PRPF8-mediated dysregulation of hBrr2 helicase disrupts human spliceosome kinetics and 5´-splice-site selection causing tissue-specific defects.

The carboxy-terminus of the spliceosomal protein PRPF8, which regulates the RNA helicase Brr2, is a hotspot for mutations causing retinitis pigmentosa-type 13, with unclear role in human splicing and tissue-specificity mechanism. We used patient induced pluripotent stem cells-derived cells, carrying the heterozygous PRPF8 c.6926 A > C (p.H2309P) mutation to demonstrate retinal-specific endophenotypes comprising photoreceptor loss, apical-basal polarity and ciliary defects. Comprehensive molecular, transcriptomic, and proteomic analyses revealed a role of the PRPF8/Brr2 regulation in 5'-splice site (5'SS) selection by spliceosomes, for which disruption impaired alternative splicing and weak/suboptimal 5'SS selection, and enhanced cryptic splicing, predominantly in ciliary and retinal-specific transcripts. Altered splicing efficiency, nuclear speckles organisation, and PRPF8 interaction with U6 snRNA, caused accumulation of active spliceosomes and poly(A)+ mRNAs in unique splicing clusters located at the nuclear periphery of photoreceptors. Collectively these elucidate the role of PRPF8/Brr2 regulatory mechanisms in splicing and the molecular basis of retinal disease, informing therapeutic approaches.

Recurrent human 16p11.2 microdeletions in type I Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome patients in Chinese Han population.

BACKGROUNDS: Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome, a severe congenital malformation of the female genital tract, is a highly heterogeneous disease which has no clear etiology. Previous studies have suggested that copy number variations (CNVs) and single-gene mutations might contribute to the development of MRKH syndrome. In particular, deletions in 16p11.2, which are suggested to be involved in several congenital diseases, have been reported in Chinese type II MRKH patients and European MRKH patients. However, few CNVs including 16p11.2 microdeletions were identified in Chinese type I MRKH cases although it accounted for the majority of MRKH patients in China. Thus, we conducted a retrospective study to identify whether CNVs at human chromosome 16p11.2 are risk factors of type I MRKH syndrome in the Chinese Han population. METHODS: We recruited 143 patients diagnosed with type I MRKH between 2012 and 2014. Five hundred unrelated Chinese without congenital malformation were enrolled in control group, consisting of 197 from the 1000 Genomes Project and 303 from Fudan University. Quantitative PCR, array comparative genomic hybridization, and sanger sequencing were conducted to screen and verify candidate variant. RESULTS: Our study identified recurrent 16p11.2 microdeletions of approximately 600 kb in two out of the 143 type I MRKH syndrome patients using high-density array-based comparative genomic hybridization (aCGH), while no 16p11.2 deletion was found in the control group. We did not find any mutations in TBX6 gene in our samples. CONCLUSIONS: The results of the study identify 16p11.2 deletion in Chinese MRKH I patients for the first time, as well as support the contention that 16p11.2 microdeletions are associated with MRKH syndrome in both types across populations. It is suggested that 16p11.2 microdeletions should be included in molecular diagnosis and genetic counseling of female reproductive tract disorders.

Effect of Sivelestat in the Treatment of Acute Lung Injury and Acute Respiratory Distress Syndrome: A Systematic Review and Meta-Analysis.

Background: The efficacy of neutrophil elastase inhibitor sivelestat in the treatment of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) remains controversial. A systematic review and meta-analysis were performed in accordance with the PRISMA guidelines assess the effect of sivelestat on ALI/ARDS patients, different studies were included. Methods: Electronic databases, National Knowledge Infrastructure (CNKI), Wan fang data, VIP, PubMed, Embase, Springer, Ovid and the Cochrane Library were searched using the following key words: ("Sivelestat" OR "Elaspol") AND ("ARDS" OR "adult respiratory distress syndrome" OR "acute lung injury"). All databases published from January 2000 to August 2022. The treatment group was treated with sivelestat and the control group was given normal saline. The outcome measurements include the mortality of 28-30 days, mechanical ventilation time, ventilation free days, intensive care unit (ICU) stays, oxygenation index (PaO2/FiO2) on day 3, the incidence of adverse events. The literature search was conducted independently by 2 researchers using standardized methods. We used the Cochrane risk-of-bias tool to assess the quality of the included studies. Mean difference (MD), Standardized mean difference (SMD) and relative risk (RR) were calculated using random effects model or fixed effects model. All statistical analyses were performed using RevMan software 5.4. Results: A total of 2050 patients were enrolled in 15 studies, including 1069 patients in treatment group and 981 patients in the control group. The results of the meta-analysis showed that: compared with the control group, sivelestat can reduce the mortality of 28-30 days (RR = 0.81, 95% CI = 0.66-0.98, p = 0.03) and the incidence of adverse events (RR = 0.91, 95% CI = 0.85-0.98, p = 0.01), shortened mechanical ventilation time (SMD = - 0.32, 95% CI = - 0.60 to - 0.04, p = 0.02) and ICU stays (SMD = - 0.72, 95% CI = - 0.92 to - 0.52, p < 0.00001), increased the ventilation free days (MD = 3.57, 95% CI = 3.42-3.73, p < 0.00001) and improve oxygenation index (PaO2/FiO2) on day 3 (SMD = 0.88, 95% CI = 0.39-1.36, p = 0.0004). Conclusions: Sivelestat can not only reduce the mortality of ALI/ARDS patients within 28-30 days and the incidence of adverse events, shorten the mechanical ventilation time and ICU stays, increase ventilation free days, but also improve the oxygenation index of patients on days 3, which has a good effect on the treatment of ALI/ARDS. These findings need to be verified in large-scale trials.

Clinical Utility of the Sivelestat for the Treatment of ALI/ARDS: Moving on in the Controversy?

Acute respiratory distress syndrome (ARDS) is a serious condition that can arise following direct or indirect acute lung injury (ALI). It is heterogeneous and has a high mortality rate. Supportive care is the mainstay of treatment and there is no definitive pharmacological treatment as yet. In nonclinical studies, neutrophil elastase inhibitor sivelestat appears to show benefit in ARDS without inhibiting the host immune defense in cases of infection. In clinical studies, the efficacy of sivelestat in the treatment of ARDS remains controversial. The currently available evidence suggests that sivelestat may show some benefit in the treatment of ARDS, although large, randomized controlled trials are needed in specific pathophysiological conditions to explore these potential benefits.

[Sivelestat protects acute kidney injury by inhibiting the PI3K/AKT pathway in septic rats].

OBJECTIVE: To explore the protective effect of sivelestat (SV) against sepsis-induced acute kidney injury (AKI) and its molecular mechanism. METHODS: According to the random number table method, 64 male Wistar rats were divided into sham operation group (Sham group), sepsis due to cecal ligation and puncture group (CLP group), low dose of SV treatment group (SL group, 50 mg/kg SV was injected into the tail vein at 12 hours and 24 hours after CLP), and high dose of SV treatment group (SH group, 100 mg/kg SV was injected into the tail vein at 12 hours and 24 hours after CLP), with 16 rats in each group. 48 hours after CLP, the 48-hour survival of rats were recorded, all rats were sacrificed and samples were harvested. Enzyme-linked immunosorbent assay (ELISA) was used to detect the serum levels of kidney injury molecule-1 (KIM-1), interleukins (IL-1ß, IL-6), tumor necrosis factor-α (TNF-α) and neutrophil elastase (NE). Hematoxylin-eosin (HE) staining was used to observe histopathological changes and assess renal tubule injury score. Masson staining was used to detect the collagen volume fraction (CVF) of kidney tissue. Western blotting was used to detect the protein expressions of phosphatidylinositol 3-kinase (PI3K), phosphorylation PI3K (p-PI3K), protein kinase B (AKT), phosphorylation AKT (p-AKT), nuclear factor-κB p65 (NF-κB p65) and NE. The protein expressions of p-PI3K, p-AKT, NF-κB p65 were detected by immunohistochemistry. RESULTS: Compared with Sham group, the 48-hour survival rate of CLP group was significantly reduced. Histopathological results showed that large tubular epithelial cells and brush margins were shed, tubular casts were formed, some tubular atrophy, glomerular hyperemia, renal interstitial inflammatory cell infiltration and increased renal tubular injury score. Renal interstitial fibrosis was obvious and CVF increased. The levels of KIM-1, IL-1ß, IL-6, TNF-α and NE in serum were significantly elevated in the CLP group. The proteins expression of inflammatory pathway-related p-PI3K/PI3K, p-AKT/AKT, NF-κB p65 and NE were significantly increased in kidney tissue. It suggested that septic rats had renal injury and the PI3K/AKT inflammatory pathway was activated. Compared with CLP group, there was no significant difference in 48-hour survival in SL group and SH group (68.75%, 75.00% vs. 56.25%, both P > 0.05), but kidney injury was significantly relieved. Specifically: renal tubular injury score and CVF significantly decreased [tubular injury score: 2 (1, 2), 1 (1, 1) vs. 2 (2, 3); CVF: (22.36±0.86)%, (18.74±1.05)% vs. (58.38±0.79)%, all P < 0.05]; the serum levels of KIM-1, IL-1ß, IL-6, TNF-α and NE also decreased significantly [KIM-1 (ng/L): 145.03±8.88, 117.58±7.02 vs. 158.22±12.00; IL-1ß (ng/L): 108.32±9.00, 92.98±8.06 vs. 133.78±8.48; IL-6 (ng/L): 124.33±10.11, 115.42±8.17 vs. 165.19±5.70; TNF-α (ng/L): 321.56±19.29, 289.68±21.57 vs. 424.88±22.76, NE (mol/L): 93.84±9.14, 75.01±10.56 vs. 113.45±6.39, all P < 0.05]; the proteins expression of inflammatory pathway-related p-PI3K/PI3K, p-AKT/AKT, NF-κB p65 and NE were significantly decreased (p-PI3K/PI3K: 0.93±0.06, 0.67±0.04 vs. 1.27±0.08; p-AKT/AKT: 0.78±0.09, 0.47±0.05 vs. 0.96±0.12; NF-κB p65/GAPDH: 1.43±0.13, 0.85±0.08 vs. 1.88±0.17; NE/GAPDH: 1.45±0.06, 0.91±0.04 vs. 1.71±0.08, all P < 0.05), the positive expressions of p-PI3K, p-AKT and NF-κB p65 in kidney tissue were decreased [p-PI3K positive expression area: (13.36±1.84)%, (8.03±1.12)% vs. (21.56±1.20)%; p-AKT positive expression area: (21.57±0.91)%, (15.21±2.76)% vs. (30.81±2.12)%; NF-κB p65 positive expression area: (25.17±1.38)%, (17.07±2.11)% vs. (37.85±2.50)%, all P < 0.05]. Serum inflammatory factor level, and PI3K/AKT pathway related protein, NF-κB p65, NE protein expression level and p-PI3K, p-AKT, NF-κB p65 positive area and other indicators in renal tissue in SH group were further lower than those in SL group (all P < 0.05). CONCLUSIONS: SV can ameliorate sepsis-induced AKI. The mechanism may be related to the inhibition of PI3K/AKT pathway, and high dose of SV has better efficacy.

Diversity and Pathogenicity of Fungi Associated with Fruit Rot of Winter Jujube in Shandong Province, China.

Winter jujube originated from China and had an extremely high nutritional value. In 2021, symptomatic winter jujube fruits were collected from eight locations in Zhanhua District of Binzhou City, Shandong Province. In total, 108 fungal isolates were obtained and grouped into 11 species based on morphological characteristics and multilocus phylogenetic analysis, including Nothophoma quercina (43.52%), Fusarium lateritium (20.37%), Alternaria alternata (12.03%), F. proliferatum (7.41%), F. graminearum (4.63%), Botryosphaeria dothidea (3.70%), Fusarium sp. (2.78%), A. tenuissima (2.78%), Diaporthe eres (1.85%), Nigrospora oryzae (0.93%), and Cercospora nicotianae (0.93%). All fungal isolates obtained in this study showed aggressiveness on detached winter jujube fruits except N. oryzae and C. nicotianae isolates, of which F. proliferatum was the most virulent, while A. alternata isolates, which have been considered the major pathogen of winter jujube fruit rot, showed a relatively low-level virulence in this study. Furthermore, D. eres, F. graminearum, F. lateritium, and an unclassified Fusarium species were first reported as causal agents of winter jujube fruit rot. The typical symptoms of winter jujube fruit rot observed in this study could be distinguished into two types. N. quercina, A. alternata, A. tenuissima, Fusarium sp., D. nobilis, and F. lateritium isolates caused reddish brown to dark gray lesions on the peel, while B. dothidea, F. graminearum, and F. proliferatum isolates caused peel and pulp decay, resulting in red to reddish brown and water-soaked lesions. In addition, haplotype analysis of N. quercina isolates obtained in this study and validly published articles showed that there were 11 haplotypes worldwide; the isolates obtained in the current study were grouped into three haplotypes (Hap 1, Hap 2, and Hap 11), and two of them (Hap 2 and Hap 11) were confirmed as new haplotypes.

[Research advance of the mechanism and treatment of septic cardiomyopathy].

Sepsis is a life-threatening organ dysfunction caused by dysregulation of the body's response to infection. It is one of the common and serious complications in clinically critical patients with trauma, burn, shock, infection, etc., with high morbidity and mortality. Although the treatment of sepsis has made great achievements in clinical practice, the mortality of patients with sepsis is still increasing due to its secondary complications. Septic cardiomyopathy (SCM) is one of the major complications that threaten septic patient's life. SCM refers to myocardial dysfunction with the aggravation of the primary disease, which is manifested by biventricular dilatation accompanied by a decrease in left ventricular ejection fraction (LVEF). It is one of the major complications that threaten the life of patients with sepsis. The existing research shows that the mechanism of SCM includes myocardial mitochondrial dysfunction, myocardial cell apoptosis, calcium circulation disorder and its treatment including conventional treatment, ß1 receptor blocker treatment and traditional Chinese medicine treatment,etc. This paper reviewed the pathogenesis of SCM and its related, in order to provide references for the rational diagnosis and treatment of SCM.

Fusarium Species Associated with Maize Leaf Blight in Heilongjiang Province, China.

Fusarium spp. are among the most important plant pathogens in the world. A survey on maize leaf blight was carried out in Heilongjiang province from 2019 to 2021. Based on morphological characteristics and a phylogenetic analysis on translation elongation factor (tef1) and second-largest subunit of RNA polymerase II (rpb2) genes, 146 Fusarium isolates were obtained and grouped into 14 Fusarium species, including F. ipomoeae (20.5%), F. compactum (17.1%), F. sporotrichioides (9.59%), F. graminearum (9.59%), F. citri (8.9%), F. asiaticum (6.85%), F. verticillioides (6.85%), F. acuminatum (5.48%), F. glycines (5.48%), F. temperatum (2.74%), F. armeniacum (2.74%), Fusarium sp. (2.05%), F. flagelliforme (1.4%), and F. annulatum (0.68%). The Fusarium incarnatum-equiseti species complex (FIESC, including F. ipomoeae, F. compactum, F. citri, and F. flagelliforme) was the most prevalent, indicating an evolving occurrence of the Fusarium species causing maize leaf blight. The typical symptoms observed on the maize leaves were oval to long strip lesions, with a gray to dark gray or brownish red coloration in the center and a chlorotic area at the edges. Based on the tef1 gene, seven haplotypes of FIESC were identified in Heilongjiang province, suggesting a population expansion. This is the first report of F. ipomoeae, F. compactum, F. flagelliforme, F. citri, F. sporotrichioides, F. graminearum, F. asiaticum, F. acuminatum, F. glycines, F. temperatum, F. armeniacum, Fusarium sp., and F. annulatum causing maize leaf blight in Heilongjiang province, China. The current research is informative for managing disease, exploring the phylogenetic relationship among Fusarium species, and clarifying the diversity of Fusarium species associated with maize leaf blight.

Identification, Pathogenicity, and Genetic Diversity of Fusarium spp. Associated with Maize Sheath Rot in Heilongjiang Province, China.

Maize sheath rot is a prevalent maize disease in China. From 2020 to 2021, symptomatic samples were collected from the main maize-growing regions of Heilongjiang province. To clarify the population and genetic diversity, as well as the virulence of pathogens responsible for maize sheath rot, a total of 132 Fusarium isolates were obtained and used for follow-up studies. Ten Fusarium species were identified based on morphological characteristics, and phylogenetic analysis was conducted using the TEF-1α gene sequences, including F. verticillioides (50.00%), F. subglutinans (18.94%), the Fusarium incarnatum-equiseti species complex (14.39%), F. temperatum (5.30%), F. acuminatum (3.03%), F. solani (2.27%), F. sporotrichioides (2.27%), F. tricinctum (1.52%), F. asiaticum (1.52%), and F. proliferatum (0.76%). All 10 Fusarium species could produce oval-to-annular lesions on maize sheath, and the lesions were grayish yellow to dark brown in the center and surrounded by a dark gray-to-dark brown halo. Of these, F. tricinctum and F. proliferatum showed significantly higher virulence than the other Fusarium species. In addition, haplotype analysis based on the concatenated sequences of the ITS and TEF-1a genes showed that 99 Fusarium isolates which belonged to the Fusarium fujikuroi species complex-consisting of F. verticillioides isolates, F. subglutinans isolates, F. temperatum isolates, and F. proliferatum isolates-could be grouped into 10 haplotypes, including 5 shared haplotypes (Haps 1, 2, 4, 5, and 6) and 5 private haplotypes (Haps 3, 7, 8, 9, and 10). Furthermore, the F. verticillioides clade in the haplotype network was radial with the center of Hap 2, suggesting that population expansion occurred. This research showed that Fusarium species associated with maize sheath rot in Heilongjiang province are more diverse than previously reported, and this is the first time that F. subglutinans, F. temperatum, F. solani, F. sporotrichioides, F. tricinctum, and F. acuminatum have been confirmed as the causal agents of maize sheath rot in Heilongjiang province.

[Predictive value of six critical illness scores for 28-day death risk in comprehensive and specialized intensive care unit patients based on MIMIC-IV database].

OBJECTIVE: To explore the basic characteristics of various types of intensive care unit (ICU) patients and the predictive value of six common disease severity scores in critically ill patients on the first day on the 28-day death risk. METHODS: The general information, disease severity scores [acute physiology score III (APS III), Oxford acute disease severity (OASIS) score, Logistic organ dysfunction score (LODS), simplified acute physiology score II (SAPS II), systemic inflammatory response syndrome (SIRS) score and sequential organ failure assessment (SOFA) score], prognosis and other indicators of critically ill patients admitted from 2008 to 2019 were extracted from Medical Information Mart for Intensive Care-IV 2.0 (MIMIC-IV 2.0). The receiver operator characteristic curve (ROC curve) of six critical illness scores for 28-day death risk of patients in various ICU, and the area under the ROC curve (AUC) was calculated, the optimal Youden index was used to determine the cut-off value, and the AUC of various ICU was verified by Delong method. RESULTS: A total of 53 150 critically ill patients were enrolled, with medical ICU (MICU) accounted for the most (19.25%, n = 10 233), followed by cardiac vascular ICU (CVICU) with 17.78%(n = 9 450), and neurological ICU (NICU) accounted for the least (6.25%, n = 3 320). The patients in coronary care unit (CCU) were the oldest [years old: 71.79 (60.27, 82.33)]. The length of ICU stay in NICU was the longest [days: 2.84 (1.51, 5.49)] and accounted for the highest proportion of total length of hospital stay [63.51% (34.61%, 97.07%)]. The patients in comprehensive ICU had the shortest length of ICU stay [days: 1.75 (0.99, 3.05)]. The patients in CVICU had the lowest proportion of length of ICU stay to total length of hospital stay [27.69% (18.68%, 45.18%)]. The six scores within the first day of ICU admission in NICU patients were lower than those in the other ICU, while APS III, LODS, OASIS, and SOFA scores in MICU patients were higher than those in the other ICU. SAP II and SIRS scores were both the highest in CVICU, respectively. In terms of prognosis, MICU patients had the highest 28-day mortality (14.14%, 1 447/10 233), while CVICU patients had the lowest (2.88%, 272/9 450). ROC curve analysis of the predictive value of each score on the 28-day death risk of various ICU patients showed that, the predictive value of APS III, LODS, and SAPS II in comprehensive ICU were higher [AUC and 95% confidence interval (95%CI) were 0.84 (0.83-0.85), 0.82 (0.81-0.84), and 0.83 (0.82-0.84), respectively]. The predictive value of OASIS, LODS, and SAPS II in surgical ICU (SICU) were higher [AUC and 95%CI were 0.80 (0.79-0.82), 0.79 (0.78-0.81), and 0.79 (0.77-0.80), respectively]. The predictive value of APS III and SAPS II in MICU were higher [AUC and 95%CI were 0.84 (0.82-0.85) and 0.82 (0.81-0.83), respectively]. The predictive value of APS III and SAPS II in CCU were higher [AUC and 95%CI were 0.86 (0.85-0.88) and 0.85 (0.83-0.86), respectively]. The predictive value of LODS and SAPS II in trauma ICU (TICU) were higher [AUC and 95%CI were 0.83 (0.82-0.83) and 0.83 (0.82-0.84), respectively]. The predictive value of OASIS and SAPS II in NICU were higher [AUC and 95%CI were 0.83 (0.80-0.85) and 0.81 (0.78-0.83), respectively]. The predictive value of APS III, LODS, and SAPS II in CVICU were higher [AUC and 95%CI were 0.84 (0.83-0.85), 0.81 (0.80-0.82), and 0.78 (0.77-0.78), respectively]. CONCLUSIONS: For the patients in comprehensive ICU, MICU, CCU, and CVICU, APS III or SAPS II can be applied for predicting 28-day death risk. For the patients in SICU and NICU, OASIS or SAPS II can be applied to predict 28-day death risk. For the patients in TICU, SAPS II or LODS can be applied for predicting 28-day death risk. For CVICU patients, APS III or LODS can be applied to predict 28-day death risk.

Comparison of the modified laparoscopic Vecchietti and Davydov colpoplasty techniques in Mayer-Rokitansky-Küster-Hauser syndrome: A long-term follow-up analysis.

AIMS: To introduce and compare the modified laparoscopic Vecchietti and Davydov techniques for vaginoplasty in patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Moreover, the long-term treatment of vaginal agenesis was followed-up. METHODS: This comparative retrospective cohort study enrolled a total of 53 women with MRKH syndrome. The patients underwent surgical creation of a neovagina including 32 patients who underwent the modified laparoscopic Vecchietti technique, and 21 patients who underwent the modified laparoscopic Davydov technique from January 2009 to February 2019. The perioperative parameters, complications, anatomical, and functional outcomes of the two groups were compared. Patients' sexual functions were evaluated over a long-term follow-up using the female sexual function index (FSFI) and the revised female sexual distress scale (FSDS-R). RESULTS: The medians (25th-75th) of the surgery duration for modified Vecchietti procedures was 50.0 (40.0-59.0) minutes, comparing to 135.0 (117.5-162.5) min for Davydov procedures (p < 0.001). The intraoperative blood loss was 20 (7.5-20.0) mL versus 50.0 (50.0-100.0) mL using the modified Vecchietti and Davydov approaches (p < 0.001), respectively. In the 39 follow-up cases, the lengths of the neovagina of the patients for Vecchietti group versus Davydov group were 7.9 ± 1.0 cm versus 8.6 ± 1.2 cm at 6 months after the vaginoplasty and 8.3 ± 0.7 cm versus 8.5 ± 0.9 cm after 2 years. There was no statistical difference in the FSFI and FSDS-R scores between the two groups. CONCLUSIONS: Both the modified Davydov and Vecchietti laparoscopic procedures successfully achieved optimal anatomic and functional outcomes in treatments of vaginal agenesis. The modified Vecchietti technique is relatively simpler than the modified Davydov technique.

Activation of autophagy reverses progressive and deleterious protein aggregation in PRPF31 patient-induced pluripotent stem cell-derived retinal pigment epithelium cells.

INTRODUCTION: Mutations in pre-mRNA processing factor 31 (PRPF31), a core protein of the spliceosomal tri-snRNP complex, cause autosomal-dominant retinitis pigmentosa (adRP). It has remained an enigma why mutations in ubiquitously expressed tri-snRNP proteins result in retina-specific disorders, and so far, the underlying mechanism of splicing factors-related RP is poorly understood. METHODS: We used the induced pluripotent stem cell (iPSC) technology to generate retinal organoids and RPE models from four patients with severe and very severe PRPF31-adRP, unaffected individuals and a CRISPR/Cas9 isogenic control. RESULTS: To fully assess the impacts of PRPF31 mutations, quantitative proteomics analyses of retinal organoids and RPE cells were carried out showing RNA splicing, autophagy and lysosome, unfolded protein response (UPR) and visual cycle-related pathways to be significantly affected. Strikingly, the patient-derived RPE and retinal cells were characterised by the presence of large amounts of cytoplasmic aggregates containing the mutant PRPF31 and misfolded, ubiquitin-conjugated proteins including key visual cycle and other RP-linked tri-snRNP proteins, which accumulated progressively with time. The mutant PRPF31 variant was not incorporated into splicing complexes, but reduction of PRPF31 wild-type levels led to tri-snRNP assembly defects in Cajal bodies of PRPF31 patient retinal cells, altered morphology of nuclear speckles and reduced formation of active spliceosomes giving rise to global splicing dysregulation. Moreover, the impaired waste disposal mechanisms further exacerbated aggregate formation, and targeting these by activating the autophagy pathway using Rapamycin reduced cytoplasmic aggregates, leading to improved cell survival. CONCLUSIONS: Our data demonstrate that it is the progressive aggregate accumulation that overburdens the waste disposal machinery rather than direct PRPF31-initiated mis-splicing, and thus relieving the RPE cells from insoluble cytoplasmic aggregates presents a novel therapeutic strategy that can be combined with gene therapy studies to fully restore RPE and retinal cell function in PRPF31-adRP patients.

Development and validation of an immune-related prognosis signature associated with hypoxia and ferroptosis in hepatocellular carcinoma.

BACKGROUND: Hypoxia and ferroptosis are crucial in the occurrence and development of hepatocellular carcinoma (HCC), and they both affect the immune status of the tumor microenvironment. Previous studies have also shown a link between hypoxia and ferroptosis. PATIENTS AND METHODS: In all, 814 HCC cases from The Cancer Genome Atlas and Gene Expression Omnibus databases were used as the discovery cohort, and 230 HCC cases from the International Cancer Genome Consortium database were used as the validation cohort. Hypoxia subtypes and ferroptosis subtypes were identified by consensus cluster analysis according to 174 hypoxia-related genes and 193 ferroptosis-related genes. The prognostic signature was constructed using the Cox and LASSO regression analyses, and two risk groups were identified. A comprehensive analysis of the clinical and immune characteristics between the two risk groups was further performed. RESULTS: Two hypoxia subtypes and two ferroptosis subtypes were distinguished and verified; subsequently, a five-gene prognostic signature was constructed and the risk score could be acquired by the following formula: risk score = 0.0604*Expression (CA9)-0.0714*Expression (ANXA10) + 0.1501*Expression (CDC20)-0.0853*Expression (CYP7A1) + 0.0530*Expression (SPP1). Compared with the low-risk group, the high-risk group had a worse prognosis. The high-risk group also showed a higher level of immune infiltration than the low-risk group, and immune checkpoints were generally upregulated in the high-risk group. The antigen presentation ability of the low-risk group was poor, which may be related to the immune escape mechanism. Drug sensitivity analysis indicated that the high- and low-risk groups were sensitive to tyrosine kinase inhibitors and chemotherapeutic drugs, respectively. CONCLUSION: The hypoxia-, ferroptosis-, and immune-associated prognostic signature we constructed could stratify patients with HCC and guide precise treatment.

Therapeutic efficacy of microwave coagulation versus liver resection for hepatocellular carcinoma within the Milan criteria: A propensity score matching analysis.

INTRODUCTION: This study aimed to compare the therapeutic efficacy of resection (RES) and microwave ablation (MWA) for hepatocellular carcinoma (HCC) within the Milan criteria. MATERIALS AND METHODS: Between 2011 and 2019, 426 HCC patients within the Milan criteria were treated at our institution (RES: n = 291; MWA: n = 135). We compared overall survival (OS), disease-free survival (DFS), complications, and hospital stay in these patients using propensity score matching (PSM) and determined the prognostic factors using multivariate Cox analysis. RESULTS: Following 1:1 matching using PSM, 121 patients were matched in each group. The 1-, 3-, and 5-year OS rates were 98.3%, 84.7%, and 69.6% for the MWA group and 96.5%, 81.8%, and 78.1% for the RES group (p = 0.667). The corresponding DFS rates for the MWA and RES groups were 81.8%, 54.4%, and 42.3% and 85.4%, 67.8%, and 57.9%, respectively (p = 0.174). The MWA group had less blood loss and shorter hospital stays (both p < 0.001) than the RES group. CONCLUSION: MWA resulted in survival outcomes that were similar to those of RES for HCC within the Milan criteria. However, it had more favorable hospital stay and blood loss outcomes than RES.

Structural, functional and molecular pathogenesis of pelvic organ prolapse in patient and Loxl1 deficient mice.

Pelvic organ prolapse is a worldwide health problem to elderly women. Understanding its pathogenesis and an ideal animal model are crucial to developing promising treatments. The present study aimed to investigate new clinical significance and detailed mechanism of pelvic organ prolapse by comparing the structural, functional and molecular dysfunctions of pelvic organ prolapse in patient and Loxl1 deficient mice. Our results showed that human vagina tissues from prolapsed site showed disarranged collagen and elastic fibers compared with the non-prolapse tissue. A gene ontology (GO) analysis of differentially expressed genes revealed molecular changes mainly related to inflammatory response and extracellular matrix (ECM) organization. While the mice lacking Loxl1 developed stable POP phenotype and disordered ECM structure in histology. Such Loxl1 knockout mice exhibited a significantly urinary dysfunction and decreased mechanical properties of the pelvic floor tissues, implying that POP in human condition might be induced by progressively decreased mechanics of pelvic tissues following ECM catabolism. Similarly, we not only identified significant up-regulated ECM catabolism processes and down-regulated ECM synthesis processes, but also characterized high level of inflammatory response in vagina tissue of the Loxl1 deficient mice. Thus, all these pathological changes in the POP mice model was consistent with those of the clinical elderly patients. These findings provide new insight into remodeling of POP by LOXL1 regulation and be of great importance to develop combination treatments of ECM metabolism and inflammation regulation strategy.

Pre-mRNA Processing Factors and Retinitis Pigmentosa: RNA Splicing and Beyond.

Retinitis pigmentosa (RP) is the most common inherited retinal disease characterized by progressive degeneration of photoreceptors and/or retinal pigment epithelium that eventually results in blindness. Mutations in pre-mRNA processing factors (PRPF3, 4, 6, 8, 31, SNRNP200, and RP9) have been linked to 15-20% of autosomal dominant RP (adRP) cases. Current evidence indicates that PRPF mutations cause retinal specific global spliceosome dysregulation, leading to mis-splicing of numerous genes that are involved in a variety of retina-specific functions and/or general biological processes, including phototransduction, retinol metabolism, photoreceptor disk morphogenesis, retinal cell polarity, ciliogenesis, cytoskeleton and tight junction organization, waste disposal, inflammation, and apoptosis. Importantly, additional PRPF functions beyond RNA splicing have been documented recently, suggesting a more complex mechanism underlying PRPF-RPs driven disease pathogenesis. The current review focuses on the key RP-PRPF genes, depicting the current understanding of their roles in RNA splicing, impact of their mutations on retinal cell's transcriptome and phenome, discussed in the context of model species including yeast, zebrafish, and mice. Importantly, information on PRPF functions beyond RNA splicing are discussed, aiming at a holistic investigation of PRPF-RP pathogenesis. Finally, work performed in human patient-specific lab models and developing gene and cell-based replacement therapies for the treatment of PRPF-RPs are thoroughly discussed to allow the reader to get a deeper understanding of the disease mechanisms, which we believe will facilitate the establishment of novel and better therapeutic strategies for PRPF-RP patients.

[Activation of NOD-like receptor protein 3 inflammasome mediates inflammatory response and apoptosis in septic intestinal injury model].

OBJECTIVE: To investigate the expression of NOD-like receptor protein 3 (NLRP3) inflammasome in intestinal injury models with different severity of sepsis and the inflammatory response and apoptosis mediated by NLRP3 inflammasome. METHODS: Human colorectal adenocarcinoma cells (Caco-2) were cultured in vitro. The logarithmic growth phase cells were divided into blank control group (normal culture in complete medium) and lipopolysaccharide (LPS) 1, 2 and 4 mg/L groups (complete medium containing 1, 2 and 4 mg/L LPS, respectively). The supernatant were collected at 6, 12 and 24 hours, and the levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1ß, IL-18) were detected by enzyme linked immunosorbent assay (ELISA). The apoptotic level of cells was detected by flow cytometry. The cells were harvested, and the real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-qPCR) was used to detect the mRNA expressions of NLRP3 and silent information regulator 1 (SIRT1). Western blotting was used to detect the protein expressions of NLRP3, SIRT1, caspase-1 and apoptosis-associated speck-like protein (ASC). RESULTS: ELISA results showed that the levels of IL-6, TNF-α, IL-1ß, and IL-18 in cell supernatant of LPS groups increased in a dose-dependent and time-dependent manner as compared with the blank control group during the same intervention period. The increase was most significant in LPS 4 mg/L group at 24 hours [IL-6 (ng/L): 3.55±0.06 vs. 0.67±0.09, TNF-α (ng/L): 15.37±0.19 vs. 5.04±0.14, IL-1ß (ng/L): 2.26±0.10 vs. 0.56±0.09, IL-18 (ng/L): 433.92±22.55 vs. 93.55±21.13, all P < 0.05]. The results of the apoptotic test showed that, compared with the blank control group, the apoptotic rate of LPS groups increased in a dose-dependent and time-dependent manner, and the apoptotic rate of LPS 4 mg/L group increased most significantly at 24 hours [(14.83±3.73)% vs. (5.87±1.17)%, P < 0.05]. RT-qPCR results showed that the expression level of NLRP3 mRNA was increased, while the expression level of SIRT1 mRNA was decreased with the increase of LPS intervention dose and the prolonging of intervention time. At 24 hours, there were significant differences between LPS 4 mg/L group and blank control group [NLRP3 mRNA (2-ΔΔCt): 8.20±2.82 vs. 1.00±0.36, SIRT1 mRNA (2-ΔΔCt): 0.58±0.01 vs. 1.03±0.06, both P < 0.05]. Western blotting showed that compared with the blank control group, the protein expression levels of NLRP3, caspase-1 and ASC in LPS groups were significantly increased, while the protein expression levels of SIRT1 were significantly decreased. During each intervention period, with the increase of LPS dose, the expressions of NLRP3, caspase-1 and ASC protein increased gradually, while the expression of SIRT1 protein decreased gradually. At 24 hours, the difference between the LPS 4 mg/L group and the blank control group was significant [NLRP3 protein (NLRP3/ß-actin): 1.48±0.03 vs. 0.90±0.12, caspase-1 protein (caspase-1/ß-actin): 1.18±0.11 vs. 0.72±0.09, ASC protein (ASC/ß-actin) : 1.09±0.01 vs. 0.82±0.03, SIRT1 protein (SIRT1/ß-actin): 0.48±0.03 vs. 0.76±0.05, all P < 0.05]. CONCLUSIONS: In vitro, in the sepsis induced intestinal inflammation model, with the increase of LPS intervention dose and the prolongation of intervention time, intestinal inflammatory response and cell apoptosis showed an increasing trend, which may be related to the up-regulation of NLRP3 inflammasome and its downstream products ASC and caspase-1, and to the down-regulation of SIRT1 expression.

Optimal Dose of Pituitrin in Laparoscopic Uterine Myomectomy: A Double-Blinded, Randomized Controlled Trial.

STUDY OBJECTIVE: To determine the optimal effective dose of pituitrin in laparoscopic myomectomy for uterine leiomyoma. DESIGN: Double-blinded, randomized controlled trial. SETTING: Tertiary women's hospital in China. PATIENTS: Total of 118 patients who underwent laparoscopic myomectomy. INTERVENTIONS: Patients randomly received 0, 2, 4, or 6 units of pituitrin injected into the myometrium surrounding the myoma. MEASUREMENTS AND MAIN RESULTS: Rate of satisfactory surgical condition, hemodynamic changes, total surgical time, and blood loss were recorded. The rates of satisfactory surgical conditions were 6.7%, 72.4%, 89.7%, and 93.3% in groups 0U, 2U, 4U, and 6U, respectively; they were higher in groups 2U, 4U, and 6U than those in group 0U, but there were no significant differences among the groups 2U, 4U, and 6U. The blood loss was higher in group 0U than that in groups 2U, 4U, and 6U (p < .01). Pituitrin was associated with a transient decrease in blood pressures and an increase in heart rate in a dose-dependent fashion, with more pronounced changes in groups 4U and 6U, and these groups also required a higher amount of vasoactive drug to correct hemodynamic changes (p < .05). CONCLUSION: Two units of pituitrin could provide a satisfactory surgical field with minimal hemodynamic changes for laparoscopic uterine myomectomy.

[Effects of resveratrol-mediated inhibition of NOD-like receptor protein 3 inflammasomevia activating silent information regulator 1 on the injury of intestinal mucosal barrier function after sepsis].

OBJECTIVE: To explore whether resveratrol (RSV) could activate silent information regulator 1 (SIRT1) to regulate the activation of NOD-like receptor protein 3 (NLRP3) inflammasome in sepsis induced intestinal injury model, and then reduce intestinal inflammation and cell apoptosis, so as to play a protective role in intestinal barrier function. METHODS: (1) In vitro experiment: human Colorectal adenocarcinoma cells (Caco-2) were cultured, which were divided into normal group (normal culture on complete medium for 48 hours), lipopolysaccharide (LPS) group (normal culture on complete medium for 24 hours, then LPS containing 2 mg/L complete medium intervention for 6 hours), RSV low, medium and high concentration groups and SIRT1 inhibitor (EX-527) group (complete medium normal culture for 24 hours, LPS containing 2 mg/L complete medium intervention for 6 hours, followed by RSV 10, 20, 40 µmol/L or EX-527 10 µmol/L intervention for 6 hours, respectively). The levels of tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-18, IL-1ß) in the cell supernatant were determined by enzyme linked immunosorbent assay (ELISA). The apoptosis level of the cells was detected by flow cytometry. Western blotting was used to detect the protein levels of NLRP3, SIRT1, caspase-1 and apoptosis-related point-like protein (ASC). (2) In vivo experiment: according to random number table method, 24 male Wistar rats were divided into sham operation group (Sham group), cecal ligation and perforation (CLP) 6 hours group (CLP 6 h group), CLP 24 h group and RSV intervention group [RSV (20 mg/kg) was intraperitoneally injected 6 hours and 12 hours after CLP], with 6 rats in each group. The levels of NLRP3, caspase-1 and ASC in the intestine of rats were detected by immunohistochemistry. RESULTS: (1) Compared with the normal group, the levels of inflammatory factors in the cell supernatant of the LPS group were increased and the expression of SIRT1 protein was decreased, while the protein expressions of NLRP3, caspase-1 and ASC were increased. Compared with LPS group, different concentrations of RSV reduced the level of inflammatory factors, increased the activity of SIRT1, inhibited the expression of NLRP3 inflammasome and its downstream products caspase-1 and ASC, and the effect of high concentration of RSV (40 µmol/L) was the most significant [TNF-α (ng/L): 8.77±0.43 vs. 12.66±0.81, IL-6 (ng/L): 1.35±0.20 vs. 1.93±0.09, IL-1ß (ng/L): 1.05±0.04 vs. 1.31±0.07, IL-18 (ng/L): 519.50±11.16 vs. 622.70±30.69, SIRT1/ß-actin: 0.80±0.05 vs. 0.58±0.02, caspase-1/ß-actin: 0.55±0.06 vs. 0.78±0.06, ASC/ß-actin: 0.78±0.08 vs. 1.04±0.15, all P < 0.05], while SIRT1 inhibitor EX-527 had the opposite effects. There was no significant difference in the apoptosis rate among normal group, LPS group, and low, medium and high concentration RSV groups, as well as EX-527 group [(7.03±0.57)%, (9.67±0.55)%, (9.57±0.70)%, (9.30±2.15)%, (9.87±0.97)%, (9.07±0.93)%, F = 2.590, P = 0.082]. (2) Immunohistochemical results showed that compared with the Sham group, the expressions of NLRP3 inflammasomes and downstream products caspase-1 and ASC in the intestinal epithelial cells in CLP 6 h group, CLP 24 h group and RSV intervention group were significantly increased. The percentage of ASC-positive area in intestinal epithelium of RSV intervention group was significantly lower than that of CLP 6 h group [(15.22±2.73)% vs. (19.88±2.67)%, P < 0.05], and the expressions of NLRP3 and caspase-1 were significantly lower than those of CLP 24 h group [(9.31±1.37)% vs. (13.19±1.92)%, (19.57±3.92)% vs. (27.28±6.33)%, both P < 0.05]. CONCLUSIONS: After sepsis, high concentration of RSV could inhibit the activation of NLRP3 inflammasome by activating SIRT1, thereby reduce the expression of caspase-1 and ASC, and inhibit the secretion of inflammatory factors to reduce the inflammatory response.

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells.

PURPOSE: Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. METHODS: Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). RESULTS: scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. CONCLUSIONS: Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.