Sub-Cluster Identification through Semi-Supervised Optimization of Rare-Cell Silhouettes (SCISSORS) in single-cell RNA-sequencing.

MOTIVATION: Single-cell RNA-sequencing (scRNA-seq) has enabled the molecular profiling of thousands to millions of cells simultaneously in biologically heterogenous samples. Currently, the common practice in scRNA-seq is to determine cell type labels through unsupervised clustering and the examination of cluster-specific genes. However, even small differences in analysis and parameter choosing can greatly alter clustering results and thus impose great influence on which cell types are identified. Existing methods largely focus on determining the optimal number of robust clusters, which can be problematic for identifying cells of extremely low abundance due to their subtle contributions toward overall patterns of gene expression. RESULTS: Here, we present a carefully designed framework, SCISSORS, which accurately profiles subclusters within broad cluster(s) for the identification of rare cell types in scRNA-seq data. SCISSORS employs silhouette scoring for the estimation of heterogeneity of clusters and reveals rare cells in heterogenous clusters by a multi-step semi-supervised reclustering process. Additionally, SCISSORS provides a method for the identification of marker genes of high specificity to the cell type. SCISSORS is wrapped around the popular Seurat R package and can be easily integrated into existing Seurat pipelines. AVAILABILITY AND IMPLEMENTATION: SCISSORS, including source code and vignettes, are freely available at https://github.com/jr-leary7/SCISSORS.

Functional gastrointestinal disorders among healthcare professionals at a tertiary Australian hospital.

Background and Aim: The aim of this study was to determine the frequency, characteristics, and associations of functional gastrointestinal disorders (FGIDs) among healthcare professionals. Methods: A qualitative survey was conducted among the staff at a tertiary Australian hospital between January 2017 and June 2018. Rome III criteria (excluding endoscopic) were used to define FGID. Multivariable logistic regression was used to explore associations. Results: Of the 274 respondents (17% doctors, 66% nurses, 17% others; 77% female), 54% had experienced GI symptoms ≥3 times per week and 23% were diagnosed with FGIDs (2% IBS, 19% FD, 2% both). GI symptoms were more common in females (58% vs. 38%), Caucasians versus Asians (59% vs. 35%), respondents who were easily (67% vs. 40%) or often stressed (58% vs. 37%), and had irregular working hours (62% vs. 46%, each P < 0.05). Independent predictors of GI symptoms included being easily stressed (OR 2.7) and female sex (OR 2.4), while Asian ethnicity was protective (OR 0.42, each P < 0.05). FGIDs were more prevalent in respondents who often felt stressed (27% vs. 10%), felt easily stressed (29% vs. 17%), and in nurses compared to others (27% vs. 16%; each P < 0.05). The only independent predictor of FGID was being often stressed (OR 4.1, P = 0.011). Conclusions: FGIDs and GI symptoms are prevalent among hospital workers. Stress, female sex, irregular working hours, and non-Asian ethnicity appeared to be associated with GI symptoms and FGIDs.

Barriers and Facilitators for Population Genetic Screening in Healthy Populations: A Systematic Review.

Studies suggest that 1-3% of the general population in the United States unknowingly carry a genetic risk factor for a common hereditary disease. Population genetic screening is the process of offering otherwise healthy patients in the general population testing for genomic variants that predispose them to diseases that are clinically actionable, meaning that they can be prevented or mitigated if they are detected early. Population genetic screening may significantly reduce morbidity and mortality from these diseases by informing risk-specific prevention or treatment strategies and facilitating appropriate participation in early detection. To better understand current barriers, facilitators, perceptions, and outcomes related to the implementation of population genetic screening, we conducted a systematic review and searched PubMed, Embase, and Scopus for articles published from date of database inception to May 2020. We included articles that 1) detailed the perspectives of participants in population genetic screening programs and 2) described the barriers, facilitators, perceptions, and outcomes related to population genetic screening programs among patients, healthcare providers, and the public. We excluded articles that 1) focused on direct-to-consumer or risk-based genetic testing and 2) were published before January 2000. Thirty articles met these criteria. Barriers and facilitators to population genetic screening were organized by the Social Ecological Model and further categorized by themes. We found that research in population genetic screening has focused on stakeholder attitudes with all included studies designed to elucidate individuals' perceptions. Additionally, inadequate knowledge and perceived limited clinical utility presented a barrier for healthcare provider uptake. There were very few studies that conducted long-term follow-up and evaluation of population genetic screening. Our findings suggest that these and other factors, such as prescreen counseling and education, may play a role in the adoption and implementation of population genetic screening. Future studies to investigate macro-level determinants, strategies to increase provider buy-in and knowledge, delivery models for prescreen counseling, and long-term outcomes of population genetic screening are needed for the effective design and implementation of such programs. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42020198198.

Low-Level Insulin Content Within Abundant Non-ß Islet Endocrine Cells in Long-standing Type 1 Diabetes.

Although most patients with type 1 diabetes (T1D) continue to produce small amounts of insulin decades after disease onset, very few ß-cells persist within their pancreata. Consequently, the source of persistent insulin secretion within T1D remains unclear. We hypothesized that low-level insulin content within non-ß-cells could underlie persistent T1D insulin secretion. We tested for low levels of insulin (insulinlow) within a large cohort of JDRF Network for Pancreatic Organ Donors With Diabetes (nPOD) human pancreata across a wide range of ages and T1D disease durations. Long exposures, high-throughput imaging, and blinded parallel examiners allowed precise quantification of insulinlow cells. Of note, abundant islet endocrine cells with low quantities of insulin were present in most T1D pancreata. Insulinlow islet abundance and composition were not influenced by age, duration of diabetes, or age of onset. Insulinlow islets also contained ß-cell markers at variable levels, including Pdx1, Nkx6.1, GLUT1, and PC1/3. Most insulinlow cells contained abundant glucagon and other α-cell markers, suggesting that α-cells drive much of the insulinlow phenotype in T1D. However, pancreatic polypeptide, somatostatin, and ghrelin cells also contributed to the insulinlow cell population. Insulinlow cells represent a potential source of persistent insulin secretion in long-standing T1D and a possible target for regenerative therapies to expand ß-cell function in disease.

Lymphocytic follicles and aggregates are a determinant of mucosal damage and duration of diarrhea.

CONTEXT: Nonspecific changes (nonspecific chronic inflammation) in patients with chronic diarrhea represent the commonest diagnosis in colorectal biopsy interpretation, but these changes are of little clinical significance. OBJECTIVE: To find, within this group, histologic and immunohistologic diagnostic criteria to predict the duration and resolution of diarrhea. DESIGN: Detailed clinical features and histologic findings were analyzed in a cohort of 47 patients with chronic diarrhea, with near-normal histology and no clear-cut known etiologic agent. Immunohistochemistry to mast cells (CD117) and Treg cells (FOXP3) was also assessed in 39 patients. RESULTS: Increased number of lymphoid follicles and aggregates, increased number of mast cells, and paucity of Treg were the statistically significant key findings (P  =  .003, P  =  .008, and P  =  .04, respectively). The duration of diarrhea was correlated with the number of large lymphoid follicles and aggregates (P  =  .001, r  =  .48), number of total lymphoid follicles and aggregates (P  =  .003, r  =  .43), density of lymphoid follicles and aggregates (P  =  .009, r  =  .38), and total lymphoid follicles and aggregates per biopsy (P  =  .004, r  =  .42) and the number of mast cells (P  =  .001, r  =  .52). The number of mast cells and Treg cells showed significant difference between resolved and unresolved cases (P  =  .001 and P  =  .01 respectively). CONCLUSIONS: Lymphocytic follicles and aggregates colitis, previously regarded as of negligible diagnostic significance, allows the prediction of the behavior of chronic diarrhea in a subset of patients with nonspecific changes on colonic biopsy. The increased number of mast cells and paucity of Treg cells further helps to identify such unresolved cases.

Experienced versus inexperienced confocal endoscopists in the diagnosis of gastric adenocarcinoma and intestinal metaplasia on confocal images.

BACKGROUND: Confocal laser endomicroscopy (CLE) may be used to diagnose gastric cancer and intestinal metaplasia, but the impact of CLE experience on the accuracy of confocal diagnosis of gastric cancer and intestinal metaplasia is not clear. OBJECTIVE: To establish the sensitivity, specificity, and intragroup interobserver agreement of CLE image interpretation by 3 experienced (group 1) and 3 inexperienced (group 2) CLE endoscopists for diagnosing gastric intestinal metaplasia (GIM) and adenocarcinoma. DESIGN: Blinded review of CLE images for the diagnosis of gastric cancer or intestinal metaplasia. SETTING: Tertiary care hospital. PATIENTS: CLE images obtained ex vivo from gastrectomy specimens with proven gastric cancer and CLE images obtained in vivo from Chinese subjects older than 50 years of age by using matched biopsy specimens as reference standards. MAIN OUTCOME MEASUREMENTS: Sensitivity, specificity, and intragroup interobserver agreement of CLE image interpretation. RESULTS: Interpretation of in vivo images by group 1 was associated with higher sensitivity (95.2% vs 61.9%, P = .039) and higher specificity (93.3% vs 62.2%, P < .001) for GIM than interpretation by group 2. The agreement between interpretation by group 1 and histology for GIM was higher than that for group 2 (κ = 0.864 vs 0.217). The sensitivity (93.3% for group 1 vs 86.7% for group 2, P = 1.000) and specificity (87.7% for group 1 vs 80.7% for group 2, P = .344) of interpretation of ex vivo CLE images for the diagnosis of gastric adenocarcinoma was similar for groups 1 and 2. LIMITATIONS: Single-center study. CONCLUSIONS: Experience in CLE was associated with greater accuracy in the diagnosis of intestinal metaplasia.

Regulation of female fertility and identification of future contraceptive targets.

Mammalian reproduction is a complex physiological process involving a tightly regulated hypothalamic-pituitary-gonadal axis and the integration of a diverse array of molecular signals. Oral contraceptives (OCs) were introduced over 40 years ago and have evolved over the years through the discovery of new estrogens and progestins, the development of progestin-only pills and the reduction of the estrogen content in combined OCs. Despite the developments that improved the safety profile of current OCs, adverse metabolic and vascular effects caused by the estrogen component and possible neoplastic effects of OCs remain and, thus, necessitate efforts to develop newer, possibly non-steroidal and non-hormonal, contraceptives. Recent advances in our understanding of ovarian endocrinology, coupled with molecular biology and transgenic technology, have enabled identification of several factors that are functionally critical in the regulation of female fertility. Progress in the area of female reproduction is showing great promise for identifying new contraceptive drug targets. In this article, the authors review the field of female contraception with emphasis on novel targets involved in reproductive function and identified through genomics and proteomics. In addition, the usefulness of these targets for contraception purposes will be discussed.

5-Alkylated thiazolidinones as follicle-stimulating hormone (FSH) receptor agonists.

We prepared analogs of potent thiazolidinone-based follicle-stimulating hormone (FSH) agonists 1, that is, 3 that contained an additional 5-alkyl substituent. This extra substituent was added to reduce synthetic problems that arose during preparation of analogs of 1. These compounds (3) were evaluated in a Chinese hamster ovary (CHO) cell line that expressed recombinant human FSH receptor (FSHR) and a luciferase reporter gene regulated by a cAMP response element (CRE). Selected compounds were also tested on a CHO-cell line that over expressed the FSHR for the ability to induce cAMP production. When the 5-alkyl substituent was a methyl group as in analog 16a, similar FSH activity (i.e., EC(50) = 51 nM, 100% efficacy relative to hFSH) to the analogous 5-hydrogen series compound (e.g., 2) was observed; thus, proving that a small 5-alkyl substituent was well tolerated. New derivatives of 3, in which the potentially hydrolytically labile secondary amide function of 1 (-CONH-) was modified to other moieties (e.g., -CH(2)NH-, -CH(2)S-, and -CH(2)OCONH-), were also prepared and evaluated. These congeners (namely 21, 22, and 24) also displayed good potency in the CRE-luciferase assay.

Allosteric activation of the follicle-stimulating hormone (FSH) receptor by selective, nonpeptide agonists.

The pituitary glycoprotein hormones, luteinizing hormone and follicle-stimulating hormone (FSH), act through their cognate receptors to initiate a series of coordinated physiological events that results in germ cell maturation. Given the importance of FSH in regulating folliculogenesis and fertility, the development of FSH mimetics has been sought to treat infertility. Currently, purified and recombinant human FSH are the only FSH receptor (FSH-R) agonists available for infertility treatment. By screening unbiased combinatorial chemistry libraries, using a cAMP-responsive luciferase reporter assay, we discovered thiazolidinone agonists (EC50's = 20 microm) of the human FSH-R. Subsequent analog library screening and parallel synthesis optimization resulted in the identification of a potent agonist (EC50 = 2 nm) with full efficacy compared with FSH that was FSH-R-selective and -dependent. The compound mediated progesterone production in Y1 cells transfected with the human FSH-R (EC50 = 980 nm) and estradiol production from primary rat ovarian granulosa cells (EC50 = 10.5 nm). This and related compounds did not compete with FSH for binding to the FSH-R. Use of human FSH/thyroid-stimulating hormone (TSH) receptor chimeras suggested a novel mechanism for receptor activation through a binding site independent of the natural hormone binding site. This study is the first report of a high affinity small molecule agonist that activates a glycoprotein hormone receptor through an allosteric mechanism. The small molecule FSH receptor agonists described here could lead to an oral alternative to the current parenteral FSH treatments used clinically to induce ovarian stimulation for both in vivo and in vitro fertilization therapy.

Preparation of highly substituted gamma-lactam follicle stimulating hormone receptor agonists.

An unusual combination of Weinreb amidation and Mitsunobu lactam formation was used to prepare highly substituted gamma-lactam analogues of a thiazolidinone follicle stimulating hormone receptor agonist. The analogue synthesis was stereoselective and the final products were chemically stable. Biological properties of the target molecules were nearly identical to those of the lead compound.

Identification and characterization of a selective, nonpeptide follicle-stimulating hormone receptor antagonist.

The glycoprotein hormones (LH, FSH, and TSH) are critical to the maintenance of physiological homeostasis and control of reproduction. However, despite an obvious utility for synthetic pharmacological agents, there are few reports of selective, nonpeptide agonists or antagonists to receptors for these hormones. We have identified and characterized a novel synthetic molecule capable of inhibiting the action of FSH. This compound, 7-[4-[Bis-(2-carbamoyl-ethyl)-amino]-6-chloro-(1,3,5)-triazin-2-ylamino)-4-hydroxy-3-(4-methoxy-phenylazo)-naphthalene]-2-sulfonic acid, sodium salt (compound 1), is a selective, noncompetitive inhibitor of the human (h) and rat (r) FSH receptors (FSHRs). Compound 1 selectively inhibited binding of [(125)I]hFSH with an IC(50) value of 5.4 +/- 2.3 micro M. Radioligand-binding assays were performed using the baculovirus expressed extracellular domain of hFSHR (BV-tFSHR) to demonstrate site-specific interaction. Compound 1 competed for [(125)I]hFSH binding to BV-tFSHR with an IC(50) value of 10 +/- 2.8 micro M. Functionally, compound 1 inhibited hFSH-induced cAMP accumulation and steroidogenesis in vitro with an IC(50) value of 3 +/- 0.6 micro M. Competition of compound 1 for binding to other glycoprotein hormone receptors and other G protein-coupled receptors demonstrated select activity for FHSRs. Compound 1 inhibited ovulation in immature and cycling adult rats. These data provide proof of concept that selective, small molecule antagonists can be designed for glycoprotein hormone receptors.

Synthesis of (bis)sulfonic acid, (bis)benzamides as follicle-stimulating hormone (FSH) antagonists.

Screening efforts identified (bis)sulfonic acid, (bis)benzamides (1-3) as compounds that interact with the follicle stimulating-hormone receptor (FSHR) and inhibit FSH-stimulated cAMP accumulation with IC(50) values in the low micromolar range. Structure-activity relationship studies using novel analogues of 1-3 revealed that two phenylsulfonic acid moieties were necessary for activity and that the carbon-carbon double bond of the stilbene sub-series was the optimum spacer connecting these groups. Selected analogues (2, 14, and 50) were also able to block FSHR-dependent estradiol production in rat primary ovarian granulosa cells and progesterone secretion in a clonal mouse adrenal Y1 cell line. IC(50) values for these compounds in these assays were in the low micromolar range. Optimization of the benzoic acid side chains of 1-3 led to gains in selectivity versus activity at the thyroid stimulating hormone (TSH) receptor (TSHR). For instance, while stilbene (bis)sulfonic acid congener 2 was only 10-fold selective for FSHR over TSHR, analogue 50 with an IC(50) value of 0.9 microM in the FSHR-cAMP assay was essentially inactive at 30 microM in the TSHR-cAMP assay.