Single Exosome Amperometric Measurements Reveal Encapsulation of Chemical Messengers for Intercellular Communication.

In multicellular organisms, cells typically communicate by sending and receiving chemical signals. Chemical messengers involved in the exocytosis of neuroendocrine cells or neurons are generally assumed to only originate from the fusing of intracellular large dense core vesicles (LDCVs) or synaptic vesicles with the cellular membrane following stimulation. Accumulated evidence suggests that exosomes─one of the main extracellular vesicles (EVs)─carrying cell-dependent DNA, mRNA, proteins, etc., play an essential role in cellular communication. Due to experimental limitations, it has been difficult to monitor the real-time release of individual exosomes; this restricts a comprehensive understanding of the basic molecular mechanisms and the functions of exosomes. In this work, we introduce amperometry with microelectrodes to capture the dynamic release of single exosomes from a single living cell, distinguish them from other EVs, and differentiate the molecules inside exosomes and those secreted from LDCVs. We show that, similar to many LDCVs and synaptic vesicles, exosomes released by neuroendocrine cells also contain catecholamine transmitters. This finding reveals a different mode of chemical communication via exosome-encapsulated chemical messengers and a potential interconnection between the two release pathways, changing the canonical view of exocytosis of neuroendocrine cells and possibly neurons. This defines a new mechanism for chemical communication at the fundamental level and opens new avenues in the research of the molecular biology of exosomes in the neuroendocrine and central nervous systems.

Passive Smoking and Oral Health of Infants, Preschoolers, and Children: A Systematic Review.

INTRODUCTION: Almost half of the world's children experience passive smoking, which is linked to numerous oral health conditions. The aim is to synthesize data on the impact of passive smoking on oral health of infants, preschoolers, and children. AIMS AND METHODS: A search was conducted across Medline (via EBSCOhost), PubMed, and Scopus up to February 2023. Risk of bias was assessed according to the Newcastle-Ottawa Scale (NOS). RESULTS: The initial search produced 1221 records and after removal of duplicates, screening by title and abstract, and full-text assessment, 25 studies were eligible for review and data extraction. The majority of studies (94.4%) found a correlation between passive smoking and increased prevalence of dental caries with three studies suggesting a dose-response relationship. Prenatal passive smoking exposure in 81.8% of studies indicated an increased dental caries experience compared to postnatal exposure. Low parental education, socioeconomic status, dietary habits, oral hygiene, and gender affected the level of environmental tobacco smoke (ETS) exposure and dental caries risk. CONCLUSIONS: The results of this systematic review strongly suggest a significant association between dental caries in the deciduous dentition and passive smoking. Early intervention and education on the effects of passive smoking on infants and children will allow for the improvement in oral health outcomes and reduction in smoking-associated systemic conditions. The results justify all health professionals paying more attention to passive smoking when conducting pediatric patient histories, contributing to improved diagnosis and appropriate treatment planning with more suitable follow-up schedules. IMPLICATIONS: The evidence from this review that environmental tobacco smoke and passive smoking is a risk factor for oral health conditions, both prenatally and postnatally during early childhood, justifies all health professionals paying more attention to passive smoking when conducting pediatric patient histories. Early intervention and appropriate parental education regarding the effects of secondhand smoke on infants and children will allow for the minimization of dental caries, improvement in oral health outcomes and overall reduction in smoking-associated systemic conditions for the children exposed.

Steroid hormones regulate genome-wide epigenetic programming and gene transcription in human endometrial cells with marked aberrancies in endometriosis.

Programmed cellular responses to cycling ovarian-derived steroid hormones are central to normal endometrial function. Abnormalities therein, as in the estrogen-dependent, progesterone-"resistant" disorder, endometriosis, predispose to infertility and poor pregnancy outcomes. The endometrial stromal fibroblast (eSF) is a master regulator of pregnancy success. However, the complex hormone-epigenome-transcriptome interplay in eSF by each individual steroid hormone, estradiol (E2) and/or progesterone (P4), under physiologic and pathophysiologic conditions, is poorly understood and was investigated herein. Genome-wide analysis in normal, early and late stage eutopic eSF revealed: i) In contrast to P4, E2 extensively affected the eSF DNA methylome and transcriptome. Importantly, E2 resulted in a more open versus closed chromatin, confirmed by histone modification analysis. Combined E2 with P4 affected a totally different landscape than E2 or P4 alone. ii) P4 responses were aberrant in early and late stage endometriosis, and mapping differentially methylated CpG sites with progesterone receptor targets from the literature revealed different but not decreased P4-targets, leading to question the P4-"resistant" phenotype in endometriosis. Interestingly, an aberrant E2-response was noted in eSF from endometriosis women; iii) Steroid hormones affected specific genomic contexts and locations, significantly enriching enhancers and intergenic regions and minimally involving proximal promoters and CpG islands, regardless of hormone type and eSF disease state. iv) In eSF from women with endometriosis, aberrant hormone-induced methylation signatures were mainly due to existing DNA methylation marks prior to hormone treatments and involved known endometriosis genes and pathways. v) Distinct DNA methylation and transcriptomic signatures revealed early and late stage endometriosis comprise unique disease subtypes. Taken together, the data herein, for the first time, provide significant insight into the hormone-epigenome-transcriptome interplay of each steroid hormone in normal eSF, and aberrant E2 response, distinct disease subtypes, and pre-existing epigenetic aberrancies in the setting of endometriosis, provide mechanistic insights into how endometriosis affects endometrial function/dysfunction.

Comparison of the radioprotective effects of the liposomal forms of five natural radioprotectants in alleviating the adverse effects of ionising irradiation on human lymphocytes and skin cells in radiotherapy.

This study aims to evaluate the radioprotective effects of liposomes encapsulating curcumin (Lip-CUR), silibinin (Lip-SIL), α-tocopherol (Lip-TOC), quercetin (Lip-QUE) and resveratrol (Lip-RES) in alleviating the adverse effects of ionising irradiation on human lymphoctyes and skin cells in radiotherapy. Liposomes encapsulating the above natural radioprotectants (Lip-NRPs) were prepared by the film hydration method combined with sonication. Their radioprotective effects for the cells against X-irradiation was evaluated using trypan-blue assay and γ-H2AX assay. All prepared Lip-NRPs had a mean diameter less than 240 nm, polydispersity index less than 0.32, and zeta potential more than -23 mV. Among them, the radioprotective effect of Lip-RES was lowest, while that of Lip-QUE was highest. Lip-SIL also exhibited a high radioprotective effect despite its low DPPH-radical scavenging activity (12.9%). The radioprotective effects of Lip-NRPs do not solely depend on the free radical scavenging activity of NRPs but also on their ability to activate cellular mechanisms.

Improvement of oocyte quality through the SIRT signaling pathway.

Background: Oocyte quality is one of the major deciding factors in female fertility competence. Methods: PubMed database was searched for reviews by using the following keyword "oocyte quality" AND "Sirtuins". The methodological quality of each literature review was assessed using the Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) 2020 statement. Main Findings: Oxidative stress has been recognized as the mechanism attenuating oocyte quality. Increasing evidence from animal experiments and clinical studies has confirmed the protective roles of the sirtuin family in improving oocyte quality via an antioxidant effect. Conclusion: The protective roles in the oocyte quality of the sirtuin family have been increasingly recognized.

Deep immunophenotyping reveals endometriosis is marked by dysregulation of the mononuclear phagocytic system in endometrium and peripheral blood.

BACKGROUND: Endometriosis is a chronic, estrogen-dependent disorder where inflammation contributes to disease-associated symptoms of pelvic pain and infertility. Immune dysfunction includes insufficient immune lesion clearance, a pro-inflammatory endometrial environment, and systemic inflammation. Comprehensive understanding of endometriosis immune pathophysiology in different hormonal milieu and disease severity has been hampered by limited direct characterization of immune populations in endometrium, blood, and lesions. Simultaneous deep phenotyping at single-cell resolution of complex tissues has transformed our understanding of the immune system and its role in many diseases. Herein, we report mass cytometry and high dimensional analyses to study immune cell phenotypes, abundance, activation states, and functions in endometrium and blood of women with and without endometriosis in different cycle phases and disease stages. METHODS: A case-control study was designed. Endometrial biopsies and blood (n = 60 total) were obtained from women with (n = 20, n = 17, respectively) and without (n = 14, n = 9) endometriosis in the proliferative and secretory cycle phases of the menstrual cycle. Two mass cytometry panels were designed: one broad panel and one specific for mononuclear phagocytic cells (MPC), and all samples were multiplexed to characterize both endometrium and blood immune composition at unprecedented resolution. We combined supervised and unsupervised analyses to finely define the immune cell subsets with an emphasis on MPC. Then, association between cell types, protein expression, disease status, and cycle phase were performed. RESULTS: The broad panel highlighted a significant modification of MPC in endometriosis; thus, they were studied in detail with an MPC-focused panel. Endometrial CD91+ macrophages overexpressed SIRPα (phagocytosis inhibitor) and CD64 (associated with inflammation) in endometriosis, and they were more abundant in mild versus severe disease. In blood, classical and intermediate monocytes were less abundant in endometriosis, whereas plasmacytoid dendritic cells and non-classical monocytes were more abundant. Non-classical monocytes were higher in severe versus mild disease. CONCLUSIONS: A greater inflammatory phenotype and decreased phagocytic capacity of endometrial macrophages in endometriosis are consistent with defective clearance of endometrial cells shed during menses and in tissue homeostasis, with implications in endometriosis pathogenesis and pathophysiology. Different proportions of monocytes and plasmacytoid dendritic cells in blood from endometriosis suggest systemically aberrant functionality of the myeloid system opening new venues for the study of biomarkers and therapies for endometriosis.

Transcriptomic analysis supports collective endometrial cell migration in the pathogenesis of adenomyosis.

RESEARCH QUESTION: Adenomyosis is a common uterine disorder of uncertain causes. Can transcriptomic analyses of the endometrium and myometrium reveal potential mechanisms underlying adenomyosis pathogenesis? DESIGN: Transcriptomic profiles of eutopic endometrium and myometrium from women with and without diffuse adenomyosis and with symptomatic FIGO type 2-5 fibroids in the proliferative phase of the menstrual cycle were assessed using RNA sequencing and bioinformatic analysis. Differentially expressed genes (DEG) and potential pathways were validated by quantitative reverse transcription polymerase chain reaction, immunoblotting and Masson staining, using additional clinical samples. RESULTS: Top biological processes in the endometrium of women with versus without adenomyosis, enriched from DEG, comprised inflammation, extracellular matrix (ECM) organization, collagen degradation and hyaluronan synthesis, which are key in cell migration and cell movement. Top biological processes enriched from DEG in the myometrium of women with versus without adenomyosis revealed ECM organization dysfunction, abnormal sensory pain perception and gamma aminobutyric acid (GABA) synaptic transmission. Dysregulation of prolactin signalling was also enriched in eutopic endometrium and in the myometrium of women with adenomyosis. CONCLUSIONS: Overall, our results support the invasive endometrium theory in the pathogenesis of adenomyosis, in which inflammation induces ECM remodelling resulting in a track for subsequent endometrial collective cell migration and onset of adenomyosis. Moreover, abnormal myometrial GABA synaptic transmission may contribute to dysmenorrhoea in women with adenomyosis and is a possible target for novel therapeutic development. Prolactin signalling abnormalities may serve as another opportunity for therapeutic intervention.

Complete response following treatment of plasma cell leukemia with venetoclax and dexamethasone: A case report.

INTRODUCTION: Plasma cell leukemia (PCL) is a rare but aggressive variant of multiple myeloma (MM) with a poor prognosis. Due to the limited number of prospective clinical trials studying PCL, treatment options are often extrapolated from data available for the treatment of MM. Venetoclax has recently demonstrated antimyeloma activity in patients with relapsed/refractory MM carrying the t(11;14) translocation. However, few cases have reported the analogous efficacy of venetoclax in PCL. CASE REPORT: A 64-year-old Caucasian male developed relapsed PCL despite treatment with hyperCD (hyperfractionated cyclophosphamide and dexamethasone) and Dara-KRd (daratumumab, carfilzomib, lenalidomide, dexamethasone). Due to the refractory nature of his disease and the presence of a t(11:14) translocation, the patient was subsequently initiated on venetoclax 400 mg daily and dexamethasone 4 mg once weekly. MANAGEMENT AND OUTCOME: The patient achieved a complete response by International Myeloma Working Group criteria three months after initiating venetoclax-dexamethasone, including a repeat bone marrow biopsy that showed no abnormal plasma cells. He successfully underwent consolidation with melphalan-based autologous stem cell transplantation. He remains disease-free 9 months after venetoclax initiation. DISCUSSION: Combination all-oral therapy with venetoclax and dexamethasone can induce deep hematologic responses in patients with relapsed/refractory PCL carrying the t(11;14) translocation.

Effects of Hochuekkito, a traditional Japanese medicine (Kampo), on reproduction of aging female mice.

PURPOSE: To determine the potentials of Hochuekkito (HET) treatment for aging infertility. METHODS: Mice at 36 weeks of age were fed without (control, n = 40) or with low (100 mg/kg/day, n = 24) and high (1000 mg/kg/day, n = 38) doses of HET for 12 weeks. Aging animals at 48 weeks of age were used for in vitro fertilization-embryo transfer (IVF-ET), and their ovaries were subjected to histological and quantitative inflammation analyses. RESULTS: HET administration decreased transcript levels of ovarian inflammatory markers, interleukin 6 (IL-6), IL-1ß, tumor necrosis factor (TNF)-α, and interferon-gamma (IFN-γ) but suppressed ovulation rates and the number of ovulated oocytes in aging mice. Furthermore, HET treatment decreased the rates of oocytes maturation and fertilization and the cumulus-cell expression of TNF-α-induced protein 6 and epidermal growth factor receptor. After IVF-ET, no improvement of declined live offspring rate by aging was achieved by HET administration, although there were no adverse effects on embryo development and implantation as well as gross morphology and bodyweight of pups. CONCLUSION: Present study indicated HET treatment interfered with ovulation and fertilization in aging mice without affecting ovarian follicle development. No improvement on the age-associated decline of live offspring rate and follicle development was achieved after HET treatment.

Quantifying Intracellular Single Vesicular Catecholamine Concentration with Open Carbon Nanopipettes to Unveil the Effect of L-DOPA on Vesicular Structure.

Understanding the regulatory mechanisms of exocytosis is essential for uncovering the pathologies of neuronal disorders and developing related pharmaceuticals. In this work intracellular vesicle impact electrochemical cytometry (IVIEC) measurements with different-sized (50-500 nm radius) open carbon nanopipettes (CNPs) were performed to quantify the vesicular content and release kinetics of specific vesicle populations grouped by orifice sizes. Intracellular vesicles with radius below 100 nm were captured and narrowed between 50 and 100 nm. On the basis of this, single vesicular catecholamine concentrations in the intracellular environment were quantified as 0.23-1.1 M. Our results with L-3,4-dihydroxyphenylalanine (L-DOPA)-exposure indicate that L-DOPA regulates exocytosis by increasing the dense core size and vesicular content while catecholamine concentrations did not show obvious alterations. These were all achieved simultaneously and relatively noninvasively with open CNPs.

In Vitro Activation Early Follicles: From the Basic Science to the Clinical Perspectives.

Development of early follicles, especially the activation of primordial follicles, is strictly modulated by a network of signaling pathways. Recent advance in ovarian physiology has been allowed the development of several therapies to improve reproductive outcomes by manipulating early folliculogenesis. Among these, in vitro activation (IVA) has been recently developed to extend the possibility of achieving genetically related offspring for patients with premature ovarian insufficiency and ovarian dysfunction. This method was established based on basic science studies of the intraovarian signaling pathways: the phosphoinositide 3-kinase (PI3K)/Akt and the Hippo signaling pathways. These two pathways were found to play crucial roles in folliculogenesis from the primordial follicle to the early antral follicle. Following the results of rodent experiments, IVA was implemented in clinical practice. There have been multiple recorded live births and ongoing pregnancies. Further investigations are essential to confirm the efficacy and safety of IVA before used widely in clinics. This review aimed to summarize the published literature on IVA and provide future perspectives for its improvement.

Correlating Molecule Count and Release Kinetics with Vesicular Size Using Open Carbon Nanopipettes.

In this work, open carbon nanopipettes (CNPs) with radius between 50 and 600 nm were used to control translocation of different-sized vesicles through the pipette orifice followed by nanoelectrochemical analysis. Vesicle impact electrochemical cytometry (VIEC) was used to determine the number of catecholamine molecules expelled from single vesicles onto an inner-wall carbon surface, where the duration of transmitter release was quantified and correlated to the vesicle size all in the same nanotip. This in turn allowed us to both size and count molecules for vesicles in a living cell. Here, small and sharp open CNPs were employed to carry out intracellular VIEC with minimal invasion and high sensitivity. Our findings with VIEC reveal that the vesicular content increases with vesicle size. The release kinetics of vesicular transmitters and dense core size have the same relation with the vesicle size, implying that the vesicular dense core size determines the speed of each release event. This direct correlation unravels one of the complexities of exocytosis.

Progestins Related to Progesterone and Testosterone Elicit Divergent Human Endometrial Transcriptomes and Biofunctions.

Progestins are widely used for the treatment of gynecologic disorders and alone, or combined with an estrogen, are used as contraceptives. While their potencies, efficacies and side effects vary due to differences in structures, doses and routes of administration, little is known about their effects on the endometrial transcriptome in the presence or absence of estrogen. Herein, we assessed the transcriptome and pathways induced by progesterone (P4) and the three most commonly used synthetic progestins, medroxyprogesterone acetate (MPA), levonorgestrel (LNG), and norethindrone acetate (NETA), on human endometrial stromal fibroblasts (eSF), key players in endometrial physiology and reproductive success. While there were similar transcriptional responses, each progestin induced unique genes and biofunctions, consistent with their structural similarities to progesterone (P4 and MPA) or testosterone (LNG and NETA), involving cellular proliferation, migration and invasion. Addition of estradiol (E2) to each progestin influenced the number of differentially expressed genes and biofunctions in P4 and MPA, while LNG and NETA signatures were more independent of E2. Together, these data suggest different mechanisms of action for different progestins, with progestin-specific altered signatures when combined with E2. Further investigation is warranted for a personalized approach in different gynecologic disorders, for contraception, and minimizing side effects associated with their use.

Initiation of direct oral anticoagulants versus warfarin for venous thromboembolism: impact on time to hospital discharge.

The objective of this project was to compare the time from initiation of oral anticoagulation to hospital discharge between warfarin and direct oral anticoagulants (DOACs) for the treatment of acute venous thromboembolism (VTE). This retrospective observational study was done at a single VA medical center. A total of 107 patients were included, with 42 patients (39%) in the DOAC group, which included rivaroxaban, dabigatran, and apixaban, and 65 patients (61%) in the warfarin group. Variables collected through chart review included comorbid conditions, time from initiation of oral anticoagulation to discharge, emergency department (ED) visits and readmission within 30 or 90 days, and bleeding events. The DOAC group had a shorter time to discharge compared to the warfarin group (28 vs. 114 h, p < 0.001). There were similar 30 and 90-day hospital readmission rates and/or ED visits for DOACs (23.8 and 33.3%) compared to warfarin (18.5 and 30.8%), including those related to bleeding of any severity (11.9% for DOACs vs. 9.2% for warfarin; p = 0.75). There was one major bleeding event in the DOAC group and two in the warfarin group. The use of DOACs for the treatment of acute VTE in hospitalized patients was associated with shorter time to hospital discharge when compared to warfarin.

Sustained intraocular pressure elevation in eyes treated with intravitreal injections of anti-vascular endothelial growth factor for diabetic macular edema in a real-life setting.

PURPOSE: The aim of this study was to investigate the sustained intraocular pressure (IOP) elevation after repeated anti-VEGF intravitreal injections (IVI) in patients with diabetic macular edema (DME). METHODS: A retrospective study included 140 eyes without prior glaucoma, treated with at least three anti-VEGF injections for DME between 2012 and 2016. IOP elevation was defined by an increase above baseline IOP by ≥6 mmHg. Baseline IOP was defined as the mean of IOP values before treatment initiation. Three groups were differentiated: group 1 without IOP elevation, groups 2 and 3 with IOP elevation and IOP <21 mmHg (group 2) and ≥21 mmHg (group 3). Rate and several risk factors of IOP elevation were assessed and compared between the three groups. RESULTS: IOP elevation occurred in ten eyes (7.1%). IOP was <21 mmHg in six eyes and ≥21 mmHg in four eyes. Statistically significant associations were found between IOP elevation and the number of injections, and HbA1c level. Two patients required local hypotonic treatment. CONCLUSIONS: In a real-life setting, we confirmed in eyes with center-involved DME without prior glaucoma or IOP elevation that repeated anti-VEGF IVI may increase the risk of sustained IOP elevation in about 7% of eyes.

Aberrant Endometrial DNA Methylome and Associated Gene Expression in Women with Endometriosis.

Endometriosis is an estrogen-dependent, progesterone-resistant disorder largely derived from retrograde transplantation of menstrual tissue/cells into the pelvis, eliciting an inflammatory response, pelvic pain, and infertility. Eutopic endometrium (within the uterus), giving rise to pelvic disease, displays cycle-dependent transcriptomic, proteomic, and signaling abnormalities, and although its DNA methylation profiles dynamically change across the cycle in healthy women, studies in endometriosis are limited. Herein, we investigated the DNA methylome and associated gene expression in three phases of the cycle in eutopic endometrium of women with severe endometriosis versus controls, matched for ethnicity, medications, smoking, and no recent contraceptive steroid use. Genome-wide DNA methylation and gene expression were coassessed in each sample. Cycle phase was determined by histology, serum hormone levels, and unsupervised principal component and hierarchical cluster analyses of microarray data. Altered endometrial DNA methylation in endometriosis was most prominent in the midsecretory phase (peak progesterone), with disruption of the normal pattern of cycle-dependent DNA methylation changes, including a bias toward methylation of CpG islands, suggesting wide-range abnormalities of the chromatin remodeling machinery in endometriosis. DNA methylation changes were associated with altered gene expression relevant to endometrial function/dysfunction, including cell proliferation, inflammation/immune response, angiogenesis, and steroid hormone response. The data provide insight into epigenetic reprogramming and steroid hormone actions in endometrium contributing to the pathogenesis and pathophysiology of endometriosis.

Regulation of hypoxia-inducible factor-1a by reactive oxygen species: new developments in an old debate.

Hypoxia-Inducible Factor-1 (HIF-1) has been largely studied for its role in cell survival in hypoxic conditions. The regulation of HIF-1 is a complex process and involves a number of molecules and pathways. Among these mechanisms a direct regulatory role of reactive oxygen species (ROS) on HIF-1 alpha subunit has received a great deal of attention and the existing body of literature includes many contradictory findings. Other intermediates such as nitric oxide (NO), specific microRNAs (miR), and transcriptional and post-translational modification have also been implicated as players in ROS mediated HIF-1a regulation. The focus of this review is to present the past conflicting evidence along with more recent findings in order to relate various aspects of this complex process. Aside from the direct role of ROS on HIF-1a regulation under hypoxia and normoxia, we analyzed the effect of different sources and concentrations of NO and the interplay between superoxide (SO) and NO in this process. We also present findings on transcriptional and translational regulation of HIF-1a via ROS and the interplay with microRNAs in this process. This review further provides insight on ERK and PI3K/AKT signaling as a common mechanism relating several pathways of ROS mediated HIF-1a regulation. Ultimately further research and discovery regarding HIF-1 regulation by oxidative stress is warranted for better understanding of disease development and potential therapeutics for pathologies such as cancer, inflammatory diseases, and ischemia-reperfusion injury.