Chemical kinetics and thermodynamics of PPO activity, colour changes and microbial degradation during blanching of the sugarcane billets.

BACKGROUND: Sugarcane juice, which has a short shelf life, is a popular thirst-quenching and rejuvenating beverage worldwide. The limited shelf life is a result of changes in polyphenol oxidase (PPO) activity, total plate count (TPC) and color attributes (L*, a* and b*-values). We hypothesized that chemical kinetics and thermodynamics of blanched sugarcane cane juice causing alterations in PPO, TPC, and L, a* and b*-values will address the challenges of sugarcane juice preservation. RESULTS: Sugarcane billets were blanched at variable time-temperature combinations in the range 0-20 min and 70-90 °C. Reaction rates increased with increasing temperature; PPO activity, TPC and colour followed first-order kinetics. PPO activity had an activation energy (Ea) of 81 kJ mol-1. The half life (t½) dropped from 16.5 to 3.47 min and decimal reduction time (D-values) dropped from 54.83 to 11.52 min. Thus reactions were temperature-sensitive. Thermodynamic studies indicated an endothermic (positive enthalpy values, ΔH > 0; 78.10 kJ mol-1) and reversible process (negative entropy values ΔS < 0; -0.044 kJmol-1 K-1). Michaeli-Menten constant (Km) and maximum velocity (Vmax) of PPO activity were determined by adding variable lemon juice concentrations in sugarcane juice. As the Km values increased (from 5.53 to 15.81 mm) and Vmax values decreased (from 666.67 to 384.61 UmL-1), a Lineweaver-Burk plot suggested decreased PPO affinity of sugarcane juice. CONCLUSION: The results of the present study indicate that studies on chemical kinetics and thermodynamics (PPO, TPC and L, a* and b*-values) of blanched sugarcane cane juice shall mitigate challenges of sugarcane juice preservation. © 2024 Society of Chemical Industry.

Reversing Uteropathies Including Cancer-Like Changes in Mice by Transplanting Mesenchymal Stromal Cells or XAR Treatment.

Pluripotent, very small embryonic-like stem cells (VSELs) and tissue-committed 'progenitors' termed endometrial stem cells (EnSCs) are reported in mouse uterus. They express gonadal and gonadotropin hormone receptors and thus are vulnerable to early-life endocrine insults. Neonatal exposure of mouse pups to endocrine disruption cause stem/progenitor cells to undergo epigenetic changes, excessive self-renewal, and blocked differentiation that results in various uteropathies including non-receptive endometrium, hyperplasia, endometriosis, adenomyosis, and cancer-like changes in adult life. Present study investigated reversal of these uteropathies, by normalizing functions of VSELs and EnSCs. Two strategies were evaluated including (i) transplanting mesenchymal stromal cells (provide paracrine support) on D60 or (ii) oral administration of XAR (epigenetic regulator) daily from days 60-100 and effects were studied later in 100 days old mice. Results show normalization of stem/progenitor cells (Oct-4, Oct-4A, Sox-2, Nanog) and Wnt signalling (Wnt-4, ß-catenin, Axin-2) specific transcripts. Flow cytometry results showed reduced numbers of 2-6 µm, LIN-CD45-SCA-1 + VSELs. Hyperplasia (Ki67) of epithelial (Pax-8, Foxa-2) and myometrial (α-Sma, Tgf-ß) cells was reduced, adenogenesis (differentiation of glands) was restored, endometrial receptivity and differentiation (LIF, c-KIT, SOX-9, NUMB) and stromal cells niche (CD90, VIMENTIN, Pdgfra, Vimentin) were improved, cancer stem cells markers (OCT-4, CD166) were reduced while tumor suppressor genes (PTEN, P53) and epigenetic regulators (Ezh-2, Sirt-1) were increased. To conclude, normalizing VSELs/EnSCs to manage uteropathies provides a novel basis for initiating clinical studies. The study falls under the umbrella of United Nations Sustainable Development Goal 3 to ensure healthy lives and well-being for all of all ages.

Mouse uterine stem cells are affected by endocrine disruption and initiate uteropathies.

In brief: Incidence of uteropathies has increased in recent times, possibly due to exposure to endocrine-disrupting chemicals during early development. The present study shows that various uteropathies like endometrial cancer, adenomyosis, and endometriosis are interlinked and occur due to the dysfunction of tissue-resident, very small embryonic-like stem cells (VSELs). Abstract: Underlying pathomechanisms leading to the initiation of uteropathies including non-receptive endometrium, hyperplasia, adenomyosis, endometriosis, fibroids, and cancer remain elusive. Two populations of stem cells exist in mouse uterus including pluripotent VSELs and 'progenitors' termed endometrial stem cells (EnSCs) which express ERα, ERß, PR, and FSHR, participate in the regular remodelling, and maintain life-long homeostasis. The present study aimed to delineate possible stem cell origins for various uteropathies. For this, mouse pups were treated with oestradiol or diethylstilbestrol and were studied for adult onset of various uteropathies. Treatment resulted in disrupted oestrous cycles, reduced uterine weights, and marked hyperplasia in both epithelial and myometrial compartments, and the stromal compartment was also affected. VSELs were increased in numbers as judged by flow cytometry and increased expression of transcripts specific for Oct-4A, Sox-2, and Nanog, but their further differentiation into a receptive endometrium was affected. Reduced 5-methyl cytosine expression suggested global hypomethylation and was associated with several oncogenic events including loss of tumour-suppressor genes (Pten, p53), dysregulated DNA mismatch repair axis, and repair enzymes. Stem cells were epigenetically altered and showed increased expression of DNMTs, loss of imprinting loci (Igf2-H19, Dlk1-Meg3), and Ezh2. Increased co-expression of CD166 and ALDHA1 with OCT-4 in stem cells was associated with increased Esr-2 and reduced Pr in the endometrium, while both were several folds upregulated in the myometrium. Study results suggest that various uteropathies ensue due to the dysfunction of tissue-resident stem cells and provide huge scope for further research.

Glycemic outcomes in patients with type 2 diabetes after bariatric surgery compared with routine care: a population-based, real-world cohort study in the United Kingdom.

BACKGROUND: Clinical trials have shown that bariatric surgery (BS) is associated with better glycemic control and diabetes remission in patients with type 2 diabetes (T2D) compared with routine care. OBJECTIVE: We conducted a real-world population-based study examining the impact of BS on glycemic control and medications in patients with T2D. SETTING AND METHODS: This was a retrospective, matched, controlled cohort study conducted between January 1, 1990, and January 31, 2018, using IQVIA Medical Research Data, a primary care electronic records database. Adults with body mass index (BMI) ≥30 kg/m2 and T2D who had BS (surgical) were matched for age, sex, BMI, and diabetes duration to two controls (with T2D and no BS). RESULTS: A total of 1126 patients in the surgical group and 2219 patients in the control group were analyzed. Mean (standard deviation) age was 50.0 (9.3) years, 67.6% were women, baseline glycocylated hemoglobin (HbA1C) was 7.8% (1.7 mmol/mol), and diabetes duration was 4.7 years (range, 2.0-8.4 years). Over a median (interquartile range) follow-up of 3.6 years (1.7-5.9 years), a higher proportion of patients in the surgical group achieved an HbA1C of ≤6.0% than the control group (65.8% versus 22.8%). The surgical group showed a decrease in mean HbA1C of 1.5% (95% confidence interval [CI]: 1.4%-1.7%), 1.4% (1.2%-1.5%), and 1.3% (1.1%-1.5%) at 1-, 2-, and 3-year follow-up, respectively, whereas HbA1C increased in the control group. The proportion of patients receiving glucose-lowering medications decreased in the surgical group (92.2% to 66.5%) but increased in the control group (85.3% to 90.2%). CONCLUSION: BS is associated with significant improvement in glycemic control, achievement of normal HbA1C levels, and reduced need for glucose-lowering therapy in patients with T2D.

Phylogenomics of the Phylum Proteobacteria: Resolving the Complex Relationships.

Proteobacteria is one of the largest and phenotypically most diverse divisions within the domain bacteria. Due to the economic importance, this phylum demands an urgent need for a clear and scientifically sound classification system to streamline their characterization. The goal of our study was to carefully reevaluate the current system of classification and suggest changes wherein necessary. Phylogenetic trees of 84 Proteobacteria were constructed using single gene-based phylogeny involving 16S rRNA genes and protein sequences of 85 conserved genes, whole genome-based phylogenetic tree using CVtree3.0, amino acid Identity matrix tree, and concatenated tree with aforementioned conserved genes. The results of our study confirm the polyphyletic relationship between Desulfurella acetivorans, a Deltaproteobacteria with Epsilonproteobacteria. The group Syntrophobacterales was found to be polyphyletic with respect to Desulfarculus baarsii and the group Thiotrichales was found to be splitting in different phylogenetic trees. Placement of phylogenetic groups belonging to Rhodocyclales, Oceonospirilalles, and Chromatiales is controversial and requires further study and revisions. Based on our analysis, we strongly support reclassification of Magnetococcales as a separate class Etaproteobacteria. From our results, we conclude that concatenated trees of conserved proteins are a more accurate method for phylogenetic analysis, as compared to other methods used.

GFP Tagged VSELs Help Delineate Novel Stem Cells Biology in Multiple Adult Tissues.

Various types of stem cells are being researched upon to exploit their potential for regenerative medicine including pluripotent human embryonic stem (hES) cells derived from spare human embryos, induced pluripotent stem (iPS) cells by reprogramming somatic cells to a pluripotent state and multipotent mesenchymal stem/stromal cells (MSCs) obtained in vitro from multiple tissues. More than 50 independent groups have reported another novel population of pluripotent stem cells in adult tissues termed very small embryonic-like stem cells (VSELs). VSELs are developmentally linked to primordial germ cells, which rather than giving rise to the germ cells and later ceasing to exist, survive throughout life in multiple organs along with tissue-specific adult stem cells better described as lineage-restricted, tissue-committed progenitors with limited plasticity. VSELs survive total body irradiation in bone marrow, oncotherapy in the gonads, bilateral ovariectomy in the uterus and partial pancreatectomy in the pancreas of mice and participate in the regeneration of multiple organs under normal physiological conditions. VSELs and tissue-specific progenitor cells work together in a subtle manner, maintain life-long tissue homeostasis and their dysfunction leads to various pathologies including cancer. However, due to their quiescent state, VSELs have invariably eluded lineage-tracing studies reported so far. Present article reviews novel insights into VSELs biology and how VSELs enriched from GFP (green fluorescent protein) mice have enabled to delineate their role in various biological processes in vivo. VSELs biology needs to be understood in-depth as this alone will help evolve the field of regenerative medicine and win the war against cancer.

Molecular Insights into Endometrial Cancer in Mice.

Pluripotent, very small embryonic-like stem cells (VSELs) and the 'progenitors' endometrial stem cells (EnSCs) along with associated molecular changes in endometrial cancer, that developed seven months after neonatal exposure to estradiol in one of the sixty mice, were studied in the present study. Endocrine disruption affected both endometrium and myometrium, there was accumulation of endometrial fluid and significant hyperplasia. Disrupted endometrial-myometrial junction resulted in mobilization of myometrial cells into endometrium and epithelial and stromal cells into myometrium suggestive of adenomyosis. Markers specific for VSELs/ EnSCs (OCT-4, NANOG, SSEA-1, SCA-1, c-KIT) showed increased expression in uterine sections and marked upregulation of corresponding transcripts (Oct-4A, Oct-4, Sox-2, Nanog, Sca-1, c-Kit) was noted in RNA extracted from both uterine tissue and stem cells enriched from endometrial fluid. Hormonal receptors (ER-α, ER-ß, PR, FSHR) were upregulated in both tumor sections and in endometrial fluid. ER-ß and FSHR (Fshr3) expression was prominent suggesting a major role in endometrial cancer. Cancer cells showed global hypomethylation (reduced expression of 5-methyl cytosine), reduced expression of tumor suppressor gene (PTEN) and increased expression of cancer stem cells marker (CD166) which suggested dysregulation and aberrant oncogenic events. Increased expression of PCNA, Ki67, SOX-9 suggested excessive proliferation and hyperplasia which are predominant signs of endometrial cancer. Results suggest that VSELs increase in numbers and possibly transform into cancer stem cells (co-express CD166 and OCT-4) in endometrial cancer. Expression of OCT-4, CD133, ALDHA1 and CD166 in side-population cells from human endometrial cancer samples suggests a possible role of VSELs in human endometrial cancer as well.

How to manage weight loss in women with obesity and PCOS seeking fertility?

Obesity exacerbates the phenotype of polycystic ovarian syndrome (PCOS) including infertility as well as reducing the efficacy and access to fertility treatments. Weight management is, therefore, a key component of treatment for women with PCOS and coexistent obesity. Many women with PCOS describe significant difficulty losing weight and treatment options are limited. The first-line treatment is lifestyle interventions though the weight loss and any impact on fertility are limited. No one dietary strategy can be preferentially recommended based on current evidence. While very low energy diets can result in significant weight loss the evidence for impact on fertility is limited. Pharmacotherapy, including a range of treatments can result in marked weight loss and there is some evidence of improved rates of conception including spontaneous and in response to assisted reproduction treatment. As with pharmacotherapy, data regarding bariatric surgery is largely from nonrandomized studies and though the significant weight loss is anticipated to improve fertility the available data prevents firm conclusions. Clinicians and patients must consider the magnitude of weight loss to be targeted as well as the anticipated fertility treatment required and the timeline of treatment when deciding upon the personalized weight loss strategy. Clinicians and patients should be confident in targeting the most appropriate treatment early in the patient's management to avoid unnecessary delays.

Additional evidence to support OCT-4 positive VSELs and EnSCs as the elusive tissue-resident stem/progenitor cells in adult mice uterus.

OBJECTIVE: True identity and specific set of markers to enrich endometrial stem cells still remains elusive. Present study was undertaken to further substantiate that very small embryonic-like stem cells (VSELs) are the true and elusive stem cells in adult mice endometrium. METHODS: This was achieved by undertaking three sets of experiments. Firstly, SSEA-1+ and Oct-4 + positive VSELs, sorted from GFP mice, were transplanted into the uterine horns of wild-type Swiss mice and GFP uptake was studied within the same estrus cycle. Secondly, uterine lumen was scratched surgically and OCT-4 expressing stem/progenitor cells were studied at the site of injury after 24-72 h. Thirdly, OCT-4 expression was studied in the endometrium and myometrium of adult mice after neonatal exposure to estradiol (20 µg/pup/day on days 5-7 after birth). RESULTS: GFP + ve VSELs expressing SSEA-1 and Oct-4 engrafted and differentiated into the epithelial cells lining the lumen as well as the glands during the estrus stage when maximum remodeling occurs. Mechanical scratching activated tissue-resident, nuclear OCT-4 positive VSELs and slightly bigger 'progenitors' endometrial stem cells (EnSCs, cytoplasmic OCT-4) which underwent clonal expansion and further differentiated into luminal and glandular epithelial cells. Neonatal exposure to endocrine disruption resulted in increased numbers of OCT-4 positive VSELs/EnSCs in adult endometrium. DISCUSSION: Results support the presence of functionally active VSELs in adult endometrium. VSELs self-renew and give rise to EnSCs that further differentiate into epithelial cells under normal physiological conditions. Also, VSELs are vulnerable to endocrine insults. To conclude VSELs are true and elusive uterine stem cells that maintain life-long uterine homeostasis and their dysregulation may result in various pathologies.

The Impact of Diabetes and Glucose-Lowering Therapies on Hepatocellular Carcinoma Incidence and Overall Survival.

PURPOSE: The incidence of hepatocellular carcinoma (HCC) in the United Kingdom has increased 60% in the past 10 years. The epidemics of obesity and type 2 diabetes are contributing factors. In this article, we examine the impact of diabetes and glucose-lowering treatments on HCC incidence and overall survival (OS). METHODS: Data from 1064 patients diagnosed with chronic liver disease (CLD) (n = 340) or HCC (n = 724) were collected from 2007 to 2012. Patients with HCC were followed up prospectively. Univariate and multivariate logistic regression determined HCC risk factors. Kaplan-Meier curves were used to examine survival and Cox proportional hazards analysis estimated hazard ratios (HRs) for death according to use of glucose-lowering therapies. FINDINGS: Diabetes prevalence was 39.6% and 10.6% within the HCC and CLD cohorts, respectively. The odds ratio for having HCC in patients with diabetes was 5.55 (P < 0.001). Univariate analysis found an increased association of HCC with age, sex, cirrhosis, hemochromatosis, alcohol abuse, diabetes, and Child's Pugh score. In multivariate analysis age, sex, cirrhosis, Child's Pugh score, diabetes status, and insulin use retained significance. Diabetes status did not significantly affect OS in HCC; however, in people with diabetes and HCC, metformin treatment was associated with improved OS (mean survival, 31 vs 24 months; P =0.016; HR for death = 0.75; P = 0.032). IMPLICATIONS: Diabetes is significantly associated with HCC in the United Kingdom. Metformin treatment is associated with improved OS after HCC diagnosis. Treatment of diabetes should be appropriately reviewed in high-risk populations, with specific consideration of the potential hepatoprotective effects of metformin in HCC.

Very small embryonic-like stem cells (VSELs) regenerate whereas mesenchymal stromal cells (MSCs) rejuvenate diseased reproductive tissues.

Compared to embryonic and induced pluripotent stem cells, mesenchymal stem/stromal cells (MSCs) have made their presence felt with good therapeutic promise and safety profile. Transplanting MSCs has successfully helped to reverse infertility and resulted in live births in animal models and also in humans. But the underlying mechanism for their therapeutic potential is not yet clear. MSCs are not pluripotent and hence lack plasticity to differentiate into multiple adult cell types. They rather act as 'paracrine providers' to the tissue-resident stem cells since similar beneficial effects are also observed when their secretome (microvesicles or exosomes) is transplanted. Cytokines, growth factors, signaling lipids, mRNAs, and miRNAs secreted by MSCs enables tissue-resident stem cells to undergo differentiation into specific cell types. Tissue-resident stem cells include pluripotent, very small embryonic-like stem cells (VSELs) and progenitors [spermatogonial (SSCs), ovarian (OSCs) and endometrial (EnSCs) stem cells in testes, ovary and uterus respectively] which function in a subtle manner to maintain life-long tissue homeostasis and regenerate damaged (non-functional) reproductive tissues by differentiating into sperm, oocytes and endometrial epithelial cells respectively. Similar to restoring spermatogenesis, primordial follicles numbers are increased upon transplanting MSCs. Published literature suggests that MSCs do not differentiate into epithelial cells in the endometrium. Nuclear OCT-4 positive VSELs and cytoplasmic OCT-4, AXIN2 and KERATIN-19 positive epithelial progenitors have a greater role during endometrial regeneration. We propose, transplantation of MSCs simply provides growth factors/cytokines essential for the tissue-resident stem/progenitor cells to undergo differentiation into sperm, eggs and endometrial epithelial cells in the reproductive tissues.

Mice Uterine Stem Cells are Affected by Neonatal Endocrine Disruption & Initiate Uteropathies in Adult Life Independent of Circulatory Ovarian Hormones.

It is generally believed that ovarian hormones regulate uterine functions and their altered levels result in various uteropathies like non-receptive uterus, endometrial hyperplasia, adenomyosis, endometriosis, leiomyomas and cancer. Uterus harbors two populations of stem cells including pluripotent, very small embryonic-like stem cells (VSELs) and tissue-specific progenitors (endometrial stem cells, EnSCs). Unlike endometrial mesenchymal stem/ stromal cells, VSELs/EnSCs express ERα, ERß and PR which makes them directly vulnerable to perinatal endocrine insults. Present study was undertaken to evaluate whether uteropathies occur due to altered hormones and/or intrinsic changes in stem/progenitor cells. Mice pups, exposed to estradiol (20 µg/pup/day) on postnatal days 3-7 or vehicle, were subjected to bilateral ovariectomy on day 30 and later exposed sequentially to estradiol and progesterone resulting in receptive uterus in control mice. Despite similar hormonal exposure, endocrine disruption resulted in non-receptive uterus with noticeable endometrial and myometrial hyperplasia and up-regulation of stem cell markers (Oct-4A, Oct-4, Sox2, Nanog). Glands were poorly formed and 'defective' epithelial progenitors were found disseminated into myometrium and blood vessels revealing how adenomyosis and endometriosis possibly initiate. Progesterone resistance and estradiol dominance due to downregulation of Erα & Pr and upregulation of Erß transcripts was observed in both intact uterus and stem cells enriched from uterus. Transcripts specific for DNA mismatch repair axis (Pcna, NP95 and Dnmt1), repair enzymes (Brca-1, Rad51 and Mlh1) were dysregulated whereas Ki67 was ten-folds increased suggestive of genomic instability. Study reveals role of stem cells in initiating uteropathies during adult life independent of circulatory ovarian hormones. Endocrine disruption affects tissue resident stem/progenitor cells (VSELs/EnSCs) in both endometrium and myometrium, result in epithelial cells hyperplasia, non-receptive endometrium, adenomyosis and defective stem cells and epithelial progenitors were detected in the perimetrium from where they can mobilize to ectopic sites to initiate endometriosis. Study shows stem cell basis for various uteropathies. VSEL: Very small embryonic like stem cell; EnSC: Endometrial stem cell; E + P: Estradiol + Progesterone; E: Endometrium; P: Perimetrium; M: Myometrium; ACD: Asymmetrical cell division; SCD: Symmetrical cell division; CE: Clonal expansion; G: Gland; S: Stromal cell; US: Undifferentiated stromal cell; LE: Luminal epithelium; GE: Glandular epithelium; EP: Epithelial progenitors; SMC: Spindle-shaped myometrial cell; OMC: Oval-shaped myometrial cell.

Prognostic Models for Predicting Remission of Diabetes Following Bariatric Surgery: A Systematic Review and Meta-analysis.

BACKGROUND: Remission of type 2 diabetes following bariatric surgery is well established, but identifying patients who will go into remission is challenging. PURPOSE: To perform a systematic review of currently available diabetes remission prediction models, compare their performance, and evaluate their applicability in clinical settings. DATA SOURCES: A comprehensive systematic literature search of MEDLINE, MEDLINE In-Process & Other Non-Indexed Citations, Embase, and Cochrane Central Register of Controlled Trials (CENTRAL) was undertaken. The search was restricted to studies published in the last 15 years and in the English language. STUDY SELECTION: All studies developing or validating a prediction model for diabetes remission in adults after bariatric surgery were included. DATA EXTRACTION: The search identified 4,165 references, of which 38 were included for data extraction. We identified 16 model development and 22 validation studies. DATA SYNTHESIS: Of the 16 model development studies, 11 developed scoring systems and 5 proposed logistic regression models. In model development studies, 10 models showed excellent discrimination with area under the receiver operating characteristic curve ≥0.800. Two of these prediction models, ABCD and DiaRem, were widely externally validated in different populations, in a variety of bariatric procedures, and for both short- and long-term diabetes remission. Newer prediction models showed excellent discrimination in test studies, but external validation was limited. LIMITATIONS: While the key messages were consistent, a large proportion of the studies were conducted in small cohorts of patients with short duration of follow-up. CONCLUSIONS: Among the prediction models identified, the ABCD and DiaRem models were the most widely validated and showed acceptable to excellent discrimination. More studies validating newer models and focusing on long-term diabetes remission are needed.

Endogenous, tissue-resident stem/progenitor cells in gonads and bone marrow express FSHR and respond to FSH via FSHR-3.

Follicle stimulating hormone (FSH) is secreted by the anterior pituitary and acts on the germ cells indirectly through Granulosa cells in ovaries and Sertoli cells in the testes. Extragonadal action of FSH has been reported but is still debated. Adult tissues harbor two populations of stem cells including a reserve population of primitive, small-sized, pluripotent very small embryonic-like stem cells (VSELs) and slightly bigger, tissue-specific progenitors which include ovarian stem cells (OSCs) in ovaries, spermatogonial stem cells (SSCs) in testes, endometrial stem cells (EnSCs) in uterus and hematopoietic stem cells (HSCs) in the bone marrow. Data has accumulated in animal models showing FSHR expression on both VSELs and progenitors in ovaries, testes, uterus and bone marrow and eventually gets lost as the cells differentiate further. FSH exerts a direct action on the stem/progenitor cells via alternatively spliced FSHR-3 rather than the canonical FSHR-1. FSH stimulates VSELs to undergo asymmetrical cell divisions to self-renew and give rise to the progenitors that in turn undergo symmetrical cell divisions and clonal expansions followed by differentiation into specific cell types. Excessive self-renewal of VSELs results in cancer and this explains ubiquitous expression of embryonic markers including nuclear OCT-4 along with FSHR in cancerous tissues. Focus of this review is to compile published data to support this concept. FSHR expression in stem/progenitor cells was confirmed by immuno-fluorescence, Western blotting, in situ hybridization and by quantitative RT-PCR. Two different commercially available antibodies (Abcam, Santacruz) were used to confirm specificity of FSHR expression along with omission of primary antibody and pre-incubation of antibody with immunizing peptide as negative controls. Western blotting allowed detection of alternatively spliced FSHR isoforms. Oligoprobes and primers specific for Fshr-1 and Fshr-3 were used to study these alternately-sliced isoforms by in situ hybridization and their differential expression upon FSH treatment by qRT-PCR. To conclude, stem/progenitor cells in adult tissues express FSHR and directly respond to FSH via FSHR-3. These findings change the field of FSH-FSHR biology, call for paradigm shift, explain FSHR expression on cancer cells in multiple organs and provide straightforward explanations for various existing conundrums including extragonadal expression of FSHR.

The Impact of Bariatric Surgery on Incident Microvascular Complications in Patients With Type 2 Diabetes: A Matched Controlled Population-Based Retrospective Cohort Study.

OBJECTIVE: To assess the impact of bariatric surgery (BS) on incident microvascular complications of diabetes-related foot disease (DFD), sight-threatening diabetic retinopathy (STDR), and chronic kidney disease (CKD) in patients with type 2 diabetes and obesity. RESEARCH DESIGN AND METHODS: A retrospective matched, controlled population-based cohort study was conducted of adults with type 2 diabetes between 1 January 1990 and 31 January 2018 using IQVIA Medical Research Data (IMRD), a database of primary care electronic records. Each patient with type 2 diabetes who subsequently had BS (surgical group) was matched on the index date with up to two patients with type 2 diabetes who did not have BS (nonsurgical group) within the same general practice by age, sex, preindex BMI, and diabetes duration. RESULTS: Included were 1,126 surgical and 2,219 nonsurgical participants. In the study population 2,261 (68%) were women. Mean (SD) age was 49.87 (9.3) years vs. 50.12 (9.3) years and BMI was 46.76 (7.96) kg/m2 vs. 46.14 (7.49) kg/m2 in the surgical versus nonsurgical group, respectively. In the surgical group, 22.1%, 22.7%, 52.2%, and 1.1% of patients had gastric band, sleeve gastrectomy, Roux-en-Y gastric bypass (RYGB), and duodenal switch, respectively. Over a median follow-up of 3.9 years (interquartile range 1.8-6.4), BS was associated with reduction in incident combined microvascular complications (adjusted hazard ratio 0.53, 95% CI 0.43-0.66, P < 0.001), DFD (0.61, 0.50-0.75, P < 0.001), STDR (0.66, 0.44-1.00, P = 0.048), and CKD (0.63, 0.51-0.78, P < 0.001). Analysis based on the type of surgery showed that all types of surgery were associated with a favorable impact on the incidence of composite microvascular complications, with the greatest reduction for RYGB. CONCLUSIONS: BS was associated with a significant reduction in incident diabetes-related microvascular complications.

Pluripotent Stem (VSELs) and Progenitor (EnSCs) Cells Exist in Adult Mouse Uterus and Show Cyclic Changes Across Estrus Cycle.

We have earlier reported pluripotent, very small embryonic-like stem cells (VSELs) and slightly bigger endometrial stem cells (EnSCs) in adult mouse uterus and their regulation by gonadotropin and steroid hormones. VSELs can differentiate into cells of all three lineages in vitro; however, they neither expand readily in vitro nor compliment a developing embryo. In the present study, a robust protocol is described to enrich uterine stem/progenitor cells along with their characterization and variation across estrus cycle. After enzymatic digestion of adult mouse uterus, single-cell suspension obtained was spun at 1000 rpm (250 g) to pellet majority of cells. Stem cells remain buoyant at this speed and were pelleted by spinning supernatant at 3000 rpm (1000 g). Spherical, darkly stained VSELs (2-6 µm) with high nucleo-cytoplasmic ratio and EnSCs (> 6 µm) expressed OCT-4, NANOG, SSEA-1, SCA-1, and c-KIT. OCT-4-positive cells co-expressed SSEA-1, ERα, ERß, PR, and FSHR. Transcripts specific for pluripotent state (Oct-4, Oct-4a, Sox-2, Nanog), primordial germ cells (Stella, Fragilis), and receptors for pituitary and steroid hormones (ERα, ERß, PR, FSHR 1 and 3) were studied by RT-PCR in 3000 rpm pellet. Cell pellet collected at 3000 rpm showed 10-fold enrichment of VSELs (2-6 µm, viable cells with surface phenotype of LIN-CD45-SCA-1+) by flow cytometry and upregulation of pluripotent transcripts by qRT-PCR compared with 1000 rpm pellet. VSELs were maximal during estrus and metestrus phases of estrus cycle. To conclude, VSELs/EnSCs can be enriched from adult uterus using the strategy described here, vary in numbers across estrus cycle, and are vulnerable to endocrine disruption as they express steroid receptors.

Gonadotropin and steroid hormones regulate pluripotent very small embryonic-like stem cells in adult mouse uterine endometrium.

BACKGROUND: Very small embryonic-like stem cells (VSELs) exist in adult organs, express pluripotent markers and have the ability to differentiate into three germ layers in vitro. Testicular, ovarian and hematopoietic stem/progenitor cells express receptors for follicle stimulating (FSH) and ovarian hormones and are activated by them to undergo proliferation/differentiation. VSELs exist in mouse uterus and are regulated by physiological dose of estradiol (E) & progesterone (P) during endometrial growth, differentiation and regeneration/remodeling. In the present study, effects of daily administration of E (2 µg/day), P (1 mg/Kg/day) or FSH (5 IU/day) for 7 days on the endometrium and stem/progenitor cells was studied in bilaterally ovariectomized mice. RESULTS: E treatment resulted in hypertrophy whereas P resulted in hyperplasia and overcrowding of epithelial cells. FSH also directly stimulated the endometrial cells. Nuclear OCT-4A positive VSELs were visualized in ovariectomized (atrophied) endometrium and cytoplasmic OCT-4B positive epithelial, stromal and endothelial cells were observed after treatment. FSH treated uterine tissue showed presence of 4 alternately spliced FSHR isoforms by Western blotting. 3-5 µm VSELs with a surface phenotype of LIN-/CD45-/SCA-1+ were enumerated by flow cytometry and were found to express ER, PR, FSHR1 and FSHR3 by RT-PCR analysis. Differential effects of treatment were observed on pluripotent (Oct4A, Sox2, Nanog), progenitors (Oct-4, Sca-1), primordial germ cells (Stella, Fragilis) and proliferation (Pcna) specific transcripts by qRT-PCR analysis. FSH and P (rather than E) exerted profound, direct stimulatory effects on uterine VSELs. Asymmetric, symmetric divisions and clonal expansion of stem/progenitor cells was confirmed by co-expression of OCT-4 and NUMB. CONCLUSIONS: Results confirm presence of VSELs and their regulation by circulatory hormones in mouse uterus. Stem cell activation was more prominent after P and FSH compared to E treatment. The results question whether epithelial cells proliferation is regulated by paracrine influence of stromal cells or due to direct action of hormones on stem cells. VSELs expressing nuclear OCT-4A are the most primitive and pluripotent stem cells, undergo asymmetric cell division to self-renew and differentiate into epithelial, stromal and endothelial cells with cytoplasmic OCT-4B. Role of follicle stimulating and steroid hormones on the stem cells needs to be studied in various uterine pathologies.

Novel Insights into Adult and Cancer Stem Cell Biology.

Adult tissues are thought to harbor two populations of "dormant" and "actively dividing" stem cells. Quiescent stem cells undergo rare asymmetric cell divisions (ACDs) through which they self-renew and give rise to tissue-committed "progenitors" of distinct fate and "progenitors" in turn undergo symmetric cell divisions (SCDs) and clonal expansion. However, quiescent stem cells have not been demonstrated in adult tissues such as skin, testis, liver, and brain. After surgical removal of part of liver and pancreas-adult differentiated cells divide and regenerate and a possible role of stem cells remains doubtful. Long-term repopulating hematopoietic stem cells are quiescent in nature but ACD has not been convincingly demonstrated even among them. Attempts by various groups to identify a common stemness program that ensures self-renewal among different kinds of stem cells have also remained futile. Uncontrolled self-renewal and compromised differentiation of stem cells possibly initiate leukemia/cancer, but the identity of leukemic stem cells and whether cancer stem cells arise by epithelial-mesenchymal transition (EMT) in solid tumors are all open-ended questions that need greater clarity. Acceptance of the presence of very small embryonic-like stem cells (VSELs) in adult tissues could clarify several of these existing dilemmas in the field. Data are compiled showing that VSELs undergo ACD in the hematopoietic system, testis, ovary, uterus, and pancreas, whereas tissue-committed progenitors undergo SCD and clonal expansion. VSELs possess similar overlapping stemness program as in embryonic stem cells, embryonic carcinoma cells, embryonic germ cells, induced pluripotent stem cells, and primordial germ cells. VSELs and leukemic and cancer cells express overlapping embryonic markers. Uncontrolled proliferation of VSELs and compromised differentiation possibly initiate leukemia. Process of EMT and initiation of solid tumor from VSELs (located among the epithelial cells) are indeed two distinct and parallel events. To conclude, VSELs provide explanation to several confounding aspects of adult stem cell biology.