Creatine kinase mitochondrial 2 promotes the growth and progression of colorectal cancer via enhancing Warburg effect through lactate dehydrogenase B.

Background: Mitochondrial creatine kinase (MtCK) plays a pivotal role in cellular energy metabolism, exhibiting enhanced expression in various tumors, including colorectal cancer (CRC). Creatine kinase mitochondrial 2 (CKMT2) is a subtype of MtCK; however, its clinical significance, biological functions, and underlying molecular mechanisms in CRC remain elusive. Methods: We employed immunohistochemical staining to discern the expression of CKMT2 in CRC and adjacent nontumor tissues of patients. The correlation between CKMT2 levels and clinical pathological factors was assessed. Additionally, we evaluated the association between CKMT2 and the prognosis of CRC patients using Kaplan-Meier survival curves and Cox regression analysis. Meanwhile, quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression levels of CKMT2 in different CRC cell lines. Finally, we explored the biological functions and potential molecular mechanisms of CKMT2 in CRC cells through various techniques, including qRT-PCR, cell culture, cell transfection, western blot, Transwell chamber assays, flow cytometry, and co-immunoprecipitation. Results: We found that CKMT2 was significantly overexpressed in CRC tissues compared with adjacent nontumor tissues. The expression of CKMT2 is correlated with pathological types, tumor size, distant metastasis, and survival in CRC patients. Importantly, CKMT2 emerged as an independent prognostic factor through Cox regression analysis. Experimental downregulation of CKMT2 expression in CRC cell lines inhibited the migration and promoted apoptosis of these cells. Furthermore, we identified a novel role for CKMT2 in promoting aerobic glycolysis in CRC cells through interaction with lactate dehydrogenase B (LDHB). Conclusion: In this study, we found the elevated expression of CKMT2 in CRC, and it was a robust prognostic indicator in CRC patients. CKMT2 regulates glucose metabolism via amplifying the Warburg effect through interaction with LDHB, which promotes the growth and progression of CRC. These insights unveil a novel regulatory mechanism by which CKMT2 influences CRC and provide promising targets for future CRC therapeutic interventions.

Analysis of treatment-related adverse events and wound complications of surgical resection after neoadjuvant chemoimmunotherapy for non-small cell lung cancer.

Neoadjuvant chemoimmunotherapy is becoming an increasingly important part of the management of lung cancer to facilitate surgical resection. This study aimed to summarize the treatment-related adverse events (TRAEs) and wound complications of neoadjuvant chemoimmunotherapy in non-small cell lung cancer (NSCLC). Eligible studies of neoadjuvant chemoimmunotherapy for NSCLC were identified from PubMed, Embase and Web of Science. The endpoints mainly included TRAEs and wound complications. Stata18 software was used for statistical analysis with p < 0.05 considered statistically significant. Twenty studies including a total of 1072 patients were eligible for this study. Among the patients who received neoadjuvant chemoimmunotherapy, the pooled prevalence of any grade TRAEs was 77% (95% confidence interval [CI] [0.64-0.86]), grade 1-2 TRAEs was 77% (95% CI [0.58-0.89]) and grade ≥3 TRAEs was 26% (95% CI [0.16-0.38]). Surgery-related complications rate was 22% (95% CI [0.14-0.33]). Among the wound complications, the pooled rate of air leakage was 10% (95% CI [0.04-0.23]), pulmonary/wound infection was 8% (95% CI [0.05-0.13]), bronchopleural fistula was 8% (95% CI [0.02-0.27]), bronchopulmonary haemorrhage was 3% (95% CI [0.01-0.05]), pneumonia was 5% (95% CI [0.02-0.10]), pulmonary embolism was 1% (95% CI [0.01-0.03]), pleural effusion was 7% (95% CI [0.03-0.14]) and chylothorax was 4% (95% CI [0.02-0.09]). Overall, neoadjuvant chemoimmunotherapy in NSCLC results a high incidence of grade 1-2 TRAEs but a low risk of increasing the incidence of ≥3 grade TRAEs and wound complications. These results need to be confirmed by more large-scale prospective randomized controlled trials and studies.

Azoramide ameliorates cadmium-induced cytotoxicity by inhibiting endoplasmic reticulum stress and suppressing oxidative stress.

Background: Cadmium (Cd) is hazardous to human health because of its cytotoxicity and long biological half-life. Azoramide is a small molecular agent that targets the endoplasmic reticulum (ER) and moderates the unfolded protein response. However, its role in Cd-induced cytotoxicity remains unclear. This study was performed to investigate the protective effect of azoramide against Cd-induced cytotoxicity and elucidate its underlying mechanisms. Methods: Inductively coupled plasma‒mass spectrometry was used to measure Cd concentrations in each tissue of ICR male mice. The human proximal tubule epithelial cell line HK-2 and the human retinal pigment epithelial cell line ARPE-19 were used in the in vitro study. Cell apoptosis was determined by DAPI staining, JC-1 staining, and annexin V/propidium iodide double staining. Intracellular oxidative stress was detected by MitoSOX red staining, western blot, and quantitative real-time PCR. Moreover, ER stress signaling, MAPK cascades, and autophagy signaling were analyzed by western blot. Results: The present data showed that Cd accumulated in various organs of ICR mice, and the concentrations of Cd in the studied organs, from high to low, were as follows: liver > kidney > testis > lung > spleen > eye. Our study demonstrated that azoramide inhibited ER stress by promoting BiP expression and suppressing the PERK-eIF2α-CHOP pathway. Additionally, we also found that azoramide significantly decreased ER stress-associated radical oxidative species production, attenuated p38 MAPK and JNK signaling, and inhibited autophagy, thus suppressing apoptosis in HK-2 and ARPE-19 cells. Conclusion: Our study investigated the effect of azoramide on Cd-induced cytotoxicity and revealed that azoramide may be a therapeutic drug for Cd poisoning.

Bowel Stiffness Assessed by Shear-Wave Ultrasound Elastography Predicts Disease Behavior Progression in Patients With Crohn's Disease.

INTRODUCTION: There is a lack of reliable predictors of disease behavior progression in patients with Crohn's disease (CD). Real-time shear-wave elastography (SWE) is a novel method for evaluating tissue stiffness. However, its value for assessing CD has not yet been investigated. We aimed to explore the value of SWE and other ultrasound parameters at diagnosis in predicting CD behavior progression. METHODS: We retrospectively collected data from patients with CD with the nonstenotic nonpenetrating disease (B1 phenotype based on the Montreal classification). All patients underwent intestinal ultrasound at baseline and were followed up. The end point was defined as disease behavior progression to stricturing (B2) or penetrating (B3) disease. Cox regression analysis was performed for the association between baseline characteristics and subsequent end points. In addition, a multivariate nomogram was established to predict the risk of disease behavior progression quantitatively. RESULTS: A total of 130 patients with CD with B1 phenotype were enrolled. Twenty-seven patients (20.8%) developed B2 or B3 disease, with a median follow-up of 33 months. Multivariate analysis identified that SWE was the only independent predictor of disease behavior progression (hazard ratio 1.08, 95% confidence interval 1.03-1.12, P = 0.001). A reverse of the HR appeared at the cutoff 12.75 kPa. The nomogram incorporating SWE and other clinical characteristics showed a good prediction performance (area under the curve = 0.792). DISCUSSION: Intestinal stiffness assessed using SWE is an independent predictor of disease behavior progression in patients with CD. Patients with CD with SWE >12.75 kPa at diagnosis are prone to progress toward stricturing or penetrating diseases.

Ovatodiolide induces autophagy-mediated cell death through the p62-Keap1-Nrf2 signaling pathway in chronic myeloid leukemia cells.

Ovatodiolide is a macrocyclic diterpenoid compound with various biological activities that displays considerable anticancer potential in different tumor models. However, the underlying mechanism for this antineoplastic activity remains unclear. The aim of the present study was to investigate the anticancer effect and possible molecular mechanism of ovatodiolide in human chronic myeloid leukemia (CML). Ovatodiolide suppressed cell colony formation and induced apoptosis in the K562 and KU812 cells. We also observed that ovatodiolide enhanced the production of reactive oxygen species (ROS), activated Nrf2 signaling, and inhibited mTOR phosphorylation. Autophagic flux was shown to be enhanced after treatment with ovatodiolide in K562 cells. Furthermore, autophagy inhibition alleviated ovatodiolide-induced cell apoptosis, whereas autophagy promotion aggravated apoptosis in CML cells. These results demonstrated that ovatodiolide activates autophagy-mediated cell death in CML cells. Additionally, ovatodiolide transcriptionally activated the expression of p62, and the p62 levels were negatively regulated by autophagy. Moreover, p62-Keap1-Nrf2 signaling was confirmed to be involved in ovatodiolide-induced cell death. Accordingly, LC3B knockdown augmented the ovatodiolide-induced p62 expression, increased the p62-Keap1 interaction, and enhanced the translocation of Nrf2 into the nucleus. In contrast, p62 inhibition abolished the effects that were induced through ovatodiolide treatment. Nrf2 inhibition with ML385 diminished the protective effect of autophagy inhibition in CML cells. Collectively, our results indicate that ovatodiolide induces oxidative stress and provokes autophagy, which effectively decreases the expression of p62 and weakens the protective effect of Nrf2 signaling activation, thus contributing to apoptosis in CML cells.

Conservative treatment of hepatic portal vein gas after transarterial chemoembolization treatment for liver metastasis of postoperative esophageal cancer: a case report.

Background: Hepatic portal vein gas (HPVG), which is a rare clinical manifestation, is usually considered a sign of critical illness. If the treatment is not timely, it will lead to intestinal ischemia, intestinal necrosis, and even death. There is still no consensus on whether to adopt surgical or conservative treatment for HPVG. Herein, we report a rare case of conservative treatment of HPVG after transarterial chemoembolization (TACE) treatment in a patient with liver metastasis of postoperative esophageal cancer, who received long-term enteral nutrition (EN). Case Description: A 69-year-old male patient, who had undergone surgery for esophageal cancer, needed long-term use of jejunal feeding tube implantation for enteral nutritional support due to postoperative complications. About 9 months after the operation, multiple metastases of the liver were detected. To control the progress of the disease, TACE was conducted. EN was restored on the second day after TACE, and the patient was discharged on the fifth day. On the night of discharge, the patient suddenly experienced abdominal pain, nausea, and vomiting. Abdominal computed tomography (CT) showed that the abdominal intestinal lumen was obviously dilated, liquid and gas plane shadowing was visible, and gas was visible in the portal vein and its branches. The physical examination showed that peritoneal irritation was present, and bowel sounds were active. Blood routine examination showed an increase in neutrophil and neutrophil. Symptomatic treatment, including gastrointestinal decompression, anti-infection, and parenteral nutritional support, was provided. On the third day after the presentation of HPVG, abdominal CT reexamination showed that HPVG had disappeared and the intestinal obstruction was relieved. Repeated blood routine shows a decrease in neutrophil and neutrophil. Conclusions: Elderly patients who require long-term EN support should avoid early EN support after TACE, as this can prevent intestinal obstruction and HPVG. If the patient suddenly experiences abdominal pain after TACE, CT scan should be performed in a timely manner to determine whether there is intestinal obstruction and HPVG. If the above type of patient experiences HPVG, conservative treatments such as early gastrointestinal decompression, fasting, and anti-infection treatment can be provided first without high-risk factors.

A novel defined cuproptosis-related gene signature for predicting the prognosis of lung adenocarcinoma.

Lung adenocarcinoma (LUAD) has become the most prevalent histologic subset of primary lung cancer, and effective innovative prognostic models are needed to enhance the feasibility of targeted therapies for the disease. Programmed cell death (PCD) performs an integral function in the origin and treatment of cancer. Some PCD-related effective signatures for predicting prognosis in LUAD patients could provide potential therapeutic options in LUAD. A copper-dependent cell death referred to as cuproptosis is distinct from known PCD. However, whether cuproptosis is associated with LUAD patients' prognoses and the potential roles of cuproptosis-related genes involved is still unknown. For the prediction of LUAD prognosis, we developed a unique cuproptosis-associated gene signature. In The Cancer Genome Atlas (TCGA) cohort, the score derived from the risk signature on the basis of six cuproptosis-related genes was found to independently serve as a risk factor for anticipating lung cancer-related death. The differentially expressed genes between the high- and low-risk groups were linked to the cilium-related function. LUAD patients' prognoses may now be predicted by a unique gene signature identified in this work. This discovery also provides a substantial foundation for future research into the links between cuproptosis-associated genes and cilium-related function in LUAD patients.

Low Female Gametophyte Fertility Contributes to the Low Seed Formation of the Diploid Loquat [Eriobotrya Japonica (Thunb.) Lindl.] Line H30-6.

Loquat is a widely grown subtropic fruit because of its unique ripening season, nutrient content, and smooth texture of its fruits. However, loquat is not well-received because the fruits contain many large seeds. Therefore, the development of seedless or few-seed loquat varieties is the main objective of loquat breeding. Polyploidization is an effective approach for few-seed loquat breeding, but the resource is rare. The few-seed loquat line H30-6 was derived from a seedy variety. Additionally, H30-6 was systematically studied for its fruit characteristics, gamete fertility, pollen mother cell (PMC) meiosis, stigma receptivity, in situ pollen germination, fruit set, and karyotype. The results were as follows. (1) H30-6 produced only 1.54 seeds per fruit and the fruit edible rate was 70.77%. The fruit setting rate was 14.44% under open pollination, and the other qualities were equivalent to those of two other seedy varieties. (2) The in vitro pollen germination rate was only 4.04 and 77.46% of the H30-6 embryo sacs were abnormal. Stigma receptivity and self-compatibility in H30-6 were verified by in situ pollen germination and artificial pollination. Furthermore, the seed numbers in the fruits of H30-6 did not significantly differ among any of the pollination treatments (from 1.59 ±0.14 to 2 ± 0.17). (3) The chromosome configuration at meiotic diakinesis of H30-6 was 6.87I + 9.99II + 1.07III +0.69IV +0.24V (H30-6), and a total of 89.55% of H30-6 PMCs presented univalent chromosomes. Furthermore, chromosome lagging was the main abnormal phenomenon. Karyotype analysis showed that chromosomes of H30-6 had no recognizable karyotype abnormalities leading to unusual synapsis on the large scale above. (4) The abnormal embryo sacs of H30-6 could be divided into three main types: those remaining in the tetrad stage (13.38%), those remaining in the binucleate embryo sac stage (1.41%), and those without embryo sacs (52.82%). Therefore, we conclude that the loquat line H30-6 is a potential few-seed loquat resource. The diploid loquat line H30-6 was with low gametophyte fertility, which may be driven by abnormal meiotic synapses. The low female gamete fertility was the main reason for the few seeds. This diploid loquat line provides a new possibility for breeding a few-seed loquat at the diploid level.

Application of Contrast-enhanced Ultrasound and Bosniak Classification to the Diagnosis of Cystic Renal Masses.

BACKGROUND: The Bosniak classification system based on contrast-enhanced computed tomography (CECT) is commonly used for the differential diagnosis of cystic renal masses. Contrastenhanced ultrasound (CEUS) is a relatively novel technique, which has gradually played an important role in the diagnosis of cystic renal cell carcinoma (CRCC) due to its safety and lowest price. OBJECTIVE: The aim of the study is to investigate the application value of CEUS and Bosniak classification into the diagnosis of cystic renal masses. METHODS: 32 cystic masses from January 2018 to December 2019 were selected. The images of conventional ultrasound (US), CEUS and CECT from subjects confirmed by surgical pathology were retrospectively analyzed. The Bosniak classification system of cystic renal masses was implemented using CEUS and CECT, and the diagnostic ability was compared. RESULTS: For the 32 cystic masses, postoperative pathology confirmed 11 cases of multilocular CRCC, 15 cases of clear cell carcinoma with hemorrhage, necrosis and cystic degeneration, 5 cases of renal cysts, and 1 case of renal tuberculosis. The Bosniak classification based on CEUS was higher than that based on CECT, and the difference was statistically significant (P = .024). The sensitivity, specificity, positive predictive value, negative predictive value and accuracy of CEUS were comparable to CECT. There was no significant difference observed in the diagnosis of CRCC (P >.05). CONCLUSION: CEUS combined with Bosniak classification greatly improves the diagnosis of CRCC. CEUS shows a comparable diagnostic ability to CECT. In daily clinical routine, patients who require multiple examinations and present contraindications for CECT can particularly benefit from CEUS.

Longitudinal Bowel Behavior Assessed by Bowel Ultrasound to Predict Early Response to Anti-TNF Therapy in Patients With Crohn's Disease: A Pilot Study.

BACKGROUND: Early changes in bowel behavior during anti-tumor necrosis factor (anti-TNF) induction therapy in Crohn's disease (CD) are relatively unknown. We determined (1) the onset of changes in bowel behavior in CD patients receiving anti-TNF therapy by ultrasound and (2) the feasibility of shear wave elastography (SWE) in predicting early response to anti-TNF therapy. METHODS: Consecutive ileal or ileocolonic CD patients programmed to initiate anti-TNF therapy were enrolled. Bowel ultrasound was performed at baseline and at weeks 2, 6, and 14. Changes in bowel wall thickness, Doppler signals of the bowel wall (Limberg score), and SWE values were compared using a linear mixed model. Early response to anti-TNF therapy was based on a composite strategy of clinical and colonoscopy assessment at week 14. RESULTS: Of the 30 patients enrolled in this study, 20 patients achieved a response to anti-TNF therapy at week 14. The bowel wall thickness and SWE value of the response group showed a significant downward trend compared with the nonresponse group (P = .003 and P = .011, respectively). Bowel wall thickness, the Limberg score, and SWE values were significantly reduced as early as week 2 compared with baseline (P < .001, P < .001, and P = .003, respectively) in the response group. Baseline SWE values (21.3 ± 8.7 kPa vs 15.3 ± 4.7 kPa; P = .022) and bowel wall thickness (8.5 ± 2.3 mm vs 6.9 ± 1.5 mm; P = .027) in the nonresponse group were significantly higher than in the response group. CONCLUSIONS: This pilot study suggested that changes in bowel ultrasound behavior could be assessed as early as week 2 after starting anti-TNF therapy. Bowel ultrasound together with elasticity imaging could predict early response to anti-TNF therapy.

This pilot study suggested that changes in bowel ultrasound behavior could be assessed as early as 2 weeks after anti-tumor necrosis factor therapy in patients with Crohn's disease. Bowel ultrasound together with elasticity imaging could predict early response to anti-tumor necrosis factor therapy.

Iron overload inhibits cell proliferation and promotes autophagy via PARP1/SIRT1 signaling in endometriosis and adenomyosis.

Emerging evidence suggests that excess iron accumulates in endometriotic and adenomyotic lesions. However, the role iron overload plays in the pathogenesis of endometriosis or adenomyosis remains unknown. Primary human eutopic endometrial stromal cells (EuESCs) from endometriosis or adenomyosis patients were used as the in vitro model of endometriosis or adenomyosis in this study. We found that iron, manifesting as ferric ammonium citrate (FAC; 0.05-4.8 mM), significantly inhibited cell growth, induced oxidative stress through the Fenton reaction, and functionally activated autophagy in EuESCs, as measured by 5-ethynyl-2'-deoxyuridine incorporation assay, MitoSOX™ Red staining, LC3 turnover assay, and tandem mCherry-eGFP-LC3B fluorescence microscopy. Immunohistochemistry analysis of Ki67 expression in proliferative-phase endometrial tissues revealed that cell proliferation in ectopic tissues was dramatically compromised, suggesting that iron overload may play a role in cell growth inhibition in vivo. We observed that autophagy may alleviate the FAC-induced inhibition of endometrial stromal cell proliferation. Furthermore, sequential FAC (0.8 mM, 24 h) and hydrogen peroxide (H2O2; 300 µM, 2 h) treatment successfully induced the Fenton reaction in EuESCs and caused extensive apoptosis, whereas the disruption of autophagy by the knockdown of BECN1 further aggravated cell death. MitoSOX™ Red staining showed that autophagy may promote the survival of EuESCs by decreasing of the Fenton reaction-induced reactive oxygen species generation. In addition, we observed that the Fenton reaction-induced oxidative stress significantly suppressed iron overload-induced autophagy. Moreover, we found that FAC treatment impaired poly(ADP-ribose)-polymerase 1 (PARP1) expression while simultaneously upregulating SIRT1 expression in EuESCs. Our data further showed that PARP1 expression decreased in endometriotic lesions, which may partially result from iron overload. We also found that PARP1 inhibition aggravated iron overload-induced cell growth suppression, and was implicated in iron overload-induced autophagy. In addition, SIRT1 silencing alleviated iron overload-induced PARP1 downregulation and autophagy activation. Overall, our data suggest that iron overload in endometrial stromal cells of endometriotic or adenomyotic lesions may be involved in the inhibition of cell proliferation, simultaneously with the activation of protective autophagy via PARP1/SIRT1 signaling.

Contrast-enhanced Ultrasound for Diagnosis of Renal Cystic Mass.

BACKGROUND: Cystic Renal Cell Carcinoma (CRCC) is often difficult to differentiate from complex cysts with sonographic manifestations of renal carcinoma. Contrast-Enhanced Ultrasound (CEUS) is a new technology, and its clinical utility in the diagnosis of renal cystic mass has not been established. OBJECTIVE: The objective of this study is to analyze the characteristics of CEUS of renal cystic masses and to explore the clinical significance and value of CEUS in the diagnosis of CRCC. METHODS: This study was a retrospective study. A total of 32 cystic masses from January 2018 to December 2019 were selected. The images of conventional Ultrasound (US) and CEUS were confirmed via surgical pathology. Routine US was used to observe the location, shape, size, boundary, cyst wall, internal echo, and blood supply of each cystic mass. CEUS observed contrast enhancement of the cyst wall, cystic septa, and solid nodules of cystic masses. RESULTS: There were 26 cases of CRCC, 5 cases of renal cysts, and 1 case of renal tuberculosis. The enhancement pattern, degree of enhancement, and pseudocapsular sign by CEUS in benign and malignant masses had statistically significant differences (P<.05). In the diagnosis of CRCC, the sensitivity, specificity, accuracy, positive predictive value, and negative predictive value were 92.3%, 83.3%, 90.6%, 96.0%, and 71.4% for CEUS; 57.6%, 66.7%, 59.3%, 88.2%, and 26.7% for conventional US, respectively. CEUS had a higher sensitivity and accuracy than the conventional US (P<.05), although the diagnostic specificity, positive predictive value and negative predictive value of the two methods were not significantly different (P>.05). CONCLUSION: CEUS is more accurate in the diagnosis of renal cystic masses, and it can be used as an effective imaging method.

miR-125a-5p-abundant exosomes derived from mesenchymal stem cells suppress chondrocyte degeneration via targeting E2F2 in traumatic osteoarthritis.

miRNAs are broad participants in vertebrate biological processes, and they are also the major players in pathological processes. miR-125a-5p was recently found a modulator in the progression of osteoarthritis (OA). Our study was aimed to explore the role and underlying mechanisms of miR-125a-5p-abundant exosomes derived from mesenchymal stem cells (MSC) on OA progression. We separated bone marrow mesenchymal stem cells (BMSCs) as well as the exosomes from traumatic OA patients. The immunofluorescence and cartilage staining were implemented for the observation and the assessment on endocytosis of chondrocytes and exosomal miR-125a-5p efficacy to cartilage degradation. Dual luciferase reporter assay was performed to verified the relationship between miR-125a-5p and E2F2. Then, the function of exosomal miR-125a-5p were examined on chondrocyte degeneration in vitro and in vivo. Our findings indicated that E2F2 expression was elevated while the miR-125a-5p was down in traumatic OA cartilage tissue, showing a negative correlation of the former and the latter. miR-125a-5p targets E2F2 in traumatic OA cartilage tissue and leads to the down-expression of E2F2. The E2F2 expression in chondrocytes was decreased after internalization of exosomes. We additionally found that BMSCs-derived exosomes were rich in miR-125a-5p content and chondrocytes can have it internalized. miR-125a-5p is endowed with a trait of accelerating chondrocytes migration, which is going along with the up-expressions of Collagen II, aggrecan and SOX9 and the down-expression of MMP-13 in vitro. Besides that, the mice model with post-traumatic OA turned out that exosomal miR-125a-5p might beget an alleviation in chondrocyte extracellular matrix degradation. All these outcomes revealed that BMSCs-derived exosomal miR-125a-5p is a positive regulator for chondrocyte migration and inhibit cartilage degeneration We thus were reasonable to believe that transferring of exosomal miR-125a-5p is a prospective strategy for OA treatment.

HPV DeepSeq: An Ultra-Fast Method of NGS Data Analysis and Visualization Using Automated Workflows and a Customized Papillomavirus Database in CLC Genomics Workbench.

Next-generation sequencing (NGS) has actualized the human papillomavirus (HPV) virome profiling for in-depth investigation of viral evolution and pathogenesis. However, viral computational analysis remains a bottleneck due to semantic discrepancies between computational tools and curated reference genomes. To address this, we developed and tested automated workflows for HPV taxonomic profiling and visualization using a customized papillomavirus database in the CLC Microbial Genomics Module. HPV genomes from Papilloma Virus Episteme were customized and incorporated into CLC "ready-to-use" workflows for stepwise data processing to include: (1) Taxonomic Analysis, (2) Estimate Alpha/Beta Diversities, and (3) Map Reads to Reference. Low-grade (n = 95) and high-grade (n = 60) Pap smears were tested with ensuing collective runtimes: Taxonomic Analysis (36 min); Alpha/Beta Diversities (5 s); Map Reads (45 min). Tabular output conversion to visualizations entailed 1-2 keystrokes. Biodiversity analysis between low- (LSIL) and high-grade squamous intraepithelial lesions (HSIL) revealed loss of species richness and gain of dominance by HPV-16 in HSIL. Integrating clinically relevant, taxonomized HPV reference genomes within automated workflows proved to be an ultra-fast method of virome profiling. The entire process named "HPV DeepSeq" provides a simple, accurate and practical means of NGS data analysis for a broad range of applications in viral research.

Exosomes Derived from Nerve Stem Cells Loaded with FTY720 Promote the Recovery after Spinal Cord Injury in Rats by PTEN/AKT Signal Pathway.

BACKGROUND: Spinal cord injury (SCI) remains a challenge owing to limited therapies. The exosome of neural stem cells (NSCs-Exos) and FTY720 transplantation could improve SCI effectively. However, the effect and mechanism of NSCs-Exos combined with FTY720 (FTY720-NSCs-Exos) transplantation in the treatment of SCI are not fully understood. METHODS: Sprague Dawley rats (8-week-old) were used to establish the SCI model, followed by the treatment of NSCs-Exos, FTY720, and FTY720-NSCs-Exos. The effect of FTY720, NSCs-Exos, and FTY720-NSCs-Exos combination treatment on hindlimb function, pathological changes, apoptosis activity, and the expression of spinal edema-related proteins and apoptosis-related proteins in SCI models were investigated by BBB scoring, HE staining, TUNEL staining and immunohistochemistry, and Western blotting. Meanwhile, the effect of these treatments on spinal cord microvascular endothelial cells (SCMECs) was detected under hypoxic circumstance. RESULTS: Our results found that FTY720-NSCs-Exos could alleviate pathological alterations and ameliorate the hindlimb function and oxygen insufficiency in model mice after SCI. In addition, exosomes could ameliorate the morphology of neurons, reduce inflammatory infiltration and edema, decrease the expression of Bax and AQP-4, upregulate the expression of claudin-5 and Bcl-2, and inhibit cell apoptosis. At the same time, in vitro experiments showed that FTY720-NSCs-Exos could protect the barrier of SCMECs under hypoxic circumstance, and the mechanism is related to PTEN/AKT pathway. CONCLUSION: FTY720-NSCs-Exos therapy displayed a positive therapeutic effect on SCI by regulating PTEN/AKT pathway and offered a new therapy for SCI.

Decreased Indian hedgehog signaling activates autophagy in endometriosis and adenomyosis.

Indian hedgehog (Ihh) signaling regulates endometrial receptivity and is an indispensable mediator of embryonic implantation. Hedgehog signaling is known to regulate autophagy, and aberrant regulation of autophagy is critically implicated in the pathogenesis of endometriosis and adenomyosis. However, potential dysregulation of Ihh signaling and its role in autophagy modulation in these diseases remain obscure. In this study, we found that components of Ihh signaling were significantly decreased, whereas the autophagy marker protein, LC3BII, was significantly increased in endometrial tissues of women with endometriosis or adenomyosis. Inhibition of Ihh signaling with the small-molecule inhibitor GANT61 or Gli1 silencing in primary endometrial stromal cells increased autophagic activity, as measured by LC3 turnover assay and tandem mCherry-eGFP-LC3B fluorescence microscopy. Furthermore, we observed that GANT61 treatment significantly attenuated hydrogen peroxide-induced cell death, whereas disruption of autophagy with chloroquine diminished this effect. Collectively, these findings reveal that Ihh signaling is suppressed in endometrial tissues of patients with endometriosis or adenomyosis. This abnormal decrease may contribute to endometrial autophagy activation, which may promote aberrant survival of endometrial cells in ectopic sites in these two gynecological diseases.

Molecular Pap Smear: Validation of HPV Genotype and Host Methylation Profiles of ADCY8, CDH8, and ZNF582 as a Predictor of Cervical Cytopathology.

Primary high-risk Human Papillomavirus (hrHPV) screening has recently become an accepted standalone or co-test with conventional cytology. Unfortunately, hrHPV singularly lacks specificity for cytopathological grade. However, mechanisms and markers of evolving virus-host interactions at the epigenome level may be harnessed as a better predictor of carcinogenesis. This study aimed to validate and expand the clinical performance of a multiparametric biomarker panel, referred to as the "Molecular Pap smear" based, on HPV genotype and ADCY8, CDH8 and ZNF582 CpG-methylation as a predictive classifier of cervical cytology. This prospective, cross-sectional study used an independent cohort of residual liquid-based cytology for HPV genotyping and epigenetic analysis. Extracted DNA underwent parallel PCR using 3 primer sets for HPV DNA amplification. HPV-infected samples were genotyped by Sanger sequencing. Promoter methylation levels of 3 tumor suppressor genes were quantified by bisulfite-pyrosequencing of genomic DNA on the newest high-resolution PyroMark Q48 platform. Logistic model performance was compared, and model parameters were used to predict and classify binary cytological outcomes. A total of 883 samples were analyzed. HPV DNA positivity correlated with worsening grade: 125/237 (53%) NILM; 136/235 (58%) ASCUS; 222/229 (97%) LSIL; and 157/182 (86%) HSIL samples. The proportion of carcinogenic HPV-types in PCR-positive sequenceable samples correlated with worsening grade: NILM 34/98 (35%); ASCUS 50/113 (44%); LSIL 92/214 (43%); HSIL 129/152 (85%). Additionally, ADCY8, CDH8, and ZNF582 methylation levels increased in direct correlation with worsening grade. Overall, the multi-marker modeling parameters predicted binarized cytological outcomes better than HPV-type alone with significantly higher area under the receiver operator curve (AUC)s, respectively: NILM vs. > NILM (AUC 0.728 vs. 0.709); NILM/ASCUS vs. LSIL/HSIL (AUC 0.805 vs. 0.776); and

All-trans-retinal induces autophagic cell death via oxidative stress and the endoplasmic reticulum stress pathway in human retinal pigment epithelial cells.

Failure of all-trans-retinal (atRAL) clearance contributes to retina degeneration. However, whether autophagy can be activated by excess atRAL accumulation in retinal pigment epithelial (RPE) cells is not known. This study showed that atRAL provoked mitochondria-associated reactive oxygen species (ROS) production, activated the nuclear factor (erythroid-derived 2)-like 2 and apoptosis in a human RPE cell line, ARPE-19 cells. Moreover, we found that autophagic flux was functionally activated after atRAL treatment. The antioxidant N-acetylcysteine attenuated the expression of autophagy markers, suggesting that ROS triggered atRAL-activated autophagy. In addition, autophagic cell death was observed in atRAL-treated RPE cells, while inhibition of autophagy with 3-methyladenine or LC3, Beclin1, p62 silencing ameliorated atRAL-induced cytotoxicity. Suppression of autophagy quenched mitochondrial ROS and inhibited HO-1 and γ-GCSh expression, indicating that atRAL-activated autophagy enhances intracellular oxidative stress, thereby promoting RPE cell apoptosis. Furthermore, we found that inhibiting endoplasmic reticulum (ER) stress suppressed atRAL-induced mitochondrial ROS generation, subsequently attenuated autophagy and apoptosis in RPE cells. Taken together, these results suggest that atRAL-induced oxidative stress and ER stress modulate autophagy, which may contribute to RPE degeneration. There may be positive feedback regulatory mechanisms between atRAL-induced oxidative stress and autophagy or ER stress.

Endoplasmic reticulum stress and autophagy contribute to cadmium-induced cytotoxicity in retinal pigment epithelial cells.

Excessive accumulation of cadmium (Cd) in retina plays an important role in tobacco smoking-associated age-related macular degeneration (AMD). Plenty of evidence has revealed that the retinal pigment epithelium (RPE) is the primary site of pathology in AMD. Our current study demonstrated that Cd induced apoptosis in a human RPE cell line ARPE-19 cells, as it dose-dependently caused cell viability loss and activated caspase-3. The reactive oxygen species (ROS) were confirmed to be important mediators for Cd-triggered cell death in ARPE-19 cells. We found that endoplasmic reticulum (ER) stress was activated as its marker BiP was remarkably upregulated by Cd-exposure. Whereas the antioxidants N-acetylcysteine (NAC) and Tempol significantly suppressed the expression of BiP and CHOP, suggesting that ROS generation is an early trigger of Cd-activated ER stress. Furthermore, we found that Cd-induced oxidative stress significantly increased autophagic flux and p62 expression. A temporal impact of Cd exposure is possibly existed in p62 expression in ARPE-19 cells. Moreover, an ER stress inhibitor salubrinal diminished Cd-induced LC3BII expression and attenuated cytotoxicity, indicating that ER stress mediates autophagy and was implicated in apoptosis of Cd-exposed ARPE-19 cells. However, CHOP expression may not exert impact on the regulation of Cd-caused autophagy. Additionally, inhibition of autophagy with si-Beclin 1 and 3-Methyladenine significantly ameliorated Cd-induced CHOP expression and cytotoxicity, indicating that autophagy was detrimental in Cd-accumulated ARPE-19 cells, and a positive feedback regulation mechanism may exist between Cd-triggered ER stress and autophagy. Taken together, these results suggest that Cd-caused ER stress and autophagy are implicated in RPE cell death associated retinopathies especially related to smoking.