Salinity Gradient Controls Microbial Community Structure and Assembly in Coastal Solar Salterns.

Salinity acts as a critical environmental filter on microbial communities in natural systems, negatively affecting microbial diversity. However, how salinity affects microbial community assembly remains unclear. This study used Wendeng multi-pond saltern as a model to evaluate the prokaryotic community composition and diversity and quantify the relative importance of ecological processes across salinity gradients. The results showed that low-saline salterns (45-80 g/L) exhibited higher bacterial diversity than high-saline salterns (175-265 g/L). The relative abundance of taxa assigned to Halomicrobiaceae, Rhodobacteraceae, Saprospiraceae, and Thiotrichaceae exhibited a hump-shaped dependence on increasing salinity. Salinity and pH were the primary environmental factors that directly or indirectly determined the composition and diversity of prokaryotic communities. Microbial co-occurrence network dynamics were more complex in the sediment than in the water of salterns. An infer Community Assembly Mechanisms by Phylogenetic-bin-based null model analysis (iCAMP) showed that microbial community assembly in sediment and water differed. Our findings provide more information about microbial community structure and the importance of various ecological processes in controlling microbial community diversity and succession along salinity gradients in water and sediment.

Growth hormone ameliorates the age-associated depletion of ovarian reserve and decline of oocyte quality via inhibiting the activation of Fos and Jun signaling.

Oocyte quality typically begins to decline with aging, which contributes to subfertility and infertility. However, there is still no effective treatment to restore the ovarian reserve and improve aged-oocyte quality. According to the present study, growth hormone (GH) secretion changes with maternal age in female mice. After intraperitoneal injection with GH (1 mg/kg body weight) every two days for two months, the 10-month-old mice showed a better ovarian reserve and oocyte quality than control mice. GH treatment decreased the occurrence rate of aneuploidy caused by spindle/chromosome defects. Additionally, the single oocyte transcriptome analysis indicated that GH decreased the expression of apoptosis-related genes in oocytes. It was also observed that GH treatment reduced the expression of γH2AX and apoptosis of aged oocytes via decreasing the activation of Fos and Jun. Collectively, our results indicate that GH treatment is an effective way to reverse the age-associated depletion of ovarian reserve and the decline of oocyte quality by decreasing apoptosis.

[Application of a multiple linear regression model of FEV1 in pulmonary function test].

OBJECTIVE: To construct a multiple linear regression model of forced expiratory volume in 1 second (FEV1) for estimating FEV1 in special populations unable to receive or uncooperative in pulmonary ventilation function tests. METHODS: The multiple linear regression model of FEV1 was constructed based on the data of 813 individuals undergoing pulmonary function tests in First Affiliated Hospital of Zhejiang Chinese Medical University between September, 2017 and September, 2019, and was validated using the data of another 94 individuals from the same hospital between January and July, 2020. FEV1 of the individuals was measured by pulmonary ventilation function test, and respiratory resistance (Rrs) was measured using forced oscillation technique (FOT). Pearson correlation analysis was used to assess the correlation between the factors, and the model equation was established by multiple stepwise regression analysis. The calculated FEV1 based on the model was compared with the measured FEV1 among both the individuals included for modeling and validation. RESULTS: FEV1 was not significantly correlated with BMI (r=-0.026, P=0.457), poorly correlated with body mass (r=0.382, P=0.000), positively correlated with height (r=0.723, P=0.000), and negatively correlated with Rrs (r=-0.503, P=0.000) with an obvious gender differences (t=18.517, P=0.000). FEV1 was positively correlated with age among individuals below 25 years of age (r=0.578, P=0.000) and was negatively correlated with age among those beyond or at the age of 25 (r=-0.589, P=0.000). For individuals beyond or at the age of 25 years, the variables of height, gender, age and Rrs were included in the model, and the calculated FEV1 did not differ significantly from the measured values in either the modeling sample (n=751; t=1.293, P=0.196) or the verification sample (n=83;t=-1.736, P=0.086), and the two values were well correlated in the verification sample (r=0.891, P=0.000). For individuals below 25 years, only height was included in the model, and the calculated FEV1 and the measured values showed no significant difference in the modeling sample (n=62; t=-0.009, P=0.993) or the verification sample (n=11; t=-0.635, P=0.540) with a good correlation in the verification sample (r=0.795, P=0.003). CONCLUSIONS: The multiple linear regression model for calculating FEV1 constructed in this study is suitable for clinical application.

In vitro fertilization-frozen embryo transfer in a patient with cytochrome P450 oxidoreductase deficiency: a case report.

Cytochrome P450 enzymes are required for the synthesis of cholesterol and steroid hormones. Cytochrome P450 oxidoreductase (POR) donates electrons to microsomal cytochrome P450 enzymes. POR deficiency (PORD) is a rare autosomal recessive disease. In patients with PORD, steroid hormone synthesis is disrupted, which can cause infertility. The objective of this study was to report on a case of in vitro fertilization-frozen embryo transfer (IVF-FET) in a patient with PORD. The patient's hormone (i.e. 17α-hydroxyprogesterone) and electrolyte levels were within normal ranges ordinarily. Upon controlled ovarian stimulation, follicle growth was normal, but serum estrogen and progesterone levels were low and high, respectively. The serum progesterone level was elevated after long-acting gonadotropin-releasing hormone agonist treatment, and an endometrial biopsy showed a change in the proliferative phase. Genetic tests detected homozygous mutations (c.976 T > G, p.Y326D) in exon 10 of the POR gene. The frozen embryo was transferred during the administration of hormone replacement therapy. No significant morphological or metabolic abnormalities were observed in the neonate. Our findings suggest that infertile women with normal hormone levels may have metabolic diseases such as PORD. Further studies are needed to determine the cause of these diseases and to assist pregnancy in such women.

Umbilical cord-derived mesenchymal stem cells on scaffolds facilitate collagen degradation via upregulation of MMP-9 in rat uterine scars.

BACKGROUND: Severe injuries of the uterus may trigger uterine scar formation, ultimately leading to infertility or obstetrical complications. To date, few methods have adequately solved the problem of collagen deposition in uterine scars. Umbilical cord-derived mesenchymal stem cells (UC-MSCs) have shown great promise in clinical applications. The objective of this study was to investigate the effect of a scaffold/UC-MSCs construct on collagen degradation and functional regeneration in rat uterine scars following full-thickness excision of uterine walls. METHODS: In order to establish a rat model of uterine scars, the uterine wall of approximately 1.0 cm in length and 0.5 cm in width (one-third of the uterine circumference) was excised from each uterine horn. A total of 128 scarred uterine horns from 64 rats were randomly assigned to four groups, including a PBS group (n = 32 uterine horns), scaffold group (n = 32 uterine horns), UC-MSCs group (n = 32 uterine horns) and scaffold/UC-MSCs group (n = 32 uterine horns) to investigate the effect of different treatments on the structure and function of uterine scars. PBS, degradable collagen fibres, UC-MSCs or UC-MSCs mixed with gelatinous degradable collagen fibres were injected into four pre-marked points surrounding each uterine scar, respectively. At days 30 and 60 post-transplantation, a subset of rats (n = 8 uterine horns) from each group was euthanized and serial sections of uterine tissues containing the operative region were prepared. Haematoxylin-eosin staining, Masson's trichrome staining, and immunohistochemical staining for MMP-2, MMP-9, α-SMA and vWF were performed. Finally, another subset of rats (n = 16 uterine horns) from each group was mated with male rats at day 60 post-transplantation and euthanized 18 days after the presence of vaginal plugs to check numbers, sizes and weights of fetuses, as well as sites of implantation. RESULTS: The scaffold/UC-MSCs group exhibited obvious collagen degradation compared with the other three groups. At day 60 post-transplantation, the number of MMP-9-positive cells in the scaffold/UC-MSCs group (25.96 ± 3.63) was significantly higher than that in the PBS group (8.19 ± 1.61, P < 0.01), the scaffold group (7.25 ± 2.17, P < 0.01) and the UC-MSCs group (8.31 ± 2.77, P < 0.01). The pregnancy rate in the scaffold/UC-MSCs group (10/16) was also significantly higher than that in the PBS group (2/16, P < 0.017), the scaffold group (1/16, P < 0.017) and the UC-MSCs group (3/16, P < 0.017). CONCLUSIONS: The scaffold/UC-MSCs system facilitated collagen degradation in uterine scars via upregulation of MMP-9, which was secreted by transplanted UC-MSCs, and promoted regeneration of the endometrium, myometrium and blood vessels in uterine scars. Furthermore, the scaffold/UC-MSCs-treated uterine scars showed nearly complete restoration of receptive fertility.

Regeneration of uterine horns in rats using collagen scaffolds loaded with human embryonic stem cell-derived endometrium-like cells.

A variety of diseases may lead to hysterectomies or uterine injuries, which may form a scar and lead to infertility. Due to the limitation of native materials, there are a few effective methods to treat such damages. Tissue engineering combines cell and molecular biology with materials and mechanical engineering to replace or repair damaged organs and tissues. The use of human embryonic stem cells (hESCs) as a donor cell source for the replacement therapy will require the development of simple and reliable cell differentiation protocols. This study aimed at efficiently generating endometrium-like cells from the hESCs and at using these cells with collagen scaffold to repair uterine damage. The hESCs were induced by co-culturing with endometrial stromal cells, and simultaneously added cytokines: epidermal growth factor (EGF), platelet-derived growth factor-b (PDGF-b), and E2. Expression of cell specific markers was analyzed by immunofluorescence and reverse trascription-polymerase chain reaction to monitor the progression toward an endometrium-like cell fate. After differentiation, the majority of cells (>80%) were positive for cytokeratin-7, and the expression of key transcription factors related to endometrial development, such as Wnt4, Wnt7a, Wnt5a, Hoxa11, and factors associated with endometrial epithelial cell function: Hoxa10, Intergrinß3, LIF, ER, and PR were also detected. Then, we established the uterine full-thickness-injury rat models to test cell function in vivo. hESC-derived cells were dropped onto collagen scaffolds and transplanted into the animal model. Twelve weeks after transplantation, we discovered that the hESC-derived cells could survive and recover the structure and function of uterine horns in a rat model of severe uterine damage. The experimental system presented here provides a reliable protocol to produce endometrium-like cells from hESCs. Our results encourage the use of hESCs in cell-replacement therapy for severe uterine damage in future.

Transplantation of bone marrow mesenchymal stem cells on collagen scaffolds for the functional regeneration of injured rat uterus.

Serious injuries of endometrium in women of reproductive age are often followed by uterine scar formation and a lack of functional endometrium predisposing to infertility or miscarriage. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have shown great promise in clinical applications. In the present study, BM-MSCs loaded onto degradable collagen membranes were constructed. Collagen membranes provided 3-dimmensional architecture for the attachment, growth and migration of rat BM-MSCs and did not impair the expression of the stemness genes. We then investigated the effect of collagen/BM-MSCs constructs in the healing of severe uterine injury in rats (partial full thickness uterine excision). At four weeks after the transplantation of collagen/BM-MSCs constructs, BM-MSCs were mainly located to the basal membrane of regenerative endometrium. The wounded tissue adjacent to collagen/BM-MSCs constructs expressed higher level of bFGF, IGF-1, TGFß1 and VEGF than the corresponding tissue in rats receiving collagen construct alone or in spontaneous regeneration group. Moreover, the collagen/BM-MSCs system increased proliferative abilities of uterine endometrial and muscular cells, facilitated microvasculature regeneration, and restored the ability of endometrium to receive the embryo and support its development to a viable stage. Our findings indicate that BM-MSCs may support uterine tissue regeneration.

The effect of collagen-binding vascular endothelial growth factor on the remodeling of scarred rat uterus following full-thickness injury.

Serious injuries of uterine which lead to scar formation will finally result in infertility or pregnancy complications. There are few effective methods to treat such damages because of the shortage of native tissues. Vascular endothelial growth factor (VEGF) is important for the formation of new vessels and re-epithelialization of endometrium. Here we produced a collagen-binding VEGF by fusing a collagen-binding domain to the N-terminal of native VEGF. After injection into a rat scarred uterus model (partial of rat uterine horn was excised and left for scar formation) the collagen targeting VEGF promoted remodeling of the scarred uterus including the regeneration of endometrium, muscular cells, and vascularization and improved pregnancy outcomes. Thus, collagen-binding VEGF may be a pragmatic solution for the treatment of severe uterine damages.

Efficient transduction of feline neural progenitor cells for delivery of glial cell line-derived neurotrophic factor using a feline immunodeficiency virus-based lentiviral construct.

Work has shown that stem cell transplantation can rescue or replace neurons in models of retinal degenerative disease. Neural progenitor cells (NPCs) modified to overexpress neurotrophic factors are one means of providing sustained delivery of therapeutic gene products in vivo. To develop a nonrodent animal model of this therapeutic strategy, we previously derived NPCs from the fetal cat brain (cNPCs). Here we use bicistronic feline lentiviral vectors to transduce cNPCs with glial cell-derived neurotrophic factor (GDNF) together with a GFP reporter gene. Transduction efficacy is assessed, together with transgene expression level and stability during induction of cellular differentiation, together with the influence of GDNF transduction on growth and gene expression profile. We show that GDNF overexpressing cNPCs expand in vitro, coexpress GFP, and secrete high levels of GDNF protein-before and after differentiation-all qualities advantageous for use as a cell-based approach in feline models of neural degenerative disease.

Enhancement of cardiomyocyte differentiation from human embryonic stem cells.

Several approaches have been used to encourage the differentiation of cardiomyocytes from human embryonic stem cells. However, the differentiation efficiency is low, and appropriate culture protocols are needed to produce adequate numbers of cardiomyocytes for therapeutic cell transplantation. This study investigated the effects of serum on cardiomyocyte differentiation in suspension culture medium during embryoid body (EB) formation by human embryonic stem cells. The addition of ascorbic acid, dimethylsulfoxide and 5-aza-2'-deoxycytidine during days 5-7 at the EB-forming stage resulted in an increase in the numbers of rhythmically contracting clusters of derived cardiomyocytes. Treatment with 0.1 mmol L(-1) ascorbic acid alone, or more notably in combination with 10 micromol L(-1) 5-aza-2'-deoxycytidine, induced the formation of beating cells within EBs. Most of the beating clusters had spontaneous contraction rates similar to those found in human adults, and their contractile activity lasted for up to 194 days.

The reversal of hyperglycaemia in diabetic mice using PLGA scaffolds seeded with islet-like cells derived from human embryonic stem cells.

Islet-like cells derived from embryonic stem (ES) cells may be a promising therapeutic option for future diabetes treatment. Here, we demonstrated a five-stage protocol with adding exendin-4 instead of nicotinamide finally could generate islet-like cells from human embryonic stem (ES) cells. Immunofluorescence analysis revealed a high percentage of c-peptide positive cells in the derivation. However, in addition to insulin/c-peptide, most cells also coexpressed PDX-1 (pancreas duodenum homeobox-1), glucagon, somatostatin or pancreatic polypeptide. Insulin and other pancreatic beta-cell-specific genes were all present in the differentiated cells. Insulin secretion could be detected and increased significantly by adding KCL in high glucose concentration in vitro. Furthermore, subcutaneous transplantation of scaffolds seeded with the islet-like cells or cell transplantation under kidney capsules for further differentiation in vivo could improve 6h fasted blood glucose levels and diabetic phenotypes in streptozotocin-induced diabetic SCID mice. More interestingly, blood vessels of host origin, characterized by mouse CD31 immunostaining, invaded the cell-scaffold complexes. This work reveals a five-stage protocol with adding exendin-4 may be an effective protocol on the differentiation of human ES cells into islet-like cells, and suggests scaffolds can serve as vehicles for islet-like cell transplantation.

Chemically defined sequential culture media for TH+ cell derivation from human embryonic stem cells.

During the past few years several differentiation protocols to derive midbrain dopamine (DA) neurons from human embryonic stem (hES) cells have been developed, but the production of sufficient amounts of the 'right' therapeutic DA cells has not yet been accomplished. The aim of this study was to efficiently generate tyrosine hydroxylase (TH)-positive cells in vitro from our hES cells using a chemically defined culture system. At the end of differentiation, the vast majority of cells (>90%) were positive for both TH and beta-tubulin isotype III (TuJ1). Other markers of dopaminergic cells, like dopamine transporter (DAT) and Nurr1 were also detected by immunofluorescence or RT-PCR. The functions of these cells were confirmed by measurements of DA release in vitro and by transplantation of derived cells into Parkinson's disease (PD) rats in vivo. We found these cells were able to release DA when depolarized by high K(+). Moreover, 4 weeks after transplantation, the hES-derived cells could survive and reduce the apomorphine-induced rotation behaviour of the rats. In conclusion, the experimental system presented here provided a reliable protocol to produce a large number of hES-derived TH(+) cells which may be used in cell therapy for PD in future.