In vivo HSC prime editing rescues sickle cell disease in a mouse model.

Sickle cell disease (SCD) is a monogenic disease caused by a nucleotide mutation in the ß-globin gene. Current gene therapy studies are mainly focused on lentiviral vector-mediated gene addition or CRISPR/Cas9-mediated fetal globin reactivation, leaving the root cause unfixed. We developed a vectorized prime editing system that can directly repair the SCD mutation in hematopoietic stem cells (HSCs) in vivo in a SCD mouse model (CD46/Townes mice). Our approach involved a single intravenous injection of a nonintegrating, prime editor-expressing viral vector into mobilized CD46/Townes mice and low-dose drug selection in vivo. This procedure resulted in the correction of ∼40% of ßS alleles in HSCs. On average, 43% of sickle hemoglobin was replaced by adult hemoglobin, thereby greatly mitigating the SCD phenotypes. Transplantation in secondary recipients demonstrated that long-term repopulating HSCs were edited. Highly efficient target site editing was achieved with minimal generation of insertions and deletions and no detectable off-target editing. Because of its simplicity and portability, our in vivo prime editing approach has the potential for application in resource-poor countries where SCD is prevalent.

Stable HIV decoy receptor expression after in vivo HSC transduction in mice and NHPs: Safety and efficacy in protection from SHIV.

We aim to develop an in vivo hematopoietic stem cell (HSC) gene therapy approach for persistent control/protection of HIV-1 infection based on the stable expression of a secreted decoy protein for HIV receptors CD4 and CCR5 (eCD4-Ig) from blood cells. HSCs in mice and a rhesus macaque were mobilized from the bone marrow and transduced by an intravenous injection of HSC-tropic, integrating HDAd5/35++ vectors expressing rhesus eCD4-Ig. In vivo HSC transduction/selection resulted in stable serum eCD4-Ig levels of ∼100 µg/mL (mice) and >20 µg/mL (rhesus) with half maximal inhibitory concentrations (IC50s) of 1 µg/mL measured by an HIV neutralization assay. After simian-human-immunodeficiency virus D (SHIV.D) challenge of rhesus macaques injected with HDAd-eCD4-Ig or a control HDAd5/35++ vector, peak plasma viral load levels were ∼50-fold lower in the eCD4-Ig-expressing animal. Furthermore, the viral load was lower in tissues with the highest eCD4-Ig expression, specifically the spleen and lymph nodes. SHIV.D challenge triggered a selective expansion of transduced CD4+CCR5+ cells, thereby increasing serum eCD4-Ig levels. The latter, however, broke immune tolerance and triggered anti-eCD4-Ig antibody responses, which could have contributed to the inability to eliminate SHIV.D. Our data will guide us in the improvement of the in vivo approach. Clearly, our conclusions need to be validated in larger animal cohorts.

Fanconi anemia complementation group D2 promotes sensitivity of endometrial cancer cells to chemotherapeutic agents by inhibiting the ferroptosis pathway.

BACKGROUND: Resistance can develop during treatment of advanced endometrial cancer (EC), leading to unsatisfactory results. Fanconi anemia complementation group D2 (Fancd2) has been shown to be closely related to drug resistance in cancer cells. Therefore, this study was designed to explore the correlation of Fancd2 with EC resistance and the mechanism of Fancd2. METHODS: Real-time quantitative PCR (RT-qPCR) was used to detect the expression of Fancd2 in EC tissues and cells. EC cells (Ishikawa) and paclitaxel-resistant EC cells (Ishikawa/TAX) were transfected to knock down Fancd2. In addition, the ferroptosis inhibitor Ferrostatin-1 was adopted to treat Ishikawa/TAX cells. The sensitivity of cancer cells to chemotherapeutic agents was observed via 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, and inhibitory concentration (IC)50 was calculated. Reactive oxygen species (ROS) levels were measured by flow cytometry, the activity of malondialdehyde (MDA) and the levels of glutathione (GSH) and Fe2+ in cells were detected by corresponding kits, and protein expression of solute farrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) was obtained through western blot. RESULTS: Compared with the normal tissues and endometrial epithelial cells, Fancd2 expression was significantly increased in EC tissues and Ishikawa cells, respectively. After knock-down of Fancd2, Ishikawa cells showed significantly increased sensitivity to chemotherapeutic agents. Besides, compared with Ishikawa cells, the levels of ROS, the activity of MDA, and the levels of GSH and Fe2+ were significantly decreased in Ishikawa/TAX cells, while the expression levels of SLC7A11 and GPX4 were significantly increased. Knock-down of Fancd2 significantly increased the ferroptosis levels in Ishikawa/TAX cells, but this effect could be reversed by Ferrostatin-1. CONCLUSION: Fancd2 increases drug resistance in EC cells by inhibiting the cellular ferroptosis pathway.

Discovery of Enzyme Inhibitors from Mangrove Sediment Derived Fungus Trichoderma harzianum SCSIO 41051.

One new fatty acid derivative, (2E,4E)-6,7-dihydroxy-2-methylocta-2,4-dienoic acid (1), and 16 known compounds (2-17) were isolated from the mangrove sediment derived fungus Trichoderma harzianum SCSIO 41051. Their structures were established by spectroscopic methods, computational ECD, and Mo2(OAc)4-induced ECD experiment. All the compounds were evaluated for their acetylcholinesterase (AChE) and pancreatic lipase (PL) inhibition. Compounds 9 and 14 exhibited moderate AChE inhibitory activities with IC50 values of 2.49 and 2.92 µM, respectively, which compounds 8 and 9 displayed moderate inhibition on PL with IC50 value of 2.30 and 2.34 µM, respectively.

Pigment Diversity in Leaves of Caladium × hortulanum Birdsey and Transcriptomic and Metabolic Comparisons between Red and White Leaves.

Leaf color is a key ornamental characteristic of cultivated caladium (Caladium × hortulanum Birdsey), a plant with diverse leaf colors. However, the genetic improvement of leaf color in cultivated caladium is hindered by the limited understanding of leaf color diversity and regulation. In this study, the chlorophyll and anthocyanin content of 137 germplasm resources were measured to explore the diversity and mechanism of leaf color formation in cultivated caladium. Association analysis of EST-SSR markers and pigment traits was performed, as well as metabolomics and transcriptomics analysis of a red leaf variety and its white leaf mutant. We found significant differences in chlorophyll and anthocyanin content among different color groups of cultivated caladium, and identified three, eight, three, and seven EST-SSR loci significantly associated with chlorophyll-a, chlorophyll-b, total chlorophyll and total anthocyanins content, respectively. The results further revealed that the white leaf mutation was caused by the down-regulation of various anthocyanins (such as cyanidin-3-O-rutinoside, quercetin-3-O-glucoside, and others). This change in concentration is likely due to the down-regulation of key genes (four PAL, four CHS, six CHI, eight F3H, one F3'H, one FLS, one LAR, four DFR, one ANS and two UFGT) involved in anthocyanin biosynthesis. Concurrently, the up-regulation of certain genes (one FLS and one LAR) that divert the anthocyanin precursors to other pathways was noted. Additionally, a significant change in the expression of numerous transcription factors (12 NAC, 12 bZIP, 23 ERF, 23 bHLH, 19 MYB_related, etc.) was observed. These results revealed the genetic and metabolic basis of leaf color diversity and change in cultivated caladium, and provided valuable information for molecular marker-assisted selection and breeding of leaf color in this ornamental plant.

A Marine Natural Product, Harzianopyridone, as an Anti-ZIKV Agent by Targeting RNA-Dependent RNA Polymerase.

The Zika virus (ZIKV) is a mosquito-borne virus that already poses a danger to worldwide human health. Patients infected with ZIKV generally have mild symptoms like a low-grade fever and joint pain. However, severe symptoms can also occur, such as Guillain-Barré syndrome, neuropathy, and myelitis. Pregnant women infected with ZIKV may also cause microcephaly in newborns. To date, we still lack conventional antiviral drugs to treat ZIKV infections. Marine natural products have novel structures and diverse biological activities. They have been discovered to have antibacterial, antiviral, anticancer, and other therapeutic effects. Therefore, marine products are important resources for compounds for innovative medicines. In this study, we identified a marine natural product, harzianopyridone (HAR), that could inhibit ZIKV replication with EC50 values from 0.46 to 2.63 µM while not showing obvious cytotoxicity in multiple cellular models (CC50 > 45 µM). Further, it also reduced the expression of viral proteins and protected cells from viral infection. More importantly, we found that HAR directly bound to the ZIKV RNA-dependent RNA polymerase (RdRp) and suppressed its polymerase activity. Collectively, our findings provide HAR as an option for the development of anti-ZIKV drugs.

Association between age, gender, and oral traumatic ulcerative lesions: a retrospective study.

BACKGROUND: Oral traumatic ulcerative lesions (OTUL) are commonly encountered in clinical practice, yet there is limited research on their clinical characteristics and traumatic etiological factors. This retrospective study aimed to analyze the age, gender, clinical characteristics, and traumatic etiological factors in a large cohort of patients with OTUL and provide valuable insights for dental clinicians to optimize patient care and prevention strategies. METHODS: A total of 1543 patients with OTUL were enrolled in this study. Age, gender, medical history, clinical characteristics and traumatic etiological factors were collected and analyzed. Logistic regression analysis was performed to determine the significance of age and gender as factors related to OTUL. RESULTS: The study revealed significant variations in clinical characteristics and traumatic etiological factors among different age groups and between genders. Logistic regression analysis demonstrated that both age and gender were significant factors related to OTUL. CONCLUSION: The clinical characteristics of OTUL and traumatic etiological factors appear to be significantly different according to age and gender. More targeted prevention strategies should be implemented for all age and gender groups.

Effectiveness of SARS-CoV-2-inactivated vaccine and the correlation to neutralizing antibodies: A test-negative case-control study.

Severe acute respiratory syndrome coronavirus 2 breakthrough infection in highly vaccinated populations raises study on the effectiveness for inactivated vaccine, including effectiveness of the vaccine dose, the continuance of effectiveness, the effectiveness against severe/critical coronavirus disease 2019 and against secondary attacks. A population of 10 870 close contacts were investigated in a Delta variant's epidemic. The effectiveness of vaccination was estimated in a test-negative case-control study. In addition, serum was used to detect neutralizing antibodies, to explore their correlation to effectiveness. The vaccine effectiveness (VE) values were estimated for populations aged 12 years or older. The overall adjusted VE was 56.2% and a two-dose vaccine was more effective than a one-dose vaccine (56.7% vs. 43.8%). In addition, the population that got the second dose vaccine within 2 months showed higher VE than the population vaccinated for longer than 2 months (61.5% vs. 52.3%). Among the population who vaccinated 2 doses or within 2 months, a higher level of neutralizing antibodies was observed. For infected cases, vaccinated populations showed lower rates of transmission (2.63% vs. 4.36%). Further, those vaccinated cases, who were not found causing transmission, had a higher level of antibodies. The study provided a full view of the effectiveness of inactivated vaccines in a real-world setting. The time-related VE against infection and lower transmission of breakthrough vaccinated cases were observed, which may indicate that a necessity of a booster vaccine to maintain the effectiveness and high level of neutralizing antibody.

Oxfendazole Induces Apoptosis in Ovarian Cancer Cells by Activating JNK/MAPK Pathway and Inducing Reactive Oxygen Species Generation.

Ovarian cancer (OC) is one of the most common and high mortality type of cancer among women worldwide. The majority of patients with OC respond to chemotherapy initially; however, most of them become resistant to chemotherapy and results in a high level of treatment failure in OC. Therefore, novel agents for the treatment of OC are urgently required. Benzimidazole anthelmintics might have the promising efficacy for cancer therapy as their selectively binding activity to ß-tubulin. Recent study has shown that one of the benzimidazole anthelmintics oxfendazole inhibited cell growth of non-small cell lung cancer cells, revealing its anti-cancer activity; however, the pharmacological action and detailed mechanism underlying the effects of oxfendazole on OC cells remain unclear. Therefore, the present study investigated the cytotoxic effects of oxfendazole on OC cells. Our results demonstrated that oxfendazole significantly decreased the viability of OC cells. Oxfendazole inhibited the proliferation, induced G2/M phase arrest and apoptotic cell death in A2780 cells. The c-Jun N-terminal kinase (JNK)/mitogen-activated protein kinase (MAPK) pathway was activated and reactive oxygen species (ROS) generation was increased in OC cells treated with oxfendazole; oxfendazole-induced apoptosis was notably abrogated when co-treated with JNK inhibitor SP600125 and ROS scavenger N-acetyl-L-cysteine (NAC), indicating that JNK/MAPK pathway activation and ROS accumulation was associated with the oxfendazole-induced apoptosis of OC cells. Moreover, oxfendazole could also induce the proliferation inhibition and apoptosis of cisplatin resistant cells. Collectively, these results revealed that oxfendazole may serve as a potential therapeutic agent for the treatment of OC.

Marine-Fungi-Derived Gliotoxin Promotes Autophagy to Suppress Mycobacteria tuberculosis Infection in Macrophage.

The Mycobacterium tuberculosis (MTB) infection causes tuberculosis (TB) and has been a long-standing public-health threat. It is urgent that we discover novel antitubercular agents to manage the increased incidence of multidrug-resistant (MDR) or extensively drug-resistant (XDR) strains of MTB and tackle the adverse effects of the first- and second-line antitubercular drugs. We previously found that gliotoxin (1), 12, 13-dihydroxy-fumitremorgin C (2), and helvolic acid (3) from the cultures of a deep-sea-derived fungus, Aspergillus sp. SCSIO Ind09F01, showed direct anti-TB effects. As macrophages represent the first line of the host defense system against a mycobacteria infection, here we showed that the gliotoxin exerted potent anti-tuberculosis effects in human THP-1-derived macrophages and mouse-macrophage-leukemia cell line RAW 264.7, using CFU assay and laser confocal scanning microscope analysis. Mechanistically, gliotoxin apparently increased the ratio of LC3-II/LC3-I and Atg5 expression, but did not influence macrophage polarization, IL-1ß, TNF-a, IL-10 production upon MTB infection, or ROS generation. Further study revealed that 3-MA could suppress gliotoxin-promoted autophagy and restore gliotoxin-inhibited MTB infection, indicating that gliotoxin-inhibited MTB infection can be treated through autophagy in macrophages. Therefore, we propose that marine fungi-derived gliotoxin holds the promise for the development of novel drugs for TB therapy.

Overexpression of MicroRNA-155 aggravates pulpitis by targeting kinesin superfamily Proteins-5C based on illumina high-throughput sequencing.

AIM: To investigate the regulatory role of miR-155 and Kinesin Superfamily Proteins-5C (KIF-5C) in the progression of pulpitis based on bioinformatic analysis. METHODOLOGY: Normal pulp tissues and pulpitis pulp tissues were collected and subjected to high-throughput sequencing and the differentially expressed miRNAs were determined. An in vitro and in vivo pulpitis model was established. HE, IHC staining and histological evaluation were used to verify the inflammatory state of human and mouse pulp tissues. The mRNA expression of IL-1ß and TGF-ß1 were determined by RT-qPCR and protein expression of IL-1α, IL-4, IL-8, IL-13, IFN-γ, IL-6, IL-10 and MCP-1 were determined by protein chip. The target genes of miR-155 were predicted by miRanda database and verified by Dual-luciferase reporter assay, RT-qPCR and western blotting. MiR-155 lentivirus were used to upregulate or downregulate miR-155 and the siRNA of KIF-5C was used to downregulate KIF-5C. The expression of miR-155 or KIF-5C was determined by RT-qPCR. All statistics were analysed using GraphPad prism 8.2. RESULTS: The high-throughput sequencing results showed that 6 miRNAs (miR-155, miR-21, miR-142, miR-223, miR-486, miR-675) were significantly upregulated in diseased human pulp tissues, and miR-155 was significantly elevated among the six miRNAs. RT-qPCR results demonstrated that miR-155 expression was upregulated in human pulpitic tissue, mice pulpitic tissue and LPS-HDPCs. IL-1ß was increased while TGF-ß1 was decreased in lenti-miR-155 transfected LPS-HDPCs. Analysis of protein chip results indicated that lenti-miR-155 transfected LPS-HDPCs produced higher levels of IL-8, IL-6, MCP-1. The opposite results were obtained when miR-155 was inhibited. Through miRanda database screen and Dual-luciferase reporter assay, the target gene (KIF-5C) of miR-155 was identified. In lenti-miR-155 transfected LPS-HDPCs, the expression of KIF-5C was downregulated. However, when shRNA-miR-155 was transfected to LPS-HDPCs, the opposite result was obtained. Silent RNA was used to knock down KIF-5C, the results showed that when both KIF-5C and miR-155 were knocked down simultaneously, the downregulated expression of inflammatory factors observed in LPS-HDPCs following miR-155 knockdown was rescued. CONCLUSION: MiR-155 plays an important role in promoting pulpitis through targeting KIF-5C and may serve as a potential therapeutic target.

Comparison of the efficacy and safety of total laparoscopic hysterectomy without and with uterine manipulator combined with pelvic lymphadenectomy for early cervical cancer.

OBJECTIVE: Some studies have reported that the prognosis of total laparoscopic hysterectomy (TLH) for early-stage cervical cancer (CC) is worse than that of open surgery. And this was associated with the use of uterine manipulator or not. Therefore, this study retrospectively analyzes the efficacy and safety of TLH without uterine manipulator combined with pelvic lymphadenectomy for early-stage CC. METHODS: Fifty-eight patients with CC (stage IB1-IIA1) who received radical hysterectomy from September 2019 to January 2020 were divided into no uterine manipulator (n = 26) and uterine manipulator group (n = 32). Then, clinical characteristics were collected and intraoperative/postoperative related indicators were compared. RESULTS: Patients in the no uterine manipulator group had significantly higher operation time and blood loss than in the uterine manipulator group. Notably, there was no significant difference in hemoglobin change, blood transfusion rate, number of pelvic nodules, anal exhaust time, complications and recurrence rate between the two groups. Additionally, patients in the uterine manipulator group were prone to urinary retention (15.6%) and lymphocyst (12.5%), while the no uterine manipulator group exhibited high probability of bladder dysfunction (23.1%) and urinary retention (15.4%). Furthermore, the 1-year disease-free survival rate and the 1-year overall survival rate were not significantly different between the two groups. CONCLUSION: There was no significant difference in the efficacy and safety of TLH with or without uterine manipulator combined with pelvic lymphadenectomy in the treatment of patients with early-stage CC. However, the latter requires consideration of the negative effects of high operation time and blood loss.

Vaginal microecological changes of different degrees of cervical lesions in Hakka women in Meizhou City.

Research shows an association between vaginal microbiota and the development of cervical cancer, but the role of altered microbiota in cancer development remains controversial. In this study, we attempted to reveal the vaginal microecological changes in cervical lesions by 16S rRNA gene sequencing. Vaginal secretions were collected from Hakka women in Meizhou City, Guangdong Province, China. The diversity, composition and the correlations among species of the vaginal microbiota were determined by sequencing the bacterial 16S rRNA gene. The microbial functional abundance was detected via KEGG and COG (Clusters of Orthologous Groups). The results showed that the Cancer group was characterised by evident changes in the composition of the vaginal microbiota, increased alpha diversity, and altered community structure distribution and microbial interaction network. Linear discriminant analysis (LDA) effect size showed that 21 bacterial species were abundant in the Cancer group. In addition, the loss of Lactobacillus stimulated other flora proliferation, resulting in a microecological disturbance. KEGG and COG analysis indicated the cancer group is mainly concentrated in energy metabolism. In short, the vaginal microecology of Hakka women in Meizhou City presents with different degrees of cervical lesions, and the flora imbalance is an important factor in the development of cervical cancer.IMPACT STATEMENTWhat is already known on this subject? Cervical cancer is one of the most common gynecological malignancies worldwide and has become a prominent public health problem.What the results of this study add? Our study showed that the type of vaginal community status of Hakka women in Meizhou area was characterised by L. Iners predominates, and the gradual loss of Lactobacillus dominance in vaginal bacteria is key to microecological imbalance.What the implications are of these findings for clinical practice and/or further research? Disturbances in vaginal microecology can stimulate energy metabolism and lipid metabolism to induce cervical cancer development.

Development and Validation of a Nomogram for Predicting Postoperative Distant Metastasis in Patients with Cervical Cancer.

BACKGROUND Cervical cancer is the fourth most commonly diagnosed malignant neoplasm among women worldwide. Despite improvements in treatment, the rate of postoperative metastasis remains a problem. Nomograms have been used to predict risk of tumor metastasis. We designed a nomogram to predict postoperative distant metastasis among cervical cancer patients, based on the SEER database, and estimated the performance of the nomogram by internal and external validations. MATERIAL AND METHODS We included 6421 participants and divided them into training (n=4495) and testing (n=1926) sets. Multivariate logistic regression was used to explore predictors. The nomogram's predictive value was assessed by internal (testing set) and external (561 Chinese patients) validations. The receiver operating characteristic curve (ROC) was plotted, and the area under the curve (AUC) value was calculated to evaluate the nomogram's discrimination. The nomogram's calibration was assessed via the Hosmer-Lemeshow test and calibration curve. RESULTS Histologic type, T stage, treatment, tumor size, and positive lymph node were identified as independent predictors of postoperative distant metastasis in surgical patients (P<0.05). The developed nomogram had an AUC of 0.866 (95% CI: 0.844 to 0.888). The AUC and the chi-square for the Hosmer-Lemeshow test of the nomogram were 0.847 (95% CI: 0.807 to 0.888) and 11.292, respectively, (P>0.05) in the internal validation, and were 0.626 (95% CI: 0.548 to 0.704) and 316.53, respectively, (P<0.05) in the external validation. CONCLUSIONS Our nomogram showed a good predictive performance for postoperative distant metastasis in cervical cancer patients based on the SEER database. It remains to be determined if it is applicable to other populations.

LncRNA DNM3OS regulates GREM2 via miR-127-5p to suppress early chondrogenic differentiation of rat mesenchymal stem cells under hypoxic conditions.

BACKGROUND: Improved chondrogenic differentiation of mesenchymal stem cells (MSCs) by genetic regulation is a potential method for regenerating articular cartilage. MiR-127-5p has been reported to promote cartilage differentiation of rat bone marrow MSCs (rMSCs); however, the regulatory mechanisms underlying hypoxia-stimulated chondrogenic differentiation remain unknown. METHODS: rMSCs were induced to undergo chondrogenic differentiation under normoxic or hypoxic conditions. Expression of lncRNA DNM3OS, miR-127-5p, and GREM2 was detected by quantitative real-time PCR. Proteoglycans were detected by Alcian blue staining. Western blot assays were performed to examine the relative levels of GREM2 and chondrogenic differentiation related proteins. Luciferase reporter assays were performed to assess the association among DNM3OS, miR-127-5p, and GREM2. RESULTS: MiR-127-5p levels were upregulated, while DNM3OS and GREM2 levels were downregulated in rMSCs induced to undergo chondrogenic differentiation, and those changes were attenuated by hypoxic conditions (1% O2). Further in vitro experiments revealed that downregulation of miR-127-5p reduced the production of proteoglycans and expression of chondrogenic differentiation markers (COL1A1, COL2A1, SOX9, and ACAN) and osteo/chondrogenic markers (BMP-2, p-SMAD1/2). MiR-127-5p overexpression produced the opposite results in rMSCs induced to undergo chondrogenic differentiation under hypoxic conditions. GREM2 was found to be a direct target of miR-127-5p, which was suppressed in rMSCs undergoing chondrogenic differentiation. Moreover, DNM3OS could directly bind to miR-127-5p and inhibit chondrogenic differentiation of rMSCs via regulating GREM2. CONCLUSIONS: Our study revealed a novel molecular pathway (DNM3OS/miR-127-5p/GREM2) that may be involved in hypoxic chondrogenic differentiation.

Special attention to nurses' protection during the COVID-19 epidemic.

As of March 8, 2020, the novel coronavirus disease 2019 (COVID-19) had caused 80,815 human infections and 3073 deaths in China, including more than 3000 infections among medical staff. Guangdong Second Provincial General Hospital (Guangzhou, Guangdong Province, China), a provincial emergency hospital, has treated more than 35 confirmed cases of COVID-19 and more than 260 suspected cases. Most of nurses' work involves direct contact with patients. As nurses have high vulnerability to COVID-19, it is necessary to establish hospital-specific protocols to reduce the risk of nurses' infection in interactions with COVID-19 patients. Our hospital has maintained a "zero nurse infection" rate while battling SARS in 2003 and during the present COVID-19 epidemic. The following are the key measures implemented in our hospital.

Letrozole Rat Model Mimics Human Polycystic Ovarian Syndrome and Changes in Insulin Signal Pathways.

BACKGROUND The aim of this study was to explore whether letrozole and high-fat diets (HFD) can induce obese insulin-resistant polycystic ovary syndrome (PCOS) with all reproductive and metabolic phenotypes in a rat model. MATERIAL AND METHODS Twenty-four 3-week-old female Sprague-Dawley rats were randomized into 4 groups: control, Letrozole, HFD, and Letrozol+HFD. The PCOS model was induced by 12 weeks of Letrozole treatment (1 mg/kg p.o. dissolved in 0.5% CMC solutions once daily) and HFD. Ovarian morphology, estrous cyclicity, hormonal status, body weight, glucose and insulin tolerance, lipid profile, and insulin signaling pathway were investigated. RESULTS The rat model manifests anovulatory cycles and PCO morphology, increased body weight, elevated testosterone levels, abnormal glucose and lipid metabolism, and insulin resistance. The rat model also expresses significantly decreased phosphorylation of 6 essential signaling proteins - INSR, IRS, PI3K, AKT, ERK1, ERK2 - in the PI3K/AKT and MAPK/ERK pathways in the classic insulin-sensitive tissues (e.g., quadriceps femoris muscle, omentum majus, and liver), as well as non-classic target ovary tissues. Disrupted insulin signaling contributes to the decrease in insulin sensitivity and compensatory hyperinsulinemia in PCOS rats. CONCLUSIONS Continuous administration of letrozole and high-fat diets can induce PCOS, metabolic phenotypes, and disrupted activation of the insulin signaling pathway.

Recurrent nonsevere hypoglycemia exacerbates imbalance of mitochondrial homeostasis leading to synapse injury and cognitive deficit in diabetes.

Recurrent nonsevere hypoglycemia (RH) can lead to cognitive dysfunction in patients with diabetes, although the involved mechanisms remain unclear. Here, we aimed to investigate the mechanism underlying RH-induced cognitive deficits with a focus on mitochondrial homeostasis. To establish a model that mimicked RH in patients with type 1 diabetes (T1DM) receiving insulin therapy, streptozotocin-induced mice with T1DM were subjected to recurrent, twice-weekly insulin injections over 4 wk. We found that RH disrupted the mitochondrial fine structure, reduced the number of mitochondria, and upregulated the expression of mitochondrial dynamics and mitophagy markers, including dynamin-related protein 1 (Drp1), Bcl-2/adenovirus E1B 19-kDa-interacting protein-3 (BNIP3), and microtubule-associated protein 1 light-chain 3 (LC3) in the hippocampus of T1DM mice. Moreover, RH and chronic hyperglycemia synergistically promoted the production of reactive oxygen species, impaired mitochondrial membrane potential, and suppressed mitochondrial energy metabolism. Under diabetic conditions, RH also altered the synaptic morphology and reduced the expression of synaptic marker proteins. Long-term recognition memory and spatial memory, assessed with the Morris water maze test, were also impaired. However, these effects were largely prevented by mitochondrial division inhibitor 1, a potent and selective Drp1 inhibitor. Thus, it appears that RH exacerbates the imbalance of mitochondrial homeostasis, leading to synapse injury and cognitive deficits in diabetes. The adjustment of mitochondrial homeostasis could serve as an effective neuroprotective approach when addressing low blood sugar conditions.

Regulatory SNPs Alter the Gene Expression of Diabetic Retinopathy Associated Secretary Factors.

OBJECTIVES: Diabetic retinopathy (DR) is a common microvascular complication in both type I and type II diabetes. Several previous reports indicated the serum centration of some secretary factors were highly associated with DR. Therefore, we hypothesis regulatory SNPs (rSNPs) genotype in secretary factors may alter these gene expression and lead to DR. METHODS: At first, pyrosequencing were applying to screen the SNPs which present allele frequency different in DR and DNR. Then individual genotyping was processed by Taqman assays in Taiwanese DR and DNR patients. To evaluate the effect of SNP allele on transcriptional activity, we measured promoter activity using luciferase reporter constructs. RESULTS: We found the frequencies of the CC, CG, and GG genotype of the rs2010963 polymorphism were 15.09%, 47.14%, and 37.74% in DR and 12.90%, 19.35%, and 67.74% in DNR, respectively (p = 0.0205). The prevalence of DR was higher (p = 0.00793) in patients with the CC or CG genotype (62.26% and 32.26% for DR and DNR, respectively) compared with the patients with the GG genotype. To evaluate the effect of rs2010963-C allele on transcriptional activity, we measured promoter activity using luciferase reporter constructs. The rs2010963-C reporter showed 1.6 to 2-fold higher luciferase activity than rs2010963-G in 3 cell lines. CONCLUSION: Our data proposed rs2010963-C altered the expression level of VEGFA in different tissues. We suggested small increase but long term exposure to VEGFA may lead to DR finally.