Integrating network pharmacology and experimental validation reveals therapeutic effects of D-mannose on NAFLD through mTOR suppression.

Non-alcoholic fatty liver disease (NAFLD), a chronic liver condition and metabolic disorder, has emerged as a significant health issue worldwide. D-mannose, a natural monosaccharide widely existing in plants and animals, has demonstrated metabolic regulatory properties. However, the effect and mechanism by which D-mannose may counteract NAFLD have not been studied. In this study, network pharmacology followed by molecular docking analysis was utilized to identify potential targets of mannose against NAFLD, and the leptin receptor-deficient, genetically obese db/db mice was employed as an animal model of NAFLD to validate the regulation of D-mannose on core targets. As a result, 67 targets of mannose are predicted associated with NAFLD, which are surprisingly centered on the mechanistic target of rapamycin (mTOR). Further analyses suggest that mTOR signaling is functionally enriched in potential targets of mannose treating NAFLD, and that mannose putatively binds to mTOR as a core mechanism. Expectedly, repeated oral gavage of supraphysiological D-mannose ameliorates liver steatosis of db/db mice, which is based on suppression of hepatic mTOR signaling. Moreover, daily D-mannose administration reduced hepatic expression of lipogenic regulatory genes in counteracting NAFLD. Together, these findings reveal D-mannose as an effective and potential NAFLD therapeutic through mTOR suppression, which holds translational promise.

Region-specific sympatho-adrenergic regulation of specialized vasculature in bone homeostasis and regeneration.

Type H vessels couple angiogenesis with osteogenesis, while sympathetic cues regulate vascular and skeletal function. The crosstalk between sympathetic nerves and type H vessels in bone remains unclear. Here, we first identify close spatial connections between sympathetic nerves and type H vessels in bone, particularly in metaphysis. Sympathoexcitation, mimicked by isoproterenol (ISO) injection, reduces type H vessels and bone mass. Conversely, beta-2-adrenergic receptor (ADRB2) deficiency maintains type H vessels and bone mass in the physiological condition. In vitro experiments reveal indirect sympathetic modulation of angiogenesis via paracrine effects of mesenchymal stem cells (MSCs), which alter the transcription of multiple angiogenic genes in endothelial cells (ECs). Furthermore, Notch signaling in ECs underlies sympathoexcitation-regulated type H vessel formation, impacting osteogenesis and bone mass. Finally, propranolol (PRO) inhibits beta-adrenergic activity and protects type H vessels and bone mass against estrogen deficiency. These findings unravel the specialized neurovascular coupling in bone homeostasis and regeneration.

Comparing the pregnancy outcomes of Re­ICSI and ICSI embryos in fresh ET and FET cycles.

Early rescue intracytoplasmic sperm injection (Re-ICSI) can prevent total fertilization failure (TFF) during conventional in vitro fertilization (IVF). However, the implantation rate of Re-ICSI embryos is lower than that of direct ICSI during fresh embryo transfer (ET). The aim of the present study was to investigate the effect of frozen ET (FET) after Re-ICSI. In the present retrospective study, primary infertility patients that underwent the first Re-ICSI and ICSI treatment, were studied. The clinical pregnancy rate, implantation rate, ectopic pregnancy, abortion rate and live birth rate were analyzed between the Re-ICSI and ICSI groups in fresh ET and FET cycles. The average age of patients between Re-ICSI and ICSI groups in fresh ET and FET cycles was (29.0±3.2 vs. 29.1±3.1, and 29.1±3.3 vs. 28.9±3.0), respectively (P>0.05). Compared with ICSI embryos, the clinical pregnancy, implantation and live birth rates of Re-ICSI embryos were lower in fresh ET cycles. By contrast, there were no significant differences in the pregnancy, implantation and live birth rates between the Re-ICSI and ICSI embryos during the FET cycles. Re-ICSI coupled with FET may overcome the impaired outcomes in fresh ET.

Mesenchymal Stem Cell Aggregation-Released Extracellular Vesicles Induce CD31+ EMCN+ Vessels in Skin Regeneration and Improve Diabetic Wound Healing.

The blood vessel system is essential for skin homeostasis and regeneration. While the heterogeneity of vascular endothelial cells has been emergingly revealed, whether a regeneration-relevant vessel subtype exists in skin remains unknown. Herein, a specialized vasculature in skin featured by simultaneous CD31 and EMCN expression contributing to the regeneration process is identified, the decline of which functionally underlies the impaired angiogenesis of diabetic nonhealing wounds. Moreover, enlightened by the developmental process that mesenchymal condensation induces angiogenesis, it is demonstrated that mesenchymal stem/stromal cell aggregates (CAs) provide an efficacious therapy to enhance regrowth of CD31+ EMCN+ vessels in diabetic wounds, which is surprisingly suppressed by pharmacological inhibition of extracellular vesicle (EV) release. It is further shown that CAs promote secretion of angiogenic protein-enriched EVs by proteomic analysis, which directly exert high efficacy in boosting CD31+ EMCN+ vessels and treating nonhealing diabetic wounds. These results add to the current knowledge on skin vasculature and help establish feasible strategies to benefit wound healing under diabetic condition.

Mesenchymal condensation in tooth development and regeneration: a focus on translational aspects of organogenesis.

The teeth are vertebrate-specific, highly specialized organs performing fundamental functions of mastication and speech, the maintenance of which is crucial for orofacial homeostasis and is further linked to systemic health and human psychosocial well-being. However, with limited ability for self-repair, the teeth can often be impaired by traumatic, inflammatory, and progressive insults, leading to high prevalence of tooth loss and defects worldwide. Regenerative medicine holds the promise to achieve physiological restoration of lost or damaged organs, and in particular an evolving framework of developmental engineering has pioneered functional tooth regeneration by harnessing the odontogenic program. As a key event of tooth morphogenesis, mesenchymal condensation dictates dental tissue formation and patterning through cellular self-organization and signaling interaction with the epithelium, which provides a representative to decipher organogenetic mechanisms and can be leveraged for regenerative purposes. In this review, we summarize how mesenchymal condensation spatiotemporally assembles from dental stem cells (DSCs) and sequentially mediates tooth development. We highlight condensation-mimetic engineering efforts and mechanisms based on ex vivo aggregation of DSCs, which have achieved functionally robust and physiologically relevant tooth regeneration after implantation in animals and in humans. The discussion of this aspect will add to the knowledge of development-inspired tissue engineering strategies and will offer benefits to propel clinical organ regeneration.

Proteomic analysis identifies Stomatin as a biological marker for psychological stress.

Psychological stress emerges to be a common health burden in the current society for its highly related risk of mental and physical disease outcomes. However, how the quickly-adaptive stress response process connects to the long-observed organismal alterations still remains unclear. Here, we investigated the profile of circulatory extracellular vesicles (EVs) after acute stress (AS) of restraint mice by phenotypic and proteomic analyses. We surprisingly discovered that AS-EVs demonstrated significant changes in size distribution and plasma concentration compared to control group (CN) EVs. AS-EVs were further characterized by various differentially expressed proteins (DEPs) closely associated with biological, metabolic and immune regulations and were functionally important in potentially underlying multiple diseases. Notably, we first identified the lipid raft protein Stomatin as an essential biomarker expressed on the surface of AS-EVs. These findings collectively reveal that EVs are a significant function-related liquid biopsy indicator that mediate circulation alterations impinged by psychological stress, while also supporting the idea that psychological stress-associated EV-stomatin can be used as a biomarker for potentially predicting acute stress responses and monitoring psychological status. Our study will pave an avenue for implementing routine plasma EV-based theranostics in the clinic.

Pregnancy outcome and follow-up of offspring of donor oocytes recipient from PCOS patients.

BACKGROUND: The use of donated oocytes (DO) for in vitro fertilization(IVF) treatment in patients with infertility is generally recognized, and females with polycystic ovarian syndrome (PCOS) can participate in oocyte donation programs as donor patients. However, the pregnancy outcomes and offspring follow-up in patients with PCOS as the recipients are unclear. This study was to compare the pregnancy outcomes and follow-up of offspring in PCOS and non-PCOS receptor. METHODS: This was a retrospective cohort study of 62 patients undergoing the oocyte reception program were separated into 2 groups: Group I, PCOS oocyte recipients (n = 30); Group II, non-PCOS recipients (n = 32). Medical records were reviewed, and rates of fertilization, cleavage, high-quality embryos and blastocysts were compared between PCOS and non-PCOS groups. Rates of implantation, pregnancy, ectopic pregnancy, early abortion, multiple pregnancies, and offspring outcomes were calculated using the first single vitrified-warmed blastocyst transfer (SVBT) analysis between the groups. RESULTS: The average recipient age and body mass index (BMI) of PCOS and non-PCOS patients was (36.3 ± 2.6 vs. 36.2 ± 2.8, and 23.4 ± 3.9 vs. 23.7 ± 4.0), respectively (P > 0.05). The fertilization, cleavage, high-quality embryos and blastocyst rates were not significantly different between the PCOS and non-PCOS groups. Rates of implantation, pregnancy, ectopic pregnancy, early abortion, and multiple pregnancies were not significantly different in SVBT between the PCOS and non-PCOS groups. The incidence of complications, such as pre-eclampsia or gestational diabetes, between PCOS and non-PCOS groups was similar (11.8% vs.11.1%, 5.9% vs.5.5%; P > 0.05). Preterm births were also similar (11.8% vs.16.7%, P > 0.05). Donor oocytes are more likely to be delivered via cesarean Sect. (80.0% vs. 86.7%: P > 0.05). The mean gestational age, birth weight, and height were comparable between the 2 groups during full-term delivery. CONCLUSION: There was no difference in the pregnancy outcomes and follow-up of the offspring between the PCOS and non-PCOS groups.

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells.

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

Optimized immunofluorescence staining protocol for identifying resident mesenchymal stem cells in bone using LacZ transgenic mice.

Glioma-associated oncogene homolog 1 (Gli1) marks a subpopulation of endogenous mesenchymal stem cells (MSCs) characterized by perivascular location. Here, we present an optimized immunofluorescence staining protocol to identify resident Gli1+ MSCs in fixed/frozen bone sections from LacZ transgenic mice. This protocol describes the preparation of fixed/frozen tissue sections and the use of LacZ immunofluorescent staining for the in vivo characterization of endogenous MSCs, regarding their specific identity and specialized niches, and is applicable to LacZ-expressing cells of diverse organs. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).

Case report: Analysis of novel compound heterozygous TPP1 variants in a Chinese patient with neuronal ceroid lipofuscinosis type 2.

Neuronal ceroid lipofuscinosis type 2 (CLN2) is an autosomal recessive neurodegenerative disease caused by variants in the TPP1 gene that lead to the deficiency of the lysosomal enzyme tripeptidyl peptidase I (TPP1) activity. Herein, we report a rare case of CLN2 caused by two novel variants of TPP1. The patient presented with seizures at onset, followed by progressive cognitive impairment, motor decline, and vision loss. Novel compound heterozygous variants, c.544_545del and c.230-3C>G, in TPP1 were identified by whole-exome sequencing. The variant assessment showed that the c.544_545del is a frameshift variant mediating mRNA decay and that c.230-3C>G is a splice variant generating aberrantly spliced TPP1 mRNA, as confirmed by a Splicing Reporter Minigene assay. In conclusion, clinical history, variant assessment, and molecular analyses demonstrate that the novel compound heterozygous variants are responsible for CLN2 disease in this patient. This study expands the mutation spectrum of TPP1.

Erratum: Resveratrol counteracts bone loss via mitofilin-mediated osteogenic improvement of mesenchymal stem cells in senescence-accelerated mice: Erratum.

[This corrects the article DOI: 10.7150/thno.23620.].

Mesenchymal stem cell-derived exosomes: A novel and potential remedy for cutaneous wound healing and regeneration.

Poor healing of cutaneous wounds is a common medical problem in the field of traumatology. Due to the intricate pathophysiological processes of wound healing, the use of conventional treatment methods, such as chemical molecule drugs and traditional dressings, have been unable to achieve satisfactory outcomes. Within recent years, explicit evidence suggests that mesenchymal stem cells (MSCs) have great therapeutic potentials on skin wound healing and regeneration. However, the direct application of MSCs still faces many challenges and difficulties. Intriguingly, exosomes as cell-secreted granular vesicles with a lipid bilayer membrane structure and containing specific components from the source cells may emerge to be excellent substitutes for MSCs. Exosomes derived from MSCs (MSC-exosomes) have been demonstrated to be beneficial for cutaneous wound healing and accelerate the process through a variety of mechanisms. These mechanisms include alleviating inflammation, promoting vascularization, and promoting proliferation and migration of epithelial cells and fibroblasts. Therefore, the application of MSC-exosomes may be a promising alternative to cell therapy in the treatment of cutaneous wounds and could promote wound healing through multiple mechanisms simultaneously. This review will provide an overview of the role and the mechanisms of MSC-derived exosomes in cutaneous wound healing, and elaborate the potentials and future perspectives of MSC-exosomes application in clinical practice.

Frozen blastocysts: Assessing the importance of day 5/day 6 blastocysts or blastocyst quality.

The aim of the present study was to analyze the high-quality blastocyst (HB) rate in all embryo frozen cycles and investigate the pregnancy outcomes for day 5/day 6 (D5/D6) blastocysts with respect to the blastocyst quality in programmed single vitrified-warmed blastocyst transfer (SVBT). We performed a retrospective study comparing D5/D6 HBs in in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) for all blastocyst frozen cycles. Patients were <35 years at the oocyte collection in their first fresh cycle without fresh transfer. A total of 1,560 IVF/ICSI cycles and 5,328 blastocysts were analyzed. The IVF HB rate was higher than that of ICSI (52.7% vs. 42.6%; P<0.05). The D5 HB rate was much higher than the D6 HB rate (61.6% vs. 29.4%; P<0.05). There were 22.4% (349/1,560) cycles that only had D6 blastocysts, of which IVF cycles were lower than ICSI (19.8% vs. 28.5%; P<0.05). The clinical pregnancy rate and implantation rate in the D5 group were significantly higher than these rates in the D6 group (57.4% vs. 46.2%, 58.9% vs. 47.3%; P<0.05). However, the clinical pregnancy rate and implantation rate of the D5 HBs were not significantly different from those of the D6 HBs (60% vs. 54.5%, 62% vs. 56.3%; P>0.05). In conclusion, the fertilization method (IVF/ICSI) directly influences the HB rate and blastocyst development rates. When we controlled for patient age, transfer frequency, and endometrium on day 5, it was not the development stage (D5/D6), rather the transfer blastocyst quality that played an important role in pregnancy outcomes.

Sympathetic Neurostress Drives Osteoblastic Exosomal MiR-21 Transfer to Disrupt Bone Homeostasis and Promote Osteopenia.

Innervation and extracellular vesicle secretion co-exist in the local tissue microenvironment for message transfer, but whether they are interconnected to regulate organ homeostasis remains unknown. Sympatho-adrenergic activation is implicated in stress-induced depression and leads to bone loss, but the mechanisms and therapeutics are incompletely elucidated. Here, it is revealed that sympathetic neurostress through the ß1/2 -adrenergic receptor (ß1/2-AR) signaling triggers the transcription response of a microRNA, miR-21, in osteoblasts, which is transferred to osteoclast progenitors via exosomes for dictating osteoclastogenesis. After confirming that miR-21 deficiency retards the ß1/2-AR agonist isoproterenol (ISO)-induced osteopenia, it is shown that the pharmacological inhibition of exosome release by two clinically-relevant drugs, dimethyl amiloride and omeprazole, suppresses osteoblastic miR-21 transfer and ameliorates bone loss under both ISO and chronic variable stress (CVS)-induced depression conditions. A targeted delivery approach to specifically silence osteoblastic miR-21 is further applied, which is effective in rescuing the bone remodeling balance and ameliorating ISO- and CVS-induced osteopenias. These results decipher a previously unrecognized paradigm that neural cues drive exosomal microRNA communication to regulate organ homeostasis and help to establish feasible strategies to counteract bone loss under psychological stresses.

Glutamine as a Potential Noninvasive Biomarker for Human Embryo Selection.

To determine whether glutamine consumption is associated with embryo quality and aneuploidy, a retrospective study was conducted in an in vitro fertilization center. Spent embryo culture media from patients undergoing assisted reproduction treatment and preimplantation genetic testing (PGT) were obtained on day 3 of in vitro culture. Embryo quality was assessed for cell number and fragmentation rate. PGT for aneuploidy was performed using whole genome amplification and DNA sequencing. Glutamine levels in spent embryo culture media were analyzed by gas chromatography-mass spectrometry. The results demonstrated that glutamine was a primary contributor to the classification of the good-quality and poor-quality embryos based on the orthogonal partial least-squares discriminant analysis model. Glutamine consumption in the poor-quality embryos was significantly higher than that in the good-quality embryos (P < 0.05). A significant increase in glutamine consumption was observed from aneuploid embryos compared with that from euploid embryos (P < 0.01). The Pearson correlation coefficients between embryo quality and glutamine consumption, and between aneuploidy and glutamine consumption, were 0.430 and 0.757, respectively. The area under the ROC curve was 0.938 (95% CI: 0.902-0.975) for identifying aneuploidy. Animal experiments demonstrate that increased glutamine consumption may be a compensatory mechanism to mitigate oxidative stress. Our data suggest that glutamine consumption is associated with embryo quality and aneuploidy. Glutamine may serve as a molecular indicator for embryo assessment and aneuploidy testing.

Glycemic control by umbilical cord-derived mesenchymal stem cells promotes effects of fasting-mimicking diet on type 2 diabetic mice.

BACKGROUND: Hepatic steatosis is a big hurdle to treat type 2 diabetes (T2D). Fasting-mimicking diet (FMD) has been shown to be an effective intervention in dyslipidemia of T2D. However, fasting may impair the normal glucose metabolism. Human umbilical cord-derived mesenchymal stem cell (UC-MSC) transplantation has been discovered to regulate immune reactions and reduce hyperglycemia in diabetes. However, the effect of UC-MSCs on improving the lipid metabolism disorder is not quite satisfactory. We have investigated the efficacy comparison and interaction between FMD and UC-MSC infusion, aiming to establish effective T2D therapies and explore its mechanism. METHODS: C57/BL6 mice were fed with high-fat diet (HFD) to induce a diet-induced obese (DIO) mouse model. Leptin receptor-deficient (db/db) mice were used for follow-up experiments. DIO or db/db mice were divided into 4 groups: phosphate buffer saline (PBS), UC-MSCs, FMD, and UC-MSCs + FMD. At the end of the study period, mice were fasted and sacrificed, with the measurement of physiological and biochemical indexes. In addition, the fresh liver, skin, and white adipose tissue were analyzed by histology. RESULTS: FMD restored the lipid metabolism in DIO mice, whereas its capacity to rescue hyperglycemia was uncertain. Infusion of UC-MSCs was effective in T2D glycemic control but the impact on dyslipidemia was insufficient. Furthermore, both the glucose and the lipid alterations of DIO and db/db mice recovered after UC-MSCs combined with FMD. It was proved that UC-MSCs promoted FMD effects on ameliorating hyperglycemia and restoring the lipid metabolism in T2D mice, while FMD had little promotion effect on UC-MSCs. Mechanistically, we discovered that UC-MSC infusion significantly modulated systematic inflammatory microenvironment, which contributed to concerted actions with FMD. CONCLUSIONS: We established a strategy that combined UC-MSC infusion and FMD and was effective in treating T2D, which provided potential approaches for developing novel clinical T2D therapies.

Lower growth arrest-specific 5 level in endometrium is related to endometriosis via promoting cell proliferation and angiogenesis.

Long noncoding RNAs are a group of more than 200 nt, nonprotein coding RNAs, some of which are dysregulated in many pathophysiological processes including endometriosis. This study aims to clarify the roles of dysregulated growth arrest-specific 5 (GAS5) in patients with endometriosis, and unveil the underlying mechanisms. We obtained endometrium samples from 37 patients with endometriosis and 23 controls without endometriosis. Primary endometrial stromal cells (ESCs) and endothelial cells were separated from the endometrium. Levels of GAS5 were quantified using quantitative real-time polymerase chain reaction, and levels of p27, cleaved caspase-3, cleaved poly (ADP-Ribose) polymerase 1, vascular endothelial growth factor A, tissue inhibitor of metalloproteinases 3 (TIMP3), and trypsin-modified soy protein 10 were assessed by immunoblotting. Cell viability was examined using MTT assays, and the cell cycle and apoptosis were analyzed by flow cytometry. Endothelial cell tube formation capacity was assayed in vitro. GAS5 and p27 levels were found lower in the endometrium samples from patients with endometriosis. Primary ESCs from patients with endometriosis had increased viability, reduced apoptosis, and a relatively uncontrolled cell cycle. Gain- and loss-of-function studies confirmed that GAS5 regulated p27 expression in ESCs. Furthermore, GAS5 level was relatively low in primary endothelial cells from patients with endometriosis and GAS5 acted as an angiogenesis inhibitor by regulating the miR-181c-TIMP3 axis. Thus, lower GAS5 level in endometrium might be related to endometriosis by regulating cell proliferation, apoptosis, cell cycle, and angiogenesis.