.

Colonic Basidiobolomycosis-An Unusual Presentation of Eosinophilic Intestinal Inflammation.

Basidiobolomycosis is a rare fungal disease caused by Basidiobolus ranarum. Involvement of the gastrointestinal tract is unusual and poses both a diagnostic and therapeutic challenge, as clinical signs are non-specific and predisposing risk factors are lacking. It can mimick inflammatory bowel disease, primary immunodeficiency, or a malignancy and should be considered in patients who do not respond to standard therapy. We present the case of a 22 months old boy with confirmed colonic Basidiobolomycosis, who presented with severe eosinophilic inflammation of the gastrointestinal tract. Panfungal PCR performed on DNA extracted directly from a tissue sample confirmed the presence of Basidiobolus. He made a full recovery with a combination of surgery and prolonged targeted antifungal medication.

Maternal Choline Supplementation Modulates Placental Markers of Inflammation, Angiogenesis, and Apoptosis in a Mouse Model of Placental Insufficiency.

Dlx3 (distal-less homeobox 3) haploinsufficiency in mice has been shown to result in restricted fetal growth and placental defects. We previously showed that maternal choline supplementation (4X versus 1X choline) in the Dlx3+/- mouse increased fetal and placental growth in mid-gestation. The current study sought to test the hypothesis that prenatal choline would modulate indicators of placenta function and development. Pregnant Dlx3+/- mice consuming 1X (control), 2X, or 4X choline from conception were sacrificed at embryonic (E) days E10.5, E12.5, E15.5, and E18.5, and placentas and embryos were harvested. Data were analyzed separately for each gestational day controlling for litter size, fetal genotype (except for models including only +/- pups), and fetal sex (except when data were stratified by this variable). 4X choline tended to increase (p < 0.1) placental labyrinth size at E10.5 and decrease (p < 0.05) placental apoptosis at E12.5. Choline supplementation decreased (p < 0.05) expression of pro-angiogenic genes Eng (E10.5, E12.5, and E15.5), and Vegf (E12.5, E15.5); and pro-inflammatory genes Il1b (at E15.5 and 18.5), Tnfα (at E12.5) and Nfκb (at E15.5) in a fetal sex-dependent manner. These findings provide support for a modulatory effect of maternal choline supplementation on biomarkers of placental function and development in a mouse model of placental insufficiency.

Synthesis of a far-red fluorophore and its use as an esterase probe in living cells.

We report the synthesis of a new far-red fluorophore, 1,3-dichloro-7-hydroxy-2H-spiro[acridine-9,1'-cyclohexane]-2',5'-diene-2,4'-dione (DSACO), which was modified to make two esterase probes: DSACO-2-AME and DSACO-7-AME. Both probes act as "turn-on" substrates for esterases and lipases. DSACO-2-AME exhibited efficient esterase-activated fluorescence inside living cells and is a stable, far-red alternative for the widely-used fluorescein diacetate.

Profiling Esterases in Mycobacterium tuberculosis Using Far-Red Fluorogenic Substrates.

Enzyme-activated, fluorogenic probes are powerful tools for studying bacterial pathogens, including Mycobacterium tuberculosis (Mtb). In prior work, we reported two 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one) (DDAO)-derived acetoxymethyl ether probes for esterase and lipase detection. Here, we report four-carbon (C4) and eight-carbon (C8) acyloxymethyl ether derivatives, which are longer-chain fluorogenic substrates. These new probes demonstrate greater stability and lipase reactivity than the two-carbon (C2) acetoxymethyl ether-masked substrates. We used these new C4 and C8 probes to profile esterases and lipases from Mtb. The C8-masked probes revealed a new esterase band in gel-resolved Mtb lysates that was not present in lysates from nonpathogenic M. bovis (bacillus Calmette-Guérin), a close genetic relative. We identified this Mtb-specific enzyme as the secreted esterase Culp1 (Rv1984c). Our C4- and C8-masked probes also produced distinct Mtb banding patterns in lysates from Mtb-infected macrophages, demonstrating the potential of these probes for detecting Mtb esterases that are active during infections.

Small-Molecule Probes Reveal Esterases with Persistent Activity in Dormant and Reactivating Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) is the deadliest bacterial pathogen in the world. An estimated one-third of humans harbor Mtb in a dormant state. These asymptomatic, latent infections impede tuberculosis eradication due to the long-term potential for reactivation. Dormant Mtb has reduced enzymatic activity, but hydrolases that remain active facilitate pathogen survival. We targeted Mtb esterases, a diverse set of enzymes in the serine hydrolase family, and studied their activities using both activity-based probes (ABPs) and fluorogenic esterase substrates. These small-molecule probes revealed functional esterases in active, dormant, and reactivating cultures. Using ABPs, we identified five esterases that remained active in dormant Mtb, including LipM (Rv2284), LipN (Rv2970c), CaeA (Rv2224c), Rv0183, and Rv1683. Three of these, CaeA, Rv0183, and Rv1683, were catalytically active in all three culture conditions. Fluorogenic probes additionally revealed LipH (Rv1399c), Culp1 (Rv1984c), and Rv3036c esterase activity in dormant and active cultures. Esterases with persistent activity are potential diagnostic biomarkers or therapeutic targets for Mtb-infected individuals with latent or active tuberculosis.

Total syntheses of (±)-securinine and (±)- allosecurinine.

Total syntheses of (±)-securinine and (±)-allosecurinine that employ a tandem rhodium carbenoid-initiated Claisen/α-ketol rearrangement sequence as a key step are described.