E. coli 6S RNA complexed to RNA polymerase maintains product RNA synthesis at low cellular ATP levels by initiation with noncanonical initiator nucleotides.

The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA promoters for binding to RNAP holoenzyme (RNAP:σ70). The 6S RNA when bound is then used as a template to synthesize a short product RNA (pRNA; usually 13-nt-long). This pRNA changes the 6S RNA structure, triggering the 6S RNA:pRNA complex to release and allowing DNA-dependent housekeeping gene expression to resume. In high nutrient conditions, 6S RNA turnover is extremely rapid but becomes very slow in low nutrient environments. This leads to a large accumulation of inhibited RNAP:σ70 in stationary phase. As pRNA initiates synthesis with ATP, we and others have proposed that the 6S RNA release rate strongly depends on ATP levels as a proxy for sensing the cellular metabolic state. By purifying endogenous 6S RNA:pRNA complexes using RNA Mango and using reverse transcriptase to generate pRNA-cDNA chimeras, we demonstrate that 6S RNA:pRNA formation can be simultaneous with 6S RNA 5' maturation. More importantly, we find a dramatic accumulation of capped pRNAs during stationary phase. This indicates that ATP levels in stationary phase are low enough for noncanonical initiator nucleotides (NCINs) such as NAD+ and NADH to initiate pRNA synthesis. In vitro, mutation of the conserved 6S RNA template sequence immediately upstream of the pRNA transcriptional start site can increase or decrease the pRNA capping efficiency, suggesting that evolution has tuned the biological 6S RNA sequence for an optimal capping rate. NCIN-initiated pRNA synthesis may therefore be essential for cell viability in low nutrient conditions.

Characterization of regulatory T cells in obese omental adipose tissue in humans.

Obesity-associated visceral adipose tissue (AT) inflammation promotes insulin resistance and type 2 diabetes (T2D). In mice, lean visceral AT is populated with anti-inflammatory cells, notably regulatory T cells (Tregs) expressing the IL-33 receptor ST2. Conversely, obese AT contains fewer Tregs and more proinflammatory cells. In humans, however, there is limited evidence for a similar pattern of obesity-associated immunomodulation. We used flow cytometry and mRNA quantification to characterize human omental AT in 29 obese subjects, 18 of whom had T2D. Patients with T2D had increased proportions of inflammatory cells, including M1 macrophages, with positive correlations to body mass index. In contrast, Treg frequencies negatively correlated to body mass index but were comparable between T2D and non-T2D individuals. Compared to human thymic Tregs, omental AT Tregs expressed similar levels of FOXP3, CD25, IKZF2, and CTLA4, but higher levels of PPARG, CCR4, PRDM1, and CXCL2. ST2, however, was not detectable on omental AT Tregs from lean or obese subjects. This is the first comprehensive investigation into how omental AT immunity changes with obesity and T2D in humans, revealing important similarities and differences to paradigms in mice. These data increase our understanding of how pathways of immune regulation could be targeted to ameliorate AT inflammation in humans.

Genetics of Diabetic Retinopathy.

PURPOSE OF REVIEW: The goal of this paper is to review the latest findings in understanding the genetics of diabetic retinopathy. We highlight recent literature using a variety of molecular genetic techniques to identify variants which contribute to genetic susceptibility for diabetic retinopathy. RECENT FINDINGS: New genome-wide association study (GWAS) and whole-exome sequencing approaches have been utilized to identify both common and rare variants associated with diabetic retinopathy. While variants have been identified in isolated studies, no variants have been replicated across multiple studies. The identification of genetic factors associated with diabetic retinopathy remains elusive. This is due to the multifactorial nature of the disease, small sample sizes for GWAS, and difficulty in controlling covariates of the disease. Larger populations as well as utilization of new sequencing and data analysis techniques may lead to new insights into genetic factors associated with diabetic retinopathy in the future.

IL-33 Reverses an Obesity-Induced Deficit in Visceral Adipose Tissue ST2+ T Regulatory Cells and Ameliorates Adipose Tissue Inflammation and Insulin Resistance.

Obesity is associated with insulin resistance and inflammation thought to be caused by a visceral adipose tissue (VAT)-localized reduction in immunoregulatory cells and increase in proinflammatory immune cells. We previously found that VAT regulatory T cells (Tregs) normally express high levels of IL-10 and that expression of this cytokine in VAT Tregs is specifically reduced in mice fed a high-fat diet. In this study, we further investigated the phenotype of VAT Tregs and found that the majority of IL-10-expressing Tregs in the VAT of lean mice also expressed the ST2 chain of the IL-33R. In addition to high expression of IL-10, ST2(+) Tregs in lean VAT expressed higher proportions of Th2-associated proteins, including GATA3 and CCR4, and Neuropillin-1 compared with ST2(-) Tregs. The proportion of ST2(+) Tregs in VAT was severely diminished in obese mice that had been fed a high-fat/sucrose diet, and this effect could be completely reversed by treatment with IL-33. IL-33 treatment also reversed VAT inflammation in obese mice and resulted in a reduction of hyperinsulinemia and insulin resistance. These data suggest that IL-33 contributes to the maintenance of the normal pool of ST2(+) Tregs in the VAT, and that therapeutic administration of IL-33 results in multiple anti-obesity effects, including the reversal of VAT inflammation and alleviation of insulin resistance.

Insulin inhibits IL-10-mediated regulatory T cell function: implications for obesity.

Chronic inflammation is known to promote metabolic dysregulation in obesity and type 2 diabetes. Although the precise origin of the unchecked inflammatory response in obesity is unclear, it is known that overproduction of proinflammatory cytokines by innate immune cells affects metabolism. For example, TNF-α contributes to the inability of cells to respond to insulin and to the increase in levels of insulin. Whether this hyperinsulinemia itself is part of a feedback loop that affects the progression of chronic adipose inflammation is unknown. In this article, we show that regulatory T cells (Tregs) express the insulin receptor, and that high levels of insulin impair the ability of Tregs to suppress inflammatory responses via effects on the AKT/mTOR signaling pathway. Insulin activated AKT signaling in Tregs, leading to inhibition of both IL-10 production and the ability of Tregs to suppress the production of TNF-α by macrophages in a contact-independent manner. The effect of insulin on Treg suppression was limited to IL-10 production and it did not alter the expression of other proteins associated with Treg function, including CTLA-4, CD39, and TGF-ß. In a model of diet-induced obesity, Tregs from the visceral adipose tissue of hyperinsulinemic, obese mice showed a similar specific decrease in IL-10 production, as well as a parallel increase in production of IFN-γ. These data suggest that hyperinsulinemia may contribute to the development of obesity-associated inflammation via a previously unknown effect of insulin on the IL-10-mediated function of Tregs.

Identification of novel PIKFYVE gene mutations associated with Fleck corneal dystrophy.

PURPOSE: To report the identification of a novel frameshift mutation and copy number variation (CNV) in PIKFYVE in two probands with fleck corneal dystrophy (FCD). METHODS: Slit-lamp examination was performed to identify characteristic features of FCD. After genomic DNA was collected, PCR amplification and automated sequencing of all 41 exons of PIKFYVE was performed. Using genomic DNA, quantitative PCR (qPCR) was performed to detect CNVs within PIKFYVE. RESULTS: In the first FCD proband, numerous panstromal punctate opacities were observed in each of the proband's corneas, consistent with the diagnosis of FCD. Screening of PIKFYVE demonstrated a novel heterozygous frameshift mutation in exon 19, c.3151dupA, which is predicted to encode for a truncated PIKFYVE protein, p.(Asp1052Argfs*18). This variant was identified in an affected sister but not in the proband's unaffected mother or brother or 200 control chromosomes. The second FCD proband presented with bilateral, discrete, punctate, grayish-white stromal opacities. Exonic screening of PIKFYVE revealed no causative variant. However, CNV analysis demonstrated the hemizygous deletion of exons 15 and 16. CONCLUSIONS: We report a novel heterozygous frameshift mutation (c.3151dupA) and a CNV in PIKFYVE, representing the first CNV and the fifth frameshift mutation associated with FCD.

Immune regulation in obesity-associated adipose inflammation.

Adipose tissue inflammation is often a consequence of obesity and is characterized by infiltration and activation of immune cells that overproduce cytokines and chemokines. This apparent loss of immune regulation in obese adipose tissue contributes to the ongoing chronic inflammation that is thought to promote the degradation of metabolic parameters in obesity. Much recent work has sought to identify the immune cell subsets that are involved in adipose tissue inflammation, understand the mechanisms by which adipose tissue inflammation develops, and develop immunotherapeutic strategies to reverse this process. In this review, we describe the known mechanisms that underlie the loss of immune regulation in obesity-associated adipose tissue inflammation and set the stage for the development of novel therapeutic approaches.

Cutting edge: PHLPP regulates the development, function, and molecular signaling pathways of regulatory T cells.

Regulatory T cells (Tregs) have a reduced capacity to activate the PI3K/Akt pathway downstream of the TCR, and the resulting low activity of Akt is necessary for their development and function. The molecular basis for the failure of Tregs to activate Akt efficiently, however, remains unknown. We show that PH-domain leucine-rich-repeat protein phosphatase (PHLPP), which dephosphorylates Akt, is upregulated in Tregs, thus suppressing Akt activation. Tregs expressed higher levels of PHLPP than those of conventional T cells, and knockdown of PHLPP1 restored TCR-mediated activation of Akt in Tregs. Consistent with their high Akt activity, the suppressive capacity of Tregs from PHLPP1(-/-) mice was significantly reduced. Moreover, the development of induced Tregs was impaired in PHLPP1(-/-) mice. The increased level of Akt's negative regulator, PHLPP, provides a novel mechanism used by T cells to control the Akt pathway and the first evidence, to our knowledge, for a molecular mechanism underlying the functionally essential reduction of Akt activity in Tregs.

Variation in Perioperative Care Practices Among Vitreoretinal Surgeons in the United States.

OBJECTIVE: To describe perioperative practice patterns among retinal surgeons managing retinal detachment (RD) repair. METHODS: This was a cross-sectional pilot survey of vitreoretinal surgeons in the United States (US), identified by a previously published web-based search and cross-referencing names from the American Society of Retina Specialists. Self-reported peri-operative practices and subgroups were analyzed. RESULTS: Of the 298 surgical retina specialists who completed the survey, 115 (39%) were in practice for ≤ 5 years, 102 (34%) were in practice for 6 to 20 years, and 81 (27%) were in practice for > 20 years; 60%, 23%, and 16% were in private, academic, and hybrid practice, respectively. Fifty-nine percent reported operating with trainees. For ocular blocks, 59% perform retrobulbar, 21% peribulbar, and 20% subtenon's (ST). Use of ST block varied significantly by years in practice and presence of trainees (P < 0.0001, P = 0.004, respectively). Sixty percent perform primary scleral buckles (SB), 55% combined SB/pars plana vitrectomy (PPV), and 11% primary PPVs under general anesthesia. Use of general anesthesia for primary SB varied significantly by years in practice (P = 0.007). Surgeons with fewer years in practice were more likely to recommend facedown positioning for macula-off RDs (P < 0.0001). Forty-six percent of surgeons do not advise stopping blood thinners before surgery and this varied significantly by years in practice (P = 0.006). CONCLUSIONS: Variation exists among US vitreoretinal surgeons in relation to anesthesia, postoperative positioning, and blood thinners restrictions. Preferences are influenced by years in practice and less by trainees and practice setting. These results serve as a basis for larger, targeted US-based surveys on perioperative care and correlation with surgical outcomes. [Ophthalmic Surg Lasers Imaging Retina 2022;53:681-690.].

Labor Complicated by Cervical Stenosis Following a Laser Cone Biopsy.

A 34-year-old lady, with three previous spontaneous vaginal deliveries, presented in labor at 37 + 0 weeks' gestation. Following the birth of her third child, she underwent a laser cone biopsy for cervical intraepithelial neoplasia (CIN) 3. Despite sustained regular contractions and augmentation with Syntocinon, progressive cervical dilatation beyond 1 cm failed to occur in this multiparous lady. A delayed diagnosis of cervical stenosis was made. She eventually underwent a cesarean section where her internal cervical os was found to be thin and fibrotic. This case describes an uncommon occurrence of cervical stenosis presenting in labor and seeks to increase awareness of this condition so as to allow preemptive counselling of similar patients, early recognition in a labor with poor progress and a swift, better-informed decision to deliver via an emergency cesarean section. We review the existing literature on cervical stenosis following cervical conization and analyze the various definitions available.

Reticular Pseudodrusen in Late-Onset Retinal Degeneration.

PURPOSE: To characterize the association of reticular pseudodrusen (RPD) with late-onset retinal degeneration (L-ORD) using multimodal imaging. DESIGN: Prospective, 2-center, longitudinal case series. PARTICIPANTS: Twenty-nine patients with L-ORD. METHODS: All patients were evaluated within a 3-year interval with near-infrared reflectance, fundus autofluorescence, and spectral-domain OCT. In addition, a subset of patients also underwent indocyanine green angiography, fundus fluorescein angiography, mesopic microperimetry, and multifocal electroretinography. MAIN OUTCOME MEASURES: Prevalence, topographic distribution, and temporal phenotypic changes of RPD in L-ORD. RESULTS: A total of 29 patients with molecularly confirmed L-ORD were included in this prospective study. Reticular pseudodrusen was detected in 18 patients (62%) at baseline, 10 of whom were men. The prevalence of RPD varied with age. The mean age of RPD patients was 57.3 ± 7.2 years. Reticular pseudodrusen was not seen in patients younger than the fifth decade of life (n = 3 patients) or in the eighth decade of life (n = 5 patients). Reticular pseudodrusen were found commonly in the macula with relative sparing of the fovea and also were identified in the peripheral retina. The morphologic features of RPD changed with follow-up. Two patients (3 eyes) demonstrated RPD regression. CONCLUSIONS: Reticular pseudodrusen is found frequently in patients with L-ORD and at a younger age than in individuals with age-related macular degeneration (AMD). Reticular pseudodrusen exhibits quick formation and collapse, change in type and morphologic features with time, and relative foveal sparing and also has a peripheral retinal location in L-ORD.

Anesthesia for Intravitreal Injection: A Systematic Review.

BACKGROUND: The intravitreal injection has become one of the most commonly performed procedures in ophthalmology; however, there is no standardized approach to anesthesia during the procedure. The goal of this systematic review is to review approaches to anesthesia for intravitreal injection and look at comparative efficacy between these different anesthetics. METHODS: A systematic review of literature was performed in the MEDLINE, PubMed, Cochrane Library, and Clinicaltrials.gov databases using the key words "anesthesia", "pain management", and "intravitreal injection". Of the initial 239 search matches, 30 articles were found to be relevant to the topic. 18 studies were excluded as they did not include primary data or did not include the visual analog scale as a primary outcome. The remaining 12 articles were assessed to look at the comparative efficacy of anesthesia and adverse events. RESULTS: The anesthesia techniques reported include topical methods such as anesthetic eyedrops, anesthetic gels, and anesthetic-soaked pledgets as well as subconjunctival injection of anesthetic. Ultimately, no single anesthetic or delivery mechanism was shown to be superior to the others in a statistically significant way and adverse events were largely insignificant. Limitations of these studies include relatively small sizes of the studies, as well as the lack of masking which may introduce bias. CONCLUSION: In the current literature, no type of anesthetic method was found to be superior to another for intravitreal injection. Future studies in this area may lead to new insights into the efficacy of different forms of intravitreal anesthesia.

T reg-specific insulin receptor deletion prevents diet-induced and age-associated metabolic syndrome.

Adipose tissue (AT) regulatory T cells (T regs) control inflammation and metabolism. Diet-induced obesity causes hyperinsulinemia and diminishes visceral AT (VAT) T reg number and function, but whether these two phenomena were mechanistically linked was unknown. Using a T reg-specific insulin receptor (Insr) deletion model, we found that diet-induced T reg dysfunction is driven by T reg-intrinsic insulin signaling. Compared with Foxp3cre mice, after 13 wk of high-fat diet, Foxp3creInsrfl/fl mice exhibited improved glucose tolerance and insulin sensitivity, effects associated with lower AT inflammation and increased numbers of ST2+ T regs in brown AT, but not VAT. Similarly, Foxp3creInsrfl/fl mice were protected from the metabolic effects of aging, but surprisingly had reduced VAT T regs and increased VAT inflammation compared with Foxp3cre mice. Thus, in both diet- and aging-associated hyperinsulinemia, excessive Insr signaling in T regs leads to undesirable metabolic outcomes. Ablation of Insr signaling in T regs represents a novel approach to mitigate the detrimental effects of hyperinsulinemia on immunoregulation of metabolic syndrome.

gRASping the redox lever to modulate cancer cell fate signaling.

RAS proteins are critical regulators of signaling networks controlling diverse cellular functions such as cell proliferation and survival and its mutation are among the most powerful oncogenic drivers in human cancers. Despite intense efforts, direct RAS-targeting strategies remain elusive due to its "undruggable" nature. To that end, bulk of the research efforts has been directed towards targeting upstream and/or downstream of RAS signaling. However, the therapeutic efficacies of these treatments are limited in the long run due to the acquired drug resistance in RAS-driven cancers. Interestingly, recent studies have uncovered a potential role of RAS in redox-regulation as well as the interplay between ROS and RAS-associated signaling networks during process of cancer initiation and progression. More specifically, these studies provide ample evidence to implicate RAS as a redox-rheostat, manipulating ROS levels to provide a redox-milieu conducive for carcinogenesis. Importantly, the understanding of RAS-ROS interplay could provide us with novel targetable vulnerabilities for designing therapeutic strategies. In this review, we provide a brief summary of the advances in the field to illustrate the dual role of RAS in redox-regulation and its implications in RAS signaling outcomes and also emerging redox-based strategies to target RAS-driven cancers.

Longitudinal circulating placental growth factor (PlGF) and soluble FMS-like tyrosine kinase-1 (sFlt-1) concentrations during pregnancy in Asian women: a prospective cohort study.

OBJECTIVES: To analyse the placental growth factor (PlGF) and soluble FMS-like tyrosine kinase-1 (sFlt-1) trends in the normal pregnant Asian population in Singapore. DESIGN: A prospective study was conducted. SETTING: The largest tertiary hospital in Singapore. METHODS: Women with single viable pregnancies, less than 14 weeks of gestation, were recruited between September 2010 and November 2013 in KK Women's and Children's Hospital. They were followed up from recruitment till their postnatal discharge from the hospital. There were four antenatal visits: gestational age (GA) less than 14+0 weeks of gestation (V1), GA 18+0 to 22+0 weeks (V2), GA 28+0 to 32+0 weeks (V3) and GA 34+0 and above (V4). Serum biochemical markers (sFlt-1, PlGF) were measured at each visit. RESULTS: There were 934 participants in the study, of which 674 had normal pregnancy outcomes. The sFlt-1 remained relatively constant till GA 28-32 weeks before it increased (p<0.001). The sFlt-1 levels increased earlier before 30 weeks' of gestation among the Malay participants and the other ethnicities. For PlGF, the levels increased from the first to the third trimester, peaking at 30-32 weeks before decreasing (p<0.001). Its serum levels significantly differed among the Indian participants and other ethnicities as compared with the Malay and Chinese participants at V3 and V4, (p=0.04 and p<0.001, respectively). CONCLUSION: There are significant differences in the PlGF and sFlt-1 concentrations during pregnancy between different ethnicities, which should be taken into consideration when using these references values for further research.

Methodological quality of clinical practice guidelines for genetic testing in children: A systematic assessment using the appraisal of guidelines for research and evaluation II instrument.

Genetic testing of children is faced with numerous problems. High-quality clinical practice guidelines (CPGs) are needed to ensure its safe, and appropriate use. This study aimed to systematically identify the current CPGs for genetic testing in children, and to assess the methodological quality of these CPGs.We searched 6 databases, 3 guideline clearinghouses, and 9 web sites of relevant academic agencies from inception to February 2019. CPGs focused on genetic testing in children were included. Four reviewers independently appraised the quality of the eligible CPGs using the appraisal of guidelines for research, and evaluation (AGREE) II instrument.Seventeen CPGs meeting our inclusion criteria were included. Among them, 16 CPGs were focused on the genetic diagnosis/evaluation of diseases, while only 1 CPG was focused on pharmacogenetics. The median domain scores from highest to lowest were: scope and purpose 80.56% (range: 56.95%-87.50%), clarity of presentation 72.22% (range: 45.83%-88.89%), stakeholder involvement 45.83% (range: 27.78%-55.56%), applicability 31.25% (range: 19.79%-54.17%), rigor of development 21.88%, (range: 13.02%-71.88%), and editorial independence 18.75% (range: 0%-83.33%). According to the overall quality, 6 (35%) CPGs were "not recommended," 8 (47%) CPGs were "recommended with modifications," and only 3 (18%) CPGs were "recommended." The clinical topics of the "recommended" CPGs were warfarin, familial Mediterranean fever, and pediatric pulmonary arterial hypertension.The quality of CPGs for genetic testing in children was generally low, and variable across different CPGs and different AGREE II domains. In future guideline development, more attention should be paid to the aspects of stakeholder involvement, rigor of development, applicability, and editorial independence. Not only will guideline users benefit from our results when determining whether to adopt related CPGs to guide genetic testing in children, but guideline developers could also take into account our results to improve the quality of future CPGs.

Naloxone Counseling for Harm Reduction and Patient Engagement.

BACKGROUND AND OBJECTIVES: The United States is experiencing an epidemic of opioid-related deaths. Naloxone, the drug of choice for reversing acute opioid overdose, is not routinely prescribed for outpatient use. The aims of this project were to improve naloxone awareness, increase naloxone prescribing, and prevent opioid overdoses. METHODS: A naloxone counseling intervention was implemented in three family health centers by an interprofessional team of providers including family medicine physicians, clinical pharmacists, and social workers. An outreach letter was designed with provider input, an electronic order set was developed to facilitate prescribing, and intranasal naloxone kits were assembled for free dispensing. Providers and staff received education about opioid overdose and naloxone prescribing. Faculty and resident physicians were surveyed before and after the intervention to assess their attitudes. Patients who received naloxone kits were surveyed to assess their attitudes and use of opioids and naloxone. RESULTS: Over 16 months, 71 outreach letters were distributed and 97 naloxone kits were dispensed. The majority of kits were prescribed for illicit opioid use. Faculty and resident physician surveys indicated improved knowledge about naloxone prescribing, and increased professional satisfaction caring for patients requesting opioids. Surveyed patients endorsed high levels of comfort discussing opioid use with their primary care physician. Five successful opioid overdose reversals were reported. CONCLUSIONS: An interprofessional naloxone counseling intervention engaged patients in opioid use discussions, increased provider satisfaction, and reversed overdoses. Improving naloxone access is an essential component of comprehensive overdose prevention programs that encourage responsible opioid prescribing and use.

Breast Cancer: A Molecular and Redox Snapshot.

SIGNIFICANCE: Breast cancer is a unique disease characterized by heterogeneous cell populations causing roadblocks in therapeutic medicine, owing to its complex etiology and primeval understanding of the biology behind its genesis, progression, and sustenance. Globocan statistics indicate over 1.7 million new breast cancer diagnoses in 2012, accounting for 25% of all cancer morbidities. RECENT ADVANCES: Despite these dismal statistics, the introduction of molecular gene signature platforms, progressive therapeutic approaches in diagnosis, and management of breast cancer has led to more effective treatment strategies and control measures concurrent with an equally reassuring decline in the mortality rate. CRITICAL ISSUES: However, an enormous body of research in this area is requisite as high mortality associated with metastatic and/or drug refractory tumors continues to present a therapeutic challenge. Despite advances in systemic chemotherapy, the median survival of patients harboring metastatic breast cancers continues to be below 2 years. FUTURE DIRECTIONS: Hence, a massive effort to scrutinize and evaluate chemotherapeutics on the basis of the molecular classification of these cancers is undertaken with the objective to devise more attractive and feasible approaches to treat breast cancers and improve patients' quality of life. This review aims to summarize the current understanding of the biology of breast cancer as well as challenges faced in combating breast cancer, with special emphasis on the current battery of treatment strategies. We will also try and gain perspective from recent encounters on novel findings responsible for the progression and metastatic transformation of breast cancer cells in an endeavor to develop more targeted treatment options. Antioxid. Redox Signal. 25, 337-370.