Single-cell sequencing provides insights into the landscape of ovary in PCOS and alterations induced by CUMS.

Polycystic ovary syndrome (PCOS) is a common endocrine disorder related to psychological distress. However, the mechanism underlying increased prevalence of depression in PCOS remained unclear. This study aimed to explore the unique transcriptional landscape of ovary and offered a platform to explore the mechanism of PCOS, as well as the influences caused by depression. The PCOS rat model was established by letrozole whereas PCOS rat model with depression was established by letrozole combined with chronic unpredicted mild stress (CUMS). Then single-cell RNA sequencing (scRNA-Seq) was applied to analyze the transcriptional features of rat ovaries. Granulosa cells (GCs) and fibroblasts (Fibros) accounted for the top two clusters of total 12 cell types. There were nine clusters in GCs, related to inflammatory response, endoplasmic reticulum (ER) stress, and steroidogenesis. The expression of differentially expressed genes (DEG) Hes1 was higher in PCOS and PCOS + CUMS groups, exhibiting enhanced expression by pseudotime and positively related to inflammation. Pseudotemporal analysis revealed that inflammation contributed to the different GCs distributions. Moreover, analysis of DEGs and gene ontology (GO) function enrichment revealed CUMS aggravated inflammation in PCOS GCs possibly via interferon signaling pathway. In theca cells (TCs), nine clusters were observed and some of them were relevant to inflammation, ER stress, and lipid metabolism. DEGs Ass1, Insl3, and Ifi27 were positively related to Cyp17a1, and Ces1d might contribute to the different trajectory of TCs. Subsequent scRNA-seq revealed a signature profile of endothelial cells (ECs) and Fibros, which suggest that inflammation-induced damage of ECs and Fibro, further exacerbated by CUMS. Finally, analysis of T cells and mononuclear phagocytes (MPs) revealed the existence of immune dysfunction, among which interferon signaling played a critical role. These findings provided more knowledge for a better understanding PCOS from the view of inflammation and identified new biomarkers and targets for the treatment of PCOS with psychological diseases.NEW & NOTEWORTHY In this study, we mapped the landscape of polycystic ovary syndrome (PCOS) ovary with rat model induced by letrozole and provided a novel insight into the molecular mechanism of PCOS accompanied by chronic unpredicted mild stress (CUMS) at single-cell transcriptomic level. These observations highlight the importance of inflammation in the pathogenesis of PCOS, which might also be the bridge between PCOS and psychological diseases.

Assessment of Instant Loss of Elbow Flexion in Children with Untreated Gartland IIA Humeral Supracondylar Fractures-A Simulation Study with Lateral Elbow Radiographs.

OBJECTIVE: The suitability of in situ cast fixation for treating Gartland IIA humeral supracondylar fractures has remained controversial due to concerns regarding loss of elbow flexion. This study aimed to assess the instant loss of elbow flexion after Gartland IIA humeral supracondylar fractures based on the relationship between the anterior marginal line of the humerus and capitellum in the lateral view. METHODS: This simulation study was conducted with normal radiographs using Adobe Photoshop 14.0, followed by verification using clinical cases. Standard lateral views of normal elbows of children were collected from January 2008 to February 2020. Adobe Photoshop was used to simulate Gartland IIA supracondylar fractures with different degrees of angulation in the sagittal plane. A formula was deduced to assess flexion loss, and this method was verified in three cases. The data were grouped by age, and the relationship between elbow flexion loss and age, as well as the angulation of the fracture, was analyzed using a one-way or multivariate ANOVA. RESULTS: There was a flexion loss of 19° (11-30°) when the anterior margin line of the humerus was tangential to the capitellum. This loss increased with age at injury (r = 0.731, P = 0.000). Moreover, the difference in angulation in the sagittal plane also influenced the extent of elbow flexion loss (r = -0.739, P = 0.000). The more horizontal the fracture line in the lateral view, the greater the loss of elbow flexion. CONCLUSION: Instant elbow flexion loss after Gartland IIA humeral supracondylar fractures increases with age at the time of injury and decreases with angulation in the sagittal plane. When the anterior margin of the humerus is tangential to the capitellum, there will be an average loss of 19° in elbow flexion. These findings provide a quantitative reference for clinical decision-making in the treatment of Gartland IIA supracondylar fractures.

Expression pattern of peritoneum IL-6 is associated with baseline peritoneal transport function in uremic patients before dialysis.

OBJECTIVE: This study aimed to investigate the biological factors associated with baseline peritoneal transport in uremic patients before dialysis. METHODS: Thirty patients with uremia were grouped according to their peritoneal dialysate creatinine/serum creatinine ratio (D/P) as high-transport (H, 16 cases) with D/P>0.65 and low-transport (L, 14 cases) with D/P≤0.65 one month after continuous ambulatory peritoneal dialysis treatment. Multi-inflammatory levels such as serum IL-6 and albumin, peritoneal IL-6 level, and microvessel density (MVD) of visceral peritoneal were compared and correlated between the two groups to determine the associated factors. RESULTS: There were no significant differences in clinical parameters between the two groups (p < 0.05). There were no significant differences in serum IL-6 and albumin between the two groups. However, peritoneal IL-6 and MVD in group H were significantly higher than group L (p=0.012, p=0.044), and they were positively correlated (r=0.368, p=0.045). Furthermore, baseline D/P was positively correlated with IL-6 expressions (r=0.640, p=0.000) and peritoneal MVD (r=0.476, p=0.008), and independently associated with peritoneal IL-6 expression (p=0.004). CONCLUSIONS: The baseline peritoneal transport performance is associated with peritoneal IL-6 expression and MVD but not circulatory IL-6.

Genes affecting ionizing radiation survival identified through combined exome sequencing and functional screening.

The study of genetic syndromes characterized by sensitivity to DNA damaging agents has provided important insights into the mechanisms that maintain genome stability and identified novel targets for cancer therapies. Here, we used exome sequencing to study 51 unrelated individuals with previously reported hypersensitivity to ionizing radiation as well as a range of neurologic, immunologic, and developmental features, but who did not clearly fit any previously defined genetic syndrome. Based on the combination of variant identification, computational evidence of deleteriousness, and functional screening, we identified three groups of subjects. Two subjects carried the bi-allelic loss of function variants in causative genes for known DNA damage response syndromes. Eight subjects carried the single loss of function variants in causative genes for DNA damage response syndromes, six of whom also carried predicted deleterious variants in other genes with DNA damage-related functions. Three subjects carried deleterious mutations in genes without obvious roles in DNA damage responses. However, treatment of U2OS cells with small interfering RNA targeting these genes resulted in significantly increased radiation sensitivity. Our results suggest that gene-gene interaction may contribute to ionizing radiation sensitivity as well as highlighting possible roles for several genes not obviously involved in the response to DNA damage.

Bifunctional, Copper-Doped, Mesoporous Silica Nanosphere-Modified, Bioceramic Scaffolds for Bone Tumor Therapy.

In the traditional surgical intervention procedure, residual tumor cells may potentially cause tumor recurrence. In addition, large bone defects caused by surgery are difficult to self-repair. Thus, it is necessary to design a bioactive scaffold that can not only kill residual tumor cells but also promote bone defect regeneration simultaneously. Here, we successfully developed Cu-containing mesoporous silica nanosphere-modified ß-tricalcium phosphate (Cu-MSN-TCP) scaffolds, with uniform and dense nanolayers with spherical morphology via 3D printing and spin coating. The scaffolds exhibited coating time- and laser power density-dependent photothermal performance, which favored the effective killing of tumor cells under near-infrared laser irradiation. Furthermore, the prepared scaffolds favored the proliferation and attachment of rabbit bone marrow-derived mesenchymal stem cells and stimulated the gene expression of osteogenic markers. Overall, Cu-MSN-TCP scaffolds can be considered for complete eradication of residual bone tumor cells and simultaneous healing of large bone defects, which may provide a novel and effective strategy for bone tumor therapy. In the future, such Cu-MSN-TCP scaffolds may function as carriers of anti-cancer drugs or immune checkpoint inhibitors in chemo-/photothermal or immune-/photothermal therapy of bone tumors, favoring for effective treatment.

Enhanced fluorescence detection of proteins using ZnO nanowires integrated inside microfluidic chips.

Nanostructure-enhanced detection is promising for a number of applications such as early cancer diagnosis, environmental monitoring and mine safety, among which nanostructures integrated microfluidic chips offers unique advantage of ultra-low quantitative analyses. Here, dense ZnO nanowires of varied diameter and length were obtained by changing the content of polyethyleneimine (PEI) and growth time via simple hydrothermal growth in microfluidic channels for protein detection. We showed that this approach was superiorly efficient compared to the conventional hydrothermal method due to the flow-induced replenishment of nutrient and the effect of shear stress. When immobilizing FITC conjugated anti-bovine immunoglobulin G (IgG) on ZnO nanowires, the fluorescence emission was significantly amplified compared to glass substrate and ZnO seed layer. Under the different growth conditions, the most remarkable fluorescence enhancement was observed on the ZnO nanowire substrate grown for 3h with 5mM PEI in solution. It is ascribed not only to the increase of the binding surface area of proteins but also the intrinsic fluorescence enhancement of ZnO nanowires as waveguides. We further used the optimized ZnO nanowires to demonstrate multiple detection of cancer biomarkers, achieving a superior limit of detection (LOD) as low as 1pg/mL in human α-fetoprotein (AFP) assay and 100 fg/mL in carcinoembryonic antigen (CEA) assay with large dynamic range of 6-7 orders, which suggests that ZnO nanowire integrated microfluidic chips are promising for high-throughput fluorescence-based diagnostic assays.

Identification of ATIC as a Novel Target for Chemoradiosensitization.

PURPOSE: Mutations in the gene encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final 2 steps of the purine de novo biosynthetic pathway, were identified in a subject referred for radiation sensitivity testing. Functional studies were performed to determine whether ATIC inhibition was radiosensitizing and, if so, to elucidate the mechanism of this effect and determine whether small molecule inhibitors of ATIC could act as effective radiosensitizing agents. METHODS AND MATERIALS: Both small interfering RNA knockdown and small molecule inhibitors were used to inactivate ATIC in cell culture. Clonogenic survival assays, the neutral comet assay, and γH2AX staining were used to assess the effects of ATIC inhibition or depletion on cellular DNA damage responses. RESULTS: Depletion of ATIC or inhibition of its transformylase activity significantly reduced the surviving fraction of cells in clonogenic survival assays in multiple cancer cell lines. In the absence of ionizing radiation exposure, ATIC knockdown or chemical inhibition activated cell cycle checkpoints, shifting cells to the more radiosensitive G2/M phase of the cell cycle, and depleted cellular adenosine triphosphate but did not result in detectable DNA damage. Cells in which ATIC was knocked down or inhibited and then treated with ionizing radiation displayed increased numbers of DNA double-strand breaks and a delay in the repair of those breaks relative to irradiated, but otherwise untreated, controls. Supplementation of culture media with exogenous adenosine triphosphate ameliorated the DNA repair phenotypes. CONCLUSIONS: These findings implicate ATIC as an effective, and previously unrecognized, target for chemoradiosensitization and, more broadly, suggest that purine levels in cells might have an underappreciated role in modulating the efficiency of DNA damage responses that could be exploited in radiosensitizing strategies.

Peritoneal microvascular endothelial function and the microinflammatory state are associated with baseline peritoneal transport characteristics in uremic patients.

PURPOSE: To investigate microvessel density (MVD), vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), and interleukin-6 (IL-6) mRNA expression in peritoneal tissues, and their relationships with baseline peritoneal transport in uremia. METHODS: Thirty uremic patients with a peritoneal dialysis catheter were selected in the Department of Nephrology in Dalian Central Hospital, Liaoning, China between 2010 and 2012. Peritoneal specimens were harvested for assessment of MVD, VEGF, eNOS, and IL-6 mRNA expression. One month after continuous ambulatory peritoneal dialysis, a peritoneal equilibration test was conducted. According to the 4-h peritoneal dialysate and plasma creatinine ratio (D/P Cr), patients were divided into high (n=16) and low (n=14) transport groups. RESULTS: General clinical data of high and low transport groups were similar (P>0.05). The MVD in peritoneal tissues was significantly higher in the high than in the low transport group (P<0.05). Correspondingly, VEGF (P<0.01), eNOS (P<0.01), and IL-6 (P<0.05) mRNA expression levels were significantly higher in the high as compared the low transport groups. Correlation analysis showed that the baseline D/P Cr was positively correlated with MVD and VEGF, eNOS, and IL-6 mRNA expression levels in the peritoneum (r=0.506, 0.646, 0.638, and 0.686, respectively; P<0.01). CONCLUSIONS: Uremic patients display differences in peritoneal microvascular endothelial function and microinflammatory states before peritoneal dialysis. Patients of the high transport group have higher MVD, increased expression of endothelial function markers (VEGF and eNOS), and the microinflammatory marker (IL-6). These observations are closely related to high baseline peritoneal transport.

Myelodysplastic syndrome and associated coagulopathy: a case report and review.

Myelodysplastic syndrome (MDS) is a primary bone marrow disorder whose hallmark is the development of peripheral cytopenias and a predilection toward the development of acute myeloid leukemia (AML). Patients often have hypercellular bone marrows with dysplastic features that may involve multiple lineages. An increased awareness of MDS has led to the reporting of a number of associated autoimmune and paraneoplastic conditions in the medical literature. We present the case of an elderly man who was transferred to our institution with persistent, refractory bleeding several weeks after the resection of a sebaceous cyst. Despite reoperation, treatment with topical and intravenous hemostatic agents, and transfusion of blood products, the patient's bleeding persisted. A comprehensive evaluation for the cause of his coagulopathy was undertaken. Bone marrow evaluation was consistent with MDS. A paraneoplastic consumptive coagulopathy or fibrinolytic process in conjunction with MDS-related platelet dysfunction was felt to be the most likely etiology of the patient's bleeding.

Repeated systemic administration of human adipose-derived stem cells attenuates overt diabetic nephropathy in rats.

Adipose-derived stem cells (ASCs) can alleviate acute kidney injury and promote kidney cell regeneration and repair. To investigate the role of ASCs in diabetic nephropathy (DN), Sprague-Dawley rats were made diabetic by intraperitoneal injection of streptozotocin (STZ) after uninephrectomy. After 12 weeks, proteinuria was well established. Five times of 5×10(6) human ASCs repeatedly injected through a tail vein at 4 weekly intervals. A reduction in proteinuria was not observed in diabetic rats until 24 weeks. However, urinary protein excretion was significantly suppressed at 28 weeks and persisted up to 32 weeks after STZ treatment. ASC treatment significantly attenuated glomerulus hypertrophy and tubular interstitial injury, and led to the downregulation of WT-1 and synaptopodin expression. CFSE labeled ASCs were injected into DN rats via the tail vein. Within 24 h after injection, the cells were detected in lung, spleen, and peritubular regions, but rarely in pancreas. Human Alu gene expression was detected in lung and spleen up to 4 weeks after ASCs injection. ASC treatment did not improve hyperglycemia or pancreatic damage. In vitro, recombinant human glial cell line-derived neurotrophic factor (GDNF) prevented podocyte injury by high glucose similarly to ASC-conditioned medium. After blocking GDNF in ASC-CM with neutralizing antibody, the therapeutic effect of ASC-CM was significantly decreased. ASCs cocultured with podocytes restored the downregulation of synaptopodin expression, which was weakened by GDNF-RNA interfering. These findings indicate that repeated intravenous ASC can reduce diabetic kidney damage in rats even at the progressive stage, and promote podocyte recovery via GDNF secretion.

PKR regulates proliferation, differentiation, and survival of murine hematopoietic stem/progenitor cells.

Protein kinase R (PKR) is an interferon (IFN)-inducible, double-stranded RNA-activated kinase that initiates apoptosis in response to cellular stress. To determine the role of PKR in hematopoiesis, we developed transgenic mouse models that express either human PKR (TgPKR) or a dominant-negative PKR (TgDNPKR) mutant specifically in hematopoietic tissues. Significantly, peripheral blood counts from TgPKR mice decrease with age in association with dysplastic marrow changes. TgPKR mice have reduced colony-forming capacity and the colonies also are more sensitive to hematopoietic stresses. Furthermore, TgPKR mice have fewer hematopoietic stem/progenitor cells (HSPCs), and the percentage of quiescent (G0) HSPCs is increased. Importantly, treatment of TgPKR bone marrow (BM) with a PKR inhibitor specifically rescues sensitivity to growth factor deprivation. In contrast, marrow from PKR knockout (PKRKO) mice has increased potential for colony formation and HSPCs are more actively proliferating and resistant to stress. Significantly, TgPKR HSPCs have increased expression of p21 and IFN regulatory factor, whereas cells from PKRKO mice display mechanisms indicative of proliferation such as reduced eukaryotic initiation factor 2α phosphorylation, increased extracellular signal-regulated protein kinases 1 and 2 phosphorylation, and increased CDK2 expression. Collectively, data reveal that PKR is an unrecognized but important regulator of HSPC cell fate and may play a role in the pathogenesis of BM failure.

Increased expression of the dsRNA-activated protein kinase PKR in breast cancer promotes sensitivity to doxorubicin.

It has been reported that the expression and activity of the interferon-inducible, dsRNA-dependent protein kinase, PKR, is increased in mammary carcinoma cell lines and primary tumor samples. To extend these findings and determine how PKR signaling may affect breast cancer cell sensitivity to chemotherapy, we measured PKR expression by immunohistochemical staining of 538 cases of primary breast cancer and normal tissues. Significantly, PKR expression was elevated in ductal, lobular and squamous cell carcinomas or lymph node metastases but not in either benign tumor specimens or cases of inflammation compared to normal tissues. Furthermore, PKR expression was increased in precancerous stages of mammary cell hyperplasia and dysplasia compared to normal tissues, indicating that PKR expression may be upregulated by the process of tumorigenesis. To test the function of PKR in breast cancer, we generated MCF7, T-47D and MDA-MB-231 breast cancer cell lines with significantly reduced PKR expression by siRNA knockdown. Importantly, while knockdown of PKR expression had no effect on cell proliferation under normal growth conditions, MCF7, T-47D or MDA-MB-231 cells with reduced PKR expression or treated with a small molecule PKR inhibitor were significantly less sensitive to doxorubicin or H(2)O(2)-induced toxicity compared to control cells. In addition, the rate of eIF2α phosphorylation following treatment with doxorubicin was delayed in breast cancer cell lines with decreased PKR expression. Significantly, treatment of breast cancer lines with reduced PKR expression with either interferon-α, which increases PKR expression, or salubrinal, which increases eIF2α phosphorylation, restored doxorubicin sensitivity to normal levels. Taken together these results indicate that increased PKR expression in primary breast cancer tissues may serve as a biomarker for response to doxorubicin-containing chemotherapy and that future therapeutic approaches to promote PKR expression/activation and eIF2α phosphorylation may be beneficial for the treatment of breast cancer.