Ovarian overexpression of ASMT gene increases follicle numbers in transgenic sheep: Association with lipid metabolism.

Melatonin plays an important role in mammalian reproductive activities, to further understand the effects of endogenous melatonin on functions of ovary, the transgenic sheep with overexpression of melatonin synthetic enzyme gene ASMT in ovary were generated. The results showed that total melatonin content in follicular fluid of transgenic sheep was significantly greater than that in the wild type. Accordingly, the follicle numbers of transgenic sheep were also significantly greater than those in the WT. The results of follicular fluid metabolites sequencing showed that compared with WT, the differential metabolites of the transgenic sheep were significantly enriched in several signaling pathways, the largest number of metabolites was lipid metabolism pathway and the main differential metabolites were lipids and lipoid molecules. SMART-seq2 were used to analyze the oocytes and granulosa cells of transgenic sheep and WT sheep. The main differential enrichment pathway was metabolic pathway, in which lipid metabolism genes accounted for the majority. In conclusion, this is the first report to show that ovary overexpression of ASMT increased local melatonin production and follicle numbers. These results may imply that ASMT plays an important role in follicle development and formation, and melatonin intervention may be a potential method to promote this process.

Melatonin improves the vitrification of sheep morulae by modulating transcriptome.

Embryo vitrification technology is widely used in livestock production, but freezing injury has been a key factor hindering the efficiency of embryo production. There is an urgent need to further analyze the molecular mechanism of embryo damage by the vitrification process. In the study, morulae were collected from Hu sheep uterine horns after superovulation and sperm transfusion. Morulae were Cryotop vitrified and warmed. Nine morulae were in the vitrified control group (frozen), and seven morulae were vitrified and warmed with 10-5 M melatonin (melatonin). Eleven non-frozen morulae were used as controls (fresh). After warming, each embryo was sequenced separately for library construction and gene expression analysis. p < 0.05 was used to differentiate differentially expressed genes (DEG). The results showed that differentiated differentially expressed genes (DEG) in vitrified morulae were mainly enriched in protein kinase activity, adhesion processes, calcium signaling pathways and Wnt, PI3K/AKT, Ras, ErbB, and MAPK signaling pathways compared to controls. Importantly, melatonin treatment upregulated the expression of key pathways that increase the resistance of morulae against vitrification induced damage. These pathways include kinase activity pathway, ErbB, and PI3K/Akt signaling pathway. It is worth mentioning that melatonin upregulates the expression of XPA, which is a key transcription factor for DNA repair. In conclusion, vitrification affected the transcriptome of in vivo-derived Hu sheep morulae, and melatonin had a protective effect on the vitrification process. For the first time, the transcriptome profiles caused by vitrification and melatonin in sheep morulae were analyzed in single embryo level. These data obtained from the single embryo level provide an important molecular mechanism for further optimizing the cryopreservation of embryos or other cells.

Effects of melatonin on rumen microorganisms and methane production in dairy cow: results from in vitro and in vivo studies.

BACKGROUND: Methane (CH4) is a major greenhouse gas, and ruminants are one of the sources of CH4 which is produced by the rumen microbiota. Modification of the rumen microbiota compositions will impact the CH4 production. In this study, the effects of melatonin on methane production in cows were investigated both in the in vitro and in vivo studies. RESULTS: Melatonin treatment significantly reduced methane production in both studies. The cows treated with melatonin reduced methane emission from their respiration by approximately 50%. The potential mechanisms are multiple. First, melatonin lowers the volatile fatty acids (VFAs) production in rumen and reduces the raw material for CH4 synthesis. Second, melatonin not only reduces the abundance of Methanobacterium which are responsible for generating methane but also inhibits the populations of protozoa to break the symbiotic relationship between Methanobacterium and protozoa in rumen to further lowers the CH4 production. The reduced VFA production is not associated with food intake, and it seems also not to jeopardize the nutritional status of the cows. This was reflected by the increased milk lipid and protein contents in melatonin treated compared to the control cows. It is likely that the energy used to synthesize methane is saved to compensate the reduced VFA production. CONCLUSION: This study enlightens the potential mechanisms by which melatonin reduces rumen methane production in dairy cows. Considering the greenhouse effects of methane on global warming, these findings provide valuable information using different approaches to achieve low carbon dairy farming to reduce the methane emission. Video Abstract.

Theoretical Approach and Scale Construction of Patient Privacy Protection Behavior of Doctors in Public Medical Institutions in China: Pilot Development Study.

BACKGROUND: Considering the high incidence of medical privacy disclosure, it is of vital importance to study doctors' privacy protection behavior and its influencing factors. OBJECTIVE: We aim to develop a scale for doctors' protection of patients' privacy in Chinese public medical institutions, following construction of a theoretical model framework through grounded theory, and subsequently to validate the scale to measure this protection behavior. METHODS: Combined with the theoretical paradigm of protection motivation theory (PMT) and semistructured interview data, the grounded theory research method, followed by the Delphi expert and group discussion methods, a theoretical framework and initial scale for doctors in Chinese public medical institutions to protect patients' privacy was formed. The adjusted scale was collected online using a WeChat electronic survey measured using a 5-point Likert scale. Exploratory and confirmatory factor analysis (EFA and CFA) and tests to analyze reliability and validity were performed on the sample data. SPSS 19.0 and Amos 26.0 statistical analysis software were used for EFA and CFA of the sample data, respectively. RESULTS: According to the internal logic of PMT, we developed a novel theoretical framework of a "storyline," which was a process from being unaware of patients' privacy to having privacy protection behavior, that affected doctors' cognitive intermediary and changed the development of doctors' awareness, finally affecting actual privacy protection behavior in Chinese public medical institutions. Ultimately, we created a scale to measure 18 variables in the theoretical model, comprising 63 measurement items, with a total of 208 doctors participating in the scaling survey, who were predominantly educated to the master's degree level (n=151, 72.6%). The department distribution was relatively balanced. Prior to EFA, the Kaiser-Meyer-Olkin (KMO) value was 0.702, indicating that the study was suitable for factor analysis. The minimum value of Cronbach α for each study variable was .754, which met the internal consistency requirements of the scale. The standard factor loading value of each potential measurement item in CFA had scores greater than 0.5, which signified that all the items in the scale could effectively converge to the corresponding potential variables. CONCLUSIONS: The theoretical framework and scale to assess doctors' patient protection behavior in public medical institutions in China fills a significant gap in the literature and can be used to further the current knowledge of physicians' thought processes and adoption decisions.

Melatonin promotes the development of sheep transgenic cloned embryos by protecting donor and recipient cells.

The yield efficiency of transgenic animal generation is relatively low[1]. To improve its efficiency has become a priority task for researchers[2]. Melatonin (N-acetyl-5-methoxytryptamine, MT) is a potent-free radical scavenger and antioxidant to protect mitochondria, lipids, protein and DNA from oxidative stress[3]. In this study, we observed that improving the quality of both donor and recipient cells by giving physiological concentration (10-7 M) of MT significantly increase the sheep transgenic embryo development in the in vitro condition. MT promotes the donor cell viability, proliferation, efficiency of monoclonal formation and the electrotransferring efficiency of fetal fibroblast cells (FFCs). The mechanistic exploration indicates that MT has the capacity for the synchronization of cell division cycle, reduction of cellular oxidative stress, apoptosis, and the increase of mitochondrial number and function. All of these render MT's ability to increase the efficiency of animal transgenic processes such as somatic cell nuclear transfer (SCNT) and electroporation. The outcomes are the increased cleavage rate and blastocyst rate of the transgenic sheep embryos after MT treatment. These beneficial effects of MT on transgenic embryo development are worth to be tested in the in vivo condition in the future.

The Improved Milk Quality and Enhanced Anti-Inflammatory Effect in Acetylserotonin-O-methyltransferase (ASMT) Overexpressed Goats: An Association with the Elevated Endogenous Melatonin Production.

BACKGROUND: Transgenic animal production is an important means of livestock breeding and can be used to model pharmaceutical applications. METHODS: In this study, to explore the biological activity of endogenously produced melatonin, Acetylserotonin-O-methyltransferase (ASMT)-overexpressed melatonin-enriched dairy goats were successfully generated through the use of pBC1-ASMT expression vector construction and prokaryotic embryo microinjection. RESULTS: These transgenic goats have the same normal phenotype as the wild-type goats (WT). However, the melatonin levels in their blood and milk were significantly increased (p < 0.05). In addition, the quality of their milk was also improved, showing elevated protein content and a reduced somatic cell number compared to the WT goats. No significant changes were detected in the intestinal microbiota patterns between groups. When the animals were challenged by the intravenous injection of E. coli, the ASMT-overexpressed goats had a lower level of pro-inflammatory cytokines and higher anti-inflammatory cytokines compared to the WT goats. Metabolic analysis uncovered a unique arachidonic acid metabolism pattern in transgenic goats. CONCLUSIONS: The increased melatonin production due to ASMT overexpression in the transgenic goats may have contributed to their improved milk quality and enhanced the anti-inflammatory ability compared to the WT goats.

Lactitol Supplementation Modulates Intestinal Microbiome in Liver Cirrhotic Patients.

Background: Cirrhosis is a common chronic liver disease characterized by irreversible diffuse liver damage. Intestinal microbiome dysbiosis and metabolite dysfunction contribute to the development of cirrhosis. Lactitol (4-ß-D-galactopyranosyl-D-glucitol) was previously reported to promote the growth of intestinal Bifidobacteria. However, the effect of lactitol on the intestinal microbiome and fecal short-chain fatty acids (SCFAs) and bile acids (BAs) and the interactions among these factors in cirrhotic patients pre- and post-lactitol treatment remain poorly understood. Methods: Here, using shotgun metagenomics and targeted metabolomics methods. Results: we found that health-promoting lactic acid bacteria, including Bifidobacterium longum, B.pseudocatenulatum, and Lactobacillus salivarius, were increased after lactitol intervention, and significant decrease of pathogen Klebsiella pneumonia and associated antibiotic resistant genes /virulence factors. Functionally, pathways including Pseudomonas aeruginosa biofilm formation, endotoxin biosynthesis, and horizontal transfer of pathogenic genes were decreased in cirrhotic patients after 4-week lactitol intervention compared with before treatment. Conclusion: We identified lactitol-associated metagenomic changes, and provide insight into the understanding of the roles of lactitol in modulating gut microbiome in cirrhotic patients.

Overexpression of ASMT likely enhances the resistance of transgenic sheep to brucellosis by influencing immune-related signaling pathways and gut microbiota.

Melatonin is a pleiotropic molecule with a variety of biological functions, which include its immunoregulatory action in mammals. Brucellosis is a worldwide endemic zoonotic disease caused by the Brucella, which not only causes huge economic losses for the livestock industry but also impacts human health. To target this problem, in current study, two marker-free transgenic sheep overexpressing melatonin synthetic enzyme ASMT (acetylserotonin O-methyltransferase) gene were generated and these melatonin enrich transgenic sheep were challenged by Brucella infection. The results showed that the serum melatonin concentration was significantly higher in transgenic sheep than that of wild type (726.92 ± 70.6074 vs 263.10 ± 34.60 pg/mL, P < .05). Brucella challenge test showed that two thirds (4/6) of the wild-type sheep had brucellosis, while none of the transgenic sheep were infected. Whole-blood RNA-seq results showed that differential expression genes (DEGs) were significantly enriched in natural killer cell-mediated cytotoxicity, phagosome, antigen processing, and presentation signaling pathways in overexpression sheep. The DEGs of toll-like receptors (TLRs) and NOD-like receptors (NLRs) families were verified by qPCR and it showed that TLR1, TLR2, TLR7, CD14, NAIP, and CXCL8 expression levels in overexpression sheep were significantly higher and NLRP1, NLRP3, and TNF expression levels were significantly lower than those of wild type. The rectal feces were subjected to 16S rDNA amplicon sequencing, and the microbial functional analysis showed that the transgenic sheep had significantly lower abundance of microbial genes related to infectious diseases compared to the wild type, indicating overexpression animals are likely more resistant to infectious diseases than wild type. Furthermore, exogenous melatonin treatment relieved brucellosis inflammation by upregulating anti-inflammatory cytokines IL-4 and downregulating pro-inflammatory IL-2, IL-6, and IFN-γ. Our preliminary results provide an informative reference for the study of the relationship between melatonin and brucellosis.

SLC39A5 dysfunction impairs extracellular matrix synthesis in high myopia pathogenesis.

High myopia is one of the leading causes of visual impairment worldwide with high heritability. We have previously identified the genetic contribution of SLC39A5 to nonsyndromic high myopia and demonstrated that disease-related mutations of SLC39A5 dysregulate the TGF-ß pathway. In this study, the mechanisms underlying SLC39A5 involvement in the pathogenesis of high myopia are determined. We observed the morphogenesis and migration abnormalities of the SLC39A5 knockout (KO) human embryonic kidney cells (HEK293) and found a significant injury of ECM constituents. RNA-seq and qRT-PCR revealed the transcription decrease in COL1A1, COL2A1, COL4A1, FN1 and LAMA1 in the KO cells. Further, we demonstrated that TGF-ß signalling, the regulator of ECM, was inhibited in SLC39A5 depletion situation, wherein the activation of receptor Smads (R-Smads) via phosphorylation was greatly blocked. SLC39A5 re-expression reversed the phenotype of TGF-ß signalling and ECM synthesis in the KO cells. The fact that TGF-ß signalling was zinc-regulated and that SLC39A5 was identified as a zinc transporter urged us to check the involvement of intracellular zinc in TGF-ß signalling impairment. Finally, we determined that insufficient zinc chelation destabilized Smad proteins, which naturally inhibited TGF-ß signalling. Overall, the SLC39A5 depletion-induced zinc deficiency destabilized Smad proteins, which inhibited the TGF-ß signalling and downstream ECM synthesis, thus contributing to the pathogenesis of high myopia. This discovery provides a deep insight into myopic development.

Microbiome-miRNA interactions in the progress from undifferentiated arthritis to rheumatoid arthritis: evidence, hypotheses, and opportunities.

The human microbiome has attracted attention for its potential utility in precision medicine. Increasingly, more researchers are recognizing changes in intestinal microbiome can upset the balance between pro- and anti-inflammatory factors of host immune system, potentially contributing to arthritis immunopathogenesis. Patients who develop rheumatoid arthritis from undifferentiated arthritis can face multiple irreversible joint lesions and even deformities. Strategies for identifying undifferentiated arthritis patients who have a tendency to develop rheumatoid arthritis and interventions to prevent rheumatoid arthritis development are urgently needed. Intestinal microbiome dysbiosis and shifts in the miRNA profile affect undifferentiated arthritis progression, and may play an important role in rheumatoid arthritis pathophysiologic process via stimulating inflammatory cytokines and disturbing host and microbial metabolic functions. However, a causal relationship between microbiome-miRNA interactions and rheumatoid arthritis development from undifferentiated arthritis has not been uncovered yet. Changes in the intestinal microbiome and miRNA profiles of undifferentiated arthritis patients with different disease outcomes should be studied together to uncover the role of the intestinal microbiome in rheumatoid arthritis development and to identify potential prognostic indicators of rheumatoid arthritis in undifferentiated arthritis patients. Herein, we discuss the possibility of microbiome-miRNA interactions contributing to rheumatoid arthritis development and describe the gaps in knowledge regarding their influence on undifferentiated arthritis prognosis that should be addressed by future studies.

Effect of Optimization of TiO2 Electron Transport Layer on Performance of Perovskite Solar Cells with Rough FTO Substrates.

The film quality of the electron transport layer (ETL) plays an important role in improving the performance of perovskite solar cells (PSCs). In order to reduce the effect of rough fluorine-doped SnO2 (FTO)substrate on the film quality of the TiO2 ETL, multiple cycles of spin-coating were employed to realize optimized TiO2 film and improve the performance of PSCs with rough FTO. The results show that TiO2 ETL was optimized most effectively using two spin-coating cycles, obtaining the best performance of PSCs with rough FTO. The carbon electrode-based PSCs were then demonstrated. Our work discusses the feasibility of low-quality rough FTO for the fabrication of PSCs and photodetectors to reduce costs.

Investigation on Low-temperature Annealing Process of Solution-processed TiO2 Electron Transport Layer for Flexible Perovskite Solar Cell.

Flexible perovskite solar cells (PSCs) have received increasing attention in wearable and portable devices over the past ten years. The low-temperature process of electron transport layer plays a key role in fabricating flexible PSCs. In this paper, we improve the performance of flexible PSCs by controlling the thermodynamic procedure in the low-temperature annealing process of solution-processed TiO2 layers and modulating the precursor concentration of (6,6)-phenyl c61 butyric acid methyl ester (PC61BM) deposited on fluorine-doped tin oxide (FTO)/TiO2 substrate. The results show that slowing down evaporation rate of residual solvent and adopting PC61BM of appropriate precursor concentration are confirmed to be effective methods to improve the performance of flexible PSCs. We also demonstrate carbon electrode-based flexible PSCs. Our work expands the feasibility of low temperature process for the development of flexible perovskite photodetectors and light-emitting diodes, as well as flexible PSCs.

Impact of Perovskite Composition on Film Formation Quality and Photophysical Properties for Flexible Perovskite Solar Cells.

In recent years, flexible perovskite solar cells have drawn tremendous attention in the field of wearable devices, and optimization of perovskite composition plays an important role in improving film quality and photophysical properties. At present, some researchers have only studied A-site organic cations mixing or X-site halide anions mixing in the ABX3 structure of perovskite, but there are few reports on co-mixing of A-site and X-site ions in flexible perovskite solar cells. In this paper, we mainly try to study the effects of different concentrations of mixed formamidine methylamine halide (FAxMA1-xBrxClyI1-x-y) precursor solutions on the quality and photophysical properties of perovskite films under low temperature process. We conclude that the film quality and photophysical properties reached the best results when the optimized precursor solution concentration was 60:6:6. The investigation on composition optimization in this experiment laid the foundation for the improvement of the performance of flexible perovskite solar cells. We also use the results of this experiment to prepare flexible perovskite solar cells based on carbon electrodes, which are expected to be applied in other flexible optoelectronic or electro-optical devices.

Applied Trace Alkali Metal Elements for Semiconductor Property Modulation of Perovskite Thin Films.

With the rapid consumption of energy, clean solar energy has become a key study and development subject, especially the when new renewable energy perovskite solar cells (PSCs) are involved. The doping method is a common means to modulate the properties of perovskite film. The main work of this paper is to incorporate trace amounts of alkali metal elements into the perovskite layer and observe the effects on the properties of the perovskite device and the majority carrier type of the perovskite film. Comparative analysis was performed by doping with Na+, K+, and Rb+ or using undoped devices in the perovskite layer. The results show that the incorporation of alkali metal ions into the perovskite layer has an important effect on the majority carrier type of the perovskite film. The majority carrier type of the undoped perovskite layer is N-type, and the majority carrier type of the perovskite layer doped with the alkali metal element is P-type. The carrier concentration of perovskite films is increased by at least two orders of magnitude after doping. That is to say, we can control the majority of the carrier type of the perovskite layer by controlling the doping subjectively. This will provide strong support for the development of future homojunction perovskite solar cells. This is of great help to improve the performance of PSC devices.

Influence of Solution Deposition Process on Modulating Majority Charge Carrier Type and Quality of Perovskite Thin Films for Solar Cells.

In the past ten years, extensive research has witnessed the rapid development of perovskite solar cells (PSCs) and diversified preparation processing craft. At present, the most widely used methods of preparing perovskite solar cells are the one-step method and the two-step method. The main work of this paper is to study the effect of the solution deposition process on the quality of perovskite thin films, as well as modulating majority charge carrier types. Perovskite film was prepared in air by designing different processes, which were then adequately analyzed with corresponding methods. It was demonstrated that the preparation process plays a crucial role in modulating the type of majority carrier and in achieving high-quality perovskite thin film. The one-step prepared perovskite layer is enriched in MA+, leading to a P type majority carrier type thin film. The two-step prepared perovskite layer is enriched in Pb2+, leading to a N type majority carrier type thin film. In addition, we found that the one-step method caused PbI2 residue due to component segregation, which seriously affects the interface and film quality of the perovskite layer. This work aims to modulate the majority carrier type of perovskite film through different preparation processes, which can lay the foundation for the study of homojunction perovskite solar cells to improve the device performance of PSCs.

Melatonin regulates the activities of ovary and delays the fertility decline in female animals via MT1/AMPK pathway.

Female fertility irreversibly declines with aging, and this is primarily associated with the decreased quality and quantity of oocytes. To evaluate whether a long-term of melatonin treatment would improve the fertility of aged mice, different concentrations of melatonin (10-3 , 10-5 , 10-7  mol/L) were supplemented into drinking water. Melatonin treatments improved the litter sizes of mice at the age of 24 weeks. Mice treated with 10-5  mol/L melatonin had the largest litter size among other concentrations. At this optimal concentration, melatonin not only significantly increased the total number of oocytes but also their quality, having more oocytes with normal morphology that could generate more blastocyst after in vitro fertilization in melatonin (10-5  mol/L)-treated group than that in the controls. When these blastocysts were transferred to recipients, the litter size was also significantly larger in melatonin treated mice than that in controls. The increases in TAOC and SOD level and decreases in MDA were detected in ovaries and uterus from melatonin-treated mice compared to the controls. Melatonin reduced ROS level and maintained mitochondrial membrane potential in the oocytes cultured in vitro. Mechanistically studies revealed that the beneficial effects of melatonin on oocytes were mediated by MT1 receptor and AMPK pathway. Thereafter, MT1 knocking out (MT1-KO) were generated and shown significantly reduced number of oocytes and litter size. The expression of SIRT1, C-myc, and CHOP were downregulated in the ovary of MT1-KO mice, but SIRT1 and p-NF-kB protein level were elevated in response to disturbed redox balance. The results have convincingly proven that melatonin administration delays ovary aging and improves fertility in mice via MT1/AMPK pathway.

Responses of Transgenic Melatonin-Enriched Goats on LPS Stimulation and the Proteogenomic Profiles of Their PBMCs.

The anti-inflammatory activity of melatonin (MT) has been well documented; however, little is known regarding endogenously occurring MT in this respect, especially for large animals. In the current study, we created a MT-enriched animal model (goats) overexpressing the MT synthetase gene Aanat. The responses of these animals to lipopolysaccharide (LPS) stimulation were systematically studied. It was found that LPS treatment exacerbated the inflammatory response in wild-type (WT) goats and increased their temperature to 40 °C. In addition, their granulocyte counts were also significantly elevated. In contrast, these symptoms were not observed in transgenic goats with LPS treatment. The rescue study with MT injection into WT goats who were treated with LPS confirmed that the protective effects in transgenic goats against LPS were attributed to a high level of endogenously produced MT. The proteomic analysis in the peripheral blood mononuclear cells (PBMCs) isolated from the transgenic animals uncovered several potential mechanisms. MT suppressed the lysosome formation as well as its function by downregulation of the lysosome-associated genes Lysosome-associated membrane protein 2 (LAMP2), Insulin-like growth factor 2 receptor (IGF2R), and Arylsulfatase B (ARSB). A high level of MT enhanced the antioxidant capacity of these cells to reduce the cell apoptosis induced by the LPS. In addition, the results also uncovered previously unknown information that showed that MT may have protective effects on some human diseases, including tuberculosis, bladder cancer, and rheumatoid arthritis, by downregulation of these disease-associated genes. All these observations warranted further investigations.