Complex evolutionary patterns within the tubulin gene family of ciliates, unicellular eukaryotes with diverse microtubular structures.

BACKGROUND: Tubulins are major components of the eukaryotic cytoskeletons that are crucial in many cellular processes. Ciliated protists comprise one of the oldest eukaryotic lineages possessing cilia over their cell surface and assembling many diverse microtubular structures. As such, ciliates are excellent model organisms to clarify the origin and evolution of tubulins in the early stages of eukaryote evolution. Nonetheless, the evolutionary history of the tubulin subfamilies within and among ciliate classes is unclear. RESULTS: We analyzed the evolutionary pattern of ciliate tubulin gene family based on genomes/transcriptomes of 60 species covering 10 ciliate classes. Results showed: (1) Six tubulin subfamilies (α_Tub, ß_Tub, γ_Tub, δ_Tub, ε_Tub, and ζ_Tub) originated from the last eukaryotic common ancestor (LECA) were observed within ciliates. Among them, α_Tub, ß_Tub, and γ_Tub were present in all ciliate species, while δ_Tub, ε_Tub, and ζ_Tub might be independently lost in some species. (2) The evolutionary history of the tubulin subfamilies varied. Evolutionary history of ciliate γ_Tub, δ_Tub, ε_Tub, and ζ_Tub showed a certain degree of consistency with the phylogeny of species after the divergence of ciliate classes, while the evolutionary history of ciliate α_Tub and ß_Tub varied among different classes. (3) Ciliate α- and ß-tubulin isoforms could be classified into an "ancestral group" present in LECA and a "divergent group" containing only ciliate sequences. Alveolata-specific expansion events probably occurred within the "ancestral group" of α_Tub and ß_Tub. The "divergent group" might be important for ciliate morphological differentiation and wide environmental adaptability. (4) Expansion events of the tubulin gene family appeared to be consistent with whole genome duplication (WGD) events in some degree. More Paramecium-specific tubulin expansions were detected than Tetrahymena-specific ones. Compared to other Paramecium species, the Paramecium aurelia complex underwent a more recent WGD which might have experienced more tubulin expansion events. CONCLUSIONS: Evolutionary history among different tubulin gene subfamilies seemed to vary within ciliated protists. And the complex evolutionary patterns of tubulins among different ciliate classes might drive functional diversification. Our investigation provided meaningful information for understanding the evolution of tubulin gene family in the early stages of eukaryote evolution.

Mitochondria-associated endoplasmic reticulum membranes involve in oxidative stress-induced intestinal barrier injury and mitochondrial dysfunction under diquat exposing.

Many factors induced by environmental toxicants have made oxidative stress a risk factor for the intestinal barrier injury and growth restriction, which is serious health threat for human and livestock and induces significant economic loss. It is well-known that diquat-induced oxidative stress is implicated in the intestinal barrier injury. Although some studies have shown that mitochondria are the primary target organelle of diquat, the underlying mechanism remains incompletely understood. Recently, mitochondria-associated endoplasmic reticulum membranes (MAMs) have aroused increasing concerns among scholars, which participate in mitochondrial dynamics and signal transduction. In this study, we investigated whether MAMs involved in intestinal barrier injury and mitochondrial dysfunction induced by diquat-induced oxidative stress in piglets and porcine intestinal epithelial cells (IPEC-J2 cells). The results showed that diquat induced growth restriction and impaired intestinal barrier. The mitochondrial reactive oxygen species (ROS) was increased and mitochondrial membrane potential was decreased following diquat exposure. The ultrastructure of mitochondria and MAMs was also disturbed. Meanwhile, diquat upregulated endoplasmic reticulum stress marker protein and activated PERK pathway. Furthermore, loosening MAMs alleviated intestinal barrier injury, decrease of antioxidant enzyme activity and mitochondrial dysfunction induced by diquat in IPEC-J2 cells, while tightening MAMs exacerbated diquat-induced mitochondrial dysfunction. These results suggested that MAMs may be associated with the intestinal barrier injury and mitochondrial dysfunction induced by diquat in the jejunum of piglets.

Comprehensive phylogenomic analyses reveal that order Armophorida is most closely related to class Armophorea (Protista, Ciliophora).

Ciliate species within the class Armophorea are widely distributed in various anaerobic environments, hence they are of great interest to researchers studying evolution and adaptation of eukaryotes to extreme habitats. However, phylogenetic relationships within the class remain largely elusive, most especially assignment of the order Armophorida and classification within the family Metopidae. In this study, we newly sequenced transcriptomes and the SSU rDNA of five armophorean species, Sulfonecta cf. uniserialis (order Armophorida), Nyctotheroides sp. (order Clevelandellida), and Metopus major, M. paraes, and Brachonella contorta (order Metopida). Comprehensive phylogenomic analyses revealed that Armophorea was most closely related to Muranotrichea and Parablepharismea. Our results indicate that the order Armophorida either belongs to Armophorea or represents a new class within APM (Armophorea-Parablepharismea-Muranotrichea). Analyses combining ecological niches and molecular trees showed that APM species might descend from an anaerobic free-living ciliate species. Existing molecular phylogenomic/phylogenetic and morphological evidence indicate that the family Metopidae is non-monophyletic and should be further classified with inclusion of additional lines of evidences. Our results provide new insights into the long-debated relationships within Armophorea.

Novel insights into molecular mechanisms of vegetative cell cycle and resting cyst formation in Apodileptus cf. visscheri (Alveolata, Ciliophora).

Ciliates usually with big cell sizes, complex morphological structures, and diverse life cycles, are good model organisms for studying cell proliferation regulation of eukaryotes. Up to date, the molecular regulation mechanisms for the vegetative cell cycle and encystment of these ciliates are poorly understood. Here, transcriptomes of Apodileptus cf. visscheri, which has an asexual vegetative cell cycle and is apt to encyst when environmental conditions become unfavorable, were sequenced to enrich our related knowledge. In this study, three replicates were sequenced for each of four cell stages, including initial period of growth, morphogenesis, cell division, and resting cyst. The significant transcription differences, involving cell cycle, biosynthesis, and energy metabolism pathways, were revealed between the resting cyst and vegetative cell cycle. Further investigations showed that the cell cycle pathway was enriched during morphogenesis stage and cell division stage. Compared to the initial period of growth stage, the differentially expressed genes involved in cellular components and molecular function were significantly enriched during cell division stage, while cellular components and biological processes were significantly enriched during morphogenesis stage. These provide novel insights into a comprehensive understanding at the molecular level of the survival and adaptive mechanism of unicellular eukaryotes.

Thoracoscopic total laryngo-pharyngo-oesophagectomy for the pharyngoesophageal junction cancer: a single-center experience of multidisciplinary team.

PURPOSE: The aims of this study were to review the surgical experience and evaluate the feasibility of thoracoscopic total laryngo-pharyngo-oesophagectomy by multidisciplinary team in the patients with pharyngoesophageal junction cancer. METHODS: A total of 31 patients with pharyngoesophageal junction cancer who underwent thoracoscopic total laryngo-pharyngo-oesophagectomy with gastric pull-up reconstruction performed by a collaborative thoracic surgery and otolaryngology surgery team in our department from January 2009 to January 2019 were retrospectively analysed. Surgical experience, Postoperative morbidity, overall survival were evaluated. RESULTS: The median age was 62 years old. Among these patients, 20 had hypopharyngeal cancer, 11 had cervical oesophageal cancer. No patients died during the perioperative period, and the median operation time was 4 h 30 min. The mean hospital stay was 13 days. The rate of complications was 32.3%. There were two cases of anastomotic leakage, four cases of moderate pulmonary infection. The median follow-up period was 31 months. Four patients were lost to follow-up in the second and fourth years and were considered to have died at that time. The 3- and 5-year overall survival rates were 52.6% and 31.6%, respectively. CONCLUSION: As a salvage surgery, thoracoscopic total laryngo-pharyngo-oesophagectomy by multidisciplinary team can be performed with an acceptable level of perioperative morbidity and mortality, relatively good recovery, and acceptable survival outcome for patients with pharyngoesophageal junction cancer.

Effects of hesperidin on mitochondrial function, mitochondria-associated endoplasmic reticulum membranes and IP3R-MCU calcium axis in the intestine of piglets exposed to deoxynivalenol.

Deoxynivalenol (DON) pollution is prevalent in crops, and can induce oxidative stress and intestinal injury. Hesperidin is one of the major flavonoids in citrus fruits that has various biological activities such as antioxidant and anti-inflammatory activities. However, whether hesperidin could alleviate DON-induced intestinal injury and the mechanism remain unclear. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) have attracted attention for their crucial signaling points to regulate ER-mitochondria calcium transfer. This study aims to evaluate the effects of hesperidin on the intestinal barrier, mitochondrial function, MAMs, and inositol 1,4,5-triphosphate receptor (IP3R)-mitochondrial calcium uniporter (MCU) calcium axis in the intestine of piglets exposed to DON. Twenty-four piglets were randomly divided into four groups in a 2 × 2 factorial arrangement for a 21-d experiment: Control: basal diet; hesperidin group: basal diet + 300 mg kg-1 hesperidin; DON: basal diet + 1.5 mg kg-1 DON; DON + hesperidin group: basal diet + 1.5 mg kg-1 DON + 300 mg kg-1 hesperidin. The data showed that when compared with the DON group, hesperidin improved growth performance and the intestinal barrier, alleviated intestinal oxidative stress and ER stress, and decreased the serum alanine aminotransferase (ALT) level (P < 0.05). Hesperidin also alleviated mitochondrial dysfunction and ferroptosis in the intestine of piglets exposed to DON (P < 0.05). Importantly, hesperidin prevented excessive MAM formation by downregulating the protein levels of Mitofusin 2 (Mfn2) and glucose-regulated protein 75 (GRP75), decreasing the ratio of the mitochondria with MAMs/total mitochondria and the ratio of MAM length/mitochondrial perimeter and lengthening the mitochondria-ER distance in MAMs (P < 0.05). Furthermore, hesperidin regulated the IP3R-glucose-regulated protein 75 (GRP75)-voltage-dependent anion channel 1 (VDAC1)-MCU calcium axis by decreasing the protein levels of GRP75 and MCU and the calcium level of the mitochondria compared with the DON group (P < 0.05). An in vitro experiment was conducted to further explore whether IP3R-mediated ER-mitochondria calcium transfer was involved in the protective effects of hesperidin on the intestinal epithelium barrier and mitochondria. Data showed that hesperidin may exert protective effects on the intestinal epithelium barrier and mitochondria via inhibiting ER-mitochondrial calcium transfer mediated by IP3Rs. These data suggested that hesperidin could alleviate MAM-mediated mitochondrial calcium overload, thereby improving mitochondrial function and alleviating oxidative stress and intestinal injury in DON-challenged piglets.

Deoxynivalenol induced intestinal barrier injury, mitochondrial dysfunction and calcium overload by inositol 1,4,5-triphosphate receptors (IP3Rs)-mitochondrial calcium uniporter (MCU) calcium axis.

Deoxynivalenol (DON) contamination is widespread in crops and could easily cause intestinal injury, which brings hazards to animals. Mitochondria are considered as an important target of DON, nevertheless, the mechanism is still unclear. Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) have gained arousing interest and are recognized as critical signaling hubs that control calcium signaling transduction between ER and mitochondria. This study aims to investigate the effects of DON on intestinal barrier, mitochondria, MAMs and inositol 1,4,5-triphosphate receptors (IP3Rs)-mitochondrial calcium uniporter (MCU) calcium axis in piglets and porcine intestinal epithelial cells (IPEC-J2). Furthermore, inhibition of IP3Rs or MCU was used to explore whether IP3Rs-MCU axis of MAMs was involved in the mitochondria dysfunction and intestinal epithelium barrier injury induced by DON in IPEC-J2. The data showed that DON induced intestinal barrier injury, mitochondrial dysfunction and ERS in piglets' jejunum and IPEC-J2. Moreover, DON increased MAMs by upregulating the protein level of Mitofusin 2 (Mfn2), increasing the percentage of mitochondria with MAMs/total mitochondria and the ratio of MAMs length/mitochondrial perimeter and shortening the distance between mitochondria and ER of MAMs. Importantly, DON influenced IP3Rs-glucose-regulated protein 75 (GRP75)-voltage-dependent anion channel 1 (VDAC1)-MCU calcium axis by increasing the protein levels of GRP75 and MCU and the interaction of VDAC1-GRP75-IP3Rs complex, which in turn induced mitochondrial calcium overload. Furthermore, inhibition of IP3Rs or MCU alleviated DON-induced intestinal epithelium barrier injury, mitochondrial dysfunction and mitochondrial calcium overload of IPEC-J2. The current investigation proposed that DON induced intestinal injury, mitochondrial dysfunction and calcium overload via IP3Rs-GRP75-VDAC1-MCU calcium axis.

Application of single-port laparoscopic retrograde gastric mobilization during McKeown esophagectomy for esophageal cancer.

BACKGROUND: As a novel alternative to the conventional minimally invasive esophagectomy (MIE) to treat esophageal cancer, single-port laparoscopic retrograde three-step gastric mobilization (SLRM) for esophageal reconstruction during MIE to treat esophageal cancer was attempted in our department. The aim of the present study was to explore the preliminary clinical outcomes and feasibility of this innovative surgery. METHODS: From March 2020 to November 2021, patients undergoing SLRM combined with four-port thoracoscopic McKeown esophagectomy for their esophageal cancers were reviewed. Gastric mobilization with abdominal lymph node dissection was performed through SLRM. The clinical characteristics and short-term outcomes were analyzed retrospectively. RESULTS: A total of 120 patients underwent R0 resection without conversion to open surgery. The mean times needed for the thoracic part, abdominal part, and total operation were 43 ± 6 min, 60 ± 18 min, and 230 ± 20 min, respectively. The numbers of mediastinal and abdominal lymph nodes harvested were 13.2 ± 2.7 and 10.2 ± 2.5, respectively. Postoperative pneumonia was encountered in 10 (8.3%) patients. Anastomotic leakage occurred in 3 (2.5%) cases. Temporary vocal cord paralysis was reported in 20 (16.6%) cases. The mean length of hospital stay was 8.5 ± 4.6 days. CONCLUSIONS: The SLRM is a technically feasible and safe treatment for patients with esophageal cancer. It can be considered an alternative method for patients, especially for the ones with obesity and gastric distension.

Effects of DON on Mitochondrial Function, Endoplasmic Reticulum Stress, and Endoplasmic Reticulum Mitochondria Contact Sites in the Jejunum of Piglets.

Recent research has emphasized the significance of investigating the interplay between organelles, with endoplasmic reticulum mitochondria contact sites (ERMCSs) being recognized as critical signaling hubs between organelles. The objective of the current study was to assess the impact of deoxynivalenol (DON) on jejunal mitochondria, ER, and ERMCSs. Twelve piglets (35 d, 10.22 ± 0.35 kg) were randomized into two groups: control group, basal diet; the DON group, basal diet + 1.5 mg/kg DON. The findings revealed that DON decreased growth performance, induced jejunal oxidative stress, and impaired jejunal barrier function. DON was also found to induce mitochondrial dysfunction, trigger endoplasmic reticulum stress (ERS) in the piglets' jejunum, and activate mitochondrial and ER apoptosis pathways by upregulating apoptosis-related proteins (Caspase-8, Caspase-12, Bax, and CHOP). To investigate the involvement of ERMCSs in DON-induced intestinal injury, we measured the protein levels of ERMCS proteins, such as mitofusin 1 (Mfn1), mitofusin 2 (Mfn2), and glucose-regulated protein 75 (GRP75) and Pearson's correlation coefficient of ERMCS proteins and ERMCS ultrastructure. Our finding showed that DON upregulated the protein level of Mfn2 and GRP75 and increased the percentage of mitochondria with ERMCSs/total mitochondria, the length of ERMCSs compared to the perimeter of mitochondria, and the Pearson's correlation coefficient of voltage-dependent anion-selective channel protein 1 (VDAC1) and inositol 1,4,5-triphosphate receptors (IP3Rs) in piglets' jejunum. Furthermore, DON shortened the distance between mitochondria and ER at ERMCSs. These findings suggested that DON impaired mitochondrial function, triggered ERS, and increased ERMCSs, indicating that the increased ERMCSs could be related to mitochondrial dysfunction and ERS involved in the intestinal injury of piglets induced by DON.

Effect of 40 Hz light flicker on cognitive impairment and transcriptome of hippocampus in right unilateral common carotid artery occlusion mice.

Vascular cognitive impairment caused by chronic cerebral hypoperfusion (CCH) seriously affects the quality of life of elderly patients. However, there is no effective treatment to control this disease. This study investigated the potential neuroprotective effect of the 40 Hz light flicker in a mouse model of CCH. CCH was induced in male C57 mice by right unilateral common carotid artery occlusion (rUCCAO), leading to chronic brain injury. The mice underwent 40 Hz light flicker stimulation for 30 days after surgery. The results showed that 40 Hz light flicker treatment ameliorated memory deficits after rUCCAO and alleviated the damage to neurons in the frontal lobe and hippocampus. Light flicker administration at 40 Hz decreased IL-1ß and TNF-α levels in the frontal lobe and hippocampus, but immunohistochemistry showed that it did not induce angiogenesis in mice with rUCCAO. Gene expression profiling revealed that the induction of genes was mainly enriched in inflammatory-related pathways. Our findings demonstrate that 40 Hz light flicker can suppress cognitive impairment caused by rUCCAO and that this effect may be involved in the attenuation of neuroinflammation.

Sodium Butyrate Ameliorates Oxidative Stress-Induced Intestinal Epithelium Barrier Injury and Mitochondrial Damage through AMPK-Mitophagy Pathway.

Sodium butyrate has gained increasing attention for its vast beneficial effects. However, whether sodium butyrate could alleviate oxidative stress-induced intestinal dysfunction and mitochondrial damage of piglets and its underlying mechanism remains unclear. The present study used a hydrogen peroxide- (H2O2-) induced oxidative stress model to study whether sodium butyrate could alleviate oxidative stress, intestinal epithelium injury, and mitochondrial dysfunction of porcine intestinal epithelial cells (IPEC-J2) in AMPK-mitophagy-dependent pathway. The results indicated that sodium butyrate alleviated the H2O2-induced oxidative stress, decreased the level of reactive oxygen species (ROS), increased mitochondrial membrane potential (MMP), mitochondrial DNA (mtDNA), and mRNA expression of genes related to mitochondrial function, and inhibited the release of mitochondrial cytochrome c (Cyt c). Sodium butyrate reduced the protein expression of recombinant NLR family, pyrin domain-containing protein 3 (NLRP3) and fluorescein isothiocyanate dextran 4 kDa (FD4) permeability and increased transepithelial resistance (TER) and the protein expression of tight junction. Sodium butyrate increased the expression of light-chain-associated protein B (LC3B) and Beclin-1, reduced the expression of P62, and enhanced mitophagy. However, the use of AMPK inhibitor or mitophagy inhibitor weakened the protective effect of sodium butyrate on mitochondrial function and intestinal epithelium barrier function and suppressed the induction effect of sodium butyrate on mitophagy. In addition, we also found that after interference with AMPKα, the protective effect of sodium butyrate on IPEC-J2 cells treated with H2O2 was suppressed, indicating that AMPKα is necessary for sodium butyrate to exert its protective effect. In summary, these results revealed that sodium butyrate induced mitophagy by activating AMPK, thereby alleviating oxidative stress, intestinal epithelium barrier injury, and mitochondrial dysfunction induced by H2O2.

LncRNA NRON negatively regulates cisplatin-induced cell apoptosis via downregulating miR-31 in esophageal squamous cell carcinomas.

LncRNA non-coding repressor of NFAT (NRON) can promote bladder cancer and suppress liver cancer, while its role in esophageal squamous cell carcinoma (ESCC) is unknown. We analyzed its involvement in ESCC. NRON and miR-31 expression in plasma before and after cisplatin treatment was detected using RT-qPCR. The correlation between NRON and miR-31 was analyzed by linear regression. Expression of NRON and miR-31 in ESCC cells was also analyzed using RT-qPCR. Overexpression of NRON and miR-31 was achieved in ESCC cells to explore their interaction. Cell apoptosis induced by cisplatin was studied using cell apoptosis assay. NRON was highly expressed in ESCC and further upregulated after cisplatin treatment. MiR-31 was downregulated in ESCC and inversely correlated with NRON. In ESCC cells, NRON overexpression decreased miR-31 expression, while miR-31 failed to alter NRON expression. Cisplatin treatment promoted NRON expression and inhibited miR-31 expression. Under cisplatin treatment, cell apoptosis was inhibited after NRON overexpression and increased after miR-31 overexpression. Moreover, NRON inhibited the effect of miR-31 on cell apoptosis. NRON might promote the development of cisplatin resistance via downregulating miR-31 in ESCC.

Effects of Curcumin on Mitochondrial Function, Endoplasmic Reticulum Stress, and Mitochondria-Associated Endoplasmic Reticulum Membranes in the Jejunum of Oxidative Stress Piglets.

Mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are not only critical for the communication between two organelles but also crucial for cellular processes such as energy metabolism, calcium signaling, and mitochondrial dynamics. The effects of curcumin on jejunal mitochondria, ER, and MAMs in piglets under diquat-induced oxidative stress were assessed. Twenty-four piglets (35 days old, weaned at 21 days, 9.54 ± 0.28 kg, six piglets per group) were used in the study: (1) control group; (2) control + curcumin group; (3) diquat group; and (4) diquat + curcumin group. Curcumin was mixed with the basic diet at 200 mg/kg and fed to piglets. Piglets were administered intraperitoneally of 0.9% saline solution or diquat at 10 mg/kg body weight on the first day. Compared with the diquat group, curcumin improved jejunal morphology and barrier function. Meanwhile, curcumin improved mitochondrial function and ultrastructure, alleviated endoplasmic reticulum stress (ERS), and inhibited apoptosis induced by diquat. Moreover, curcumin prevented excessive MAM formation and alleviated MAM disorder. In conclusion, dietary curcumin ameliorated jejunal damage and mitochondrial dysfunction, attenuated ERS, and alleviated MAM disorder in oxidative stress piglets induced by diquat.

Utility of single-port laparoscopic retrograde gastric mobilization during McKeown esophagectomy for esophageal cancer: a 2-year experience with 120 cases in a single institution.

Background: As a novel alternative to the conventional minimally invasive esophagectomy (MIE), more minimally invasive single-port laparoscopic retrograde 3-step gastric mobilization (SLRM) for esophageal reconstruction during MIE to treat esophageal cancer was attempted by our department. This study explored the preliminary clinical outcomes and feasibility of this innovative surgery. Methods: The data of 120 patients who had undergone SLRM combined with 4-port thoracoscopic McKeown esophagectomy for their esophageal cancers from March 2020 to November 2021 were reviewed. Gastric mobilization with abdominal lymph node dissection was performed via SLRM. The clinical characteristics and short-term outcomes were retrospectively analyzed. The data of operating time, blood loss, harvested lymph nodes, postoperative hospital stay and complications are presented as the mean and standard deviation. Results: A total of 120 patients underwent R0 resection without conversion to open surgery. The mean times for the thoracic procedure, abdominal procedure, and total operation were 43±6, 60±18, and 195±20 min, respectively. The numbers of mediastinal and abdominal lymph nodes harvested were 13.2±2.7, and 10.2±2.5, respectively. Postoperative pneumonia occurred in 10 patients (8.3%). Anastomotic leakage occurred in 3 patients (2.5%). Temporary vocal cord paralysis was reported in 20 patients (16.6%). The mean length of hospital stay was 8.5±4.6 days. Conclusions: SLRM is a technically feasible and safe treatment for patients with esophageal cancer. It can be considered an alternative method for patients, especially those with obesity and gastric distension.

Clostridium butyricum and Its Derived Extracellular Vesicles Modulate Gut Homeostasis and Ameliorate Acute Experimental Colitis.

Microbiological treatments are expected to have a role in the future management of inflammatory bowel disease (IBD). Clostridium butyricum (C. butyricum) is a probiotic microorganism that exhibits beneficial effects on various disease conditions. Although many studies have revealed that C. butyricum provides protective effects in mice with colitis, the way C. butyricum establishes beneficial results in the host remains unclear. In this study, we investigated the mechanisms by which C. butyricum modifies the gut microbiota, produces bacterial metabolites that may be involved, and, specifically, how microbial extracellular vesicles (EVs) positively influence IBD, using a dextran sulfate sodium (DSS)-induced colitis murine model in mice. First, we showed that C. butyricum provides a protective effect against colitis, as evidenced by the prevention of body weight loss, a reduction in the disease activity index (DAI) score, a shortened colon length, decreased histology score, and an improved gut barrier function, accompanied by reduced levels of pathogenic bacteria, including Escherichia/Shigella, and an increased relative abundance of butyrate-producing Clostridium sensu stricto-1 and Butyricicoccus. Second, we also confirmed that the gut microbiota and metabolites produced by C. butyricum played key roles in the attenuation of DSS-induced experimental colitis, as supported by the profound alleviation of colitis effects following fecal transplantation or fecal filtrate insertion supplied from C. butyricum-treated mice. Finally, C. butyricum-derived EVs protected the gut barrier function, improved gut microbiota homeostasis in ulcerative colitis, and contributed to overall colitis alleviation. IMPORTANCE This study indicated that C. butyricum provided a prevention effect against colitis mice, which involved protection of the intestinal barrier and positively regulating gut microbiota. Furthermore, we confirmed that the gut microbiota and metabolites that were induced by C. butyricum also contributed to the attenuation of DSS-induced colitis. Importantly, C. butyricum-derived EVs showed an effective impact in alleviating colitis.

Transcriptome Comparison of Chorion-Attached and Non-chorion-attached Endometrium in Mid-gestation of Rabbit.

Background: The chorion from the placenta is directly attached to the endometrium (CA) after embryo implantation while some parts of the endometrium are not chorion-attached (NCA). The differences in gene expression between the CA and NCA endometrium mid-gestation are unknown. Our objective was to compare the gene expression profiles of the CA and NCA endometrium of rabbit, to identify the differentially expressed genes (DEGs), and correlate the differences with the physiological state of the endometrium at mid-gestation of rabbit. Methods: We used transcriptome sequencing to reveal the differences in gene expression between CA and NCA endometrium (n = 3), and then determined the concentration of inflammatory cytokines in CA and NCA tissue and serum by ELISA. Results: Six Hundred and Forty-Six DEGs were identified between the CA and NCA endometrium [p < 0.05, |log2 (fold change) |≥ 2], The expression levels of 590 DEGs were higher in the NCA endometrium than in the CA endometrium, while the expression level of only 56 DEGs were higher in CA than in NCA. The DEGs were enriched in gene ontology (GO) terms and pathways related to immune regulation and cellular adhesions. Six hub-genes related to inflammatory mediator regulation of transient receptor potential (TRP) channels and chemokine signaling pathways had a lower expression level in the CA endometrium compared to the NCA endometrium, and the expression levels of genes related to focal adhesion and extracellular matrix (ECM)-receptors were significantly higher in NCA endometrium than in CA endometrium. The level of pro-inflammatory cytokines accumulated in the CA endometrium, and high abundance of integrin-ß and THBS1 were localized in the luminal epithelium of the NCA endometrium, but not in the CA endometrium. Conclusions: Our study reveals differences in gene expression between the CA and NCA endometrium at mid-gestation of rabbit, and suggests implications for endometrial physiological function. The CA endometrium showed relative low-level gene expression compared to the NCA endometrium, while the NCA endometrium performed physiological functions related to focal adhesion and ECM-receptor interaction.

MICAL2 Facilitates Gastric Cancer Cell Migration via MRTF-A-Mediated CDC42 Activation.

Aims and Hypothesis: Cell migration is driven by the reorganization of the actin cytoskeleton. Although MICAL2 is known to mediate the oxidation of actin filaments to regulate F-actin dynamics, relatively few studies have investigated the potential role of MICAL2 during cancer cell migration. Methods: The migratory ability of gastric cancer cells was measured by wound healing and transwell assays. The relationship between MICAL2 expression and MRTF-A nuclear localization was analyzed using gene overexpression and knockdown strategies. The production of reactive oxygen species (ROS) was evaluated by DCFH-DA staining. mRNA and protein levels of MMP9 were measured using qPCR and immunoblotting analysis. The activities of CDC42 and RhoA were assessed using pulldown assays. Results: Depletion of MICAL2 markedly reduced gastric cancer cell migration. Mechanistically, silencing of MICAL2 inhibited the nuclear translocation of MRTF-A in response to EGF and serum stimulation, whereas the contents of MRTF-A remained unchanged. Further analysis showed that silencing of MICAL2 decreased the activation of CDC42 as well as mRNA and protein levels of MMP9. Ectopic expression of MICAL2 augmented MRTF-A levels in the nucleus, and promoted the activation of CDC42, MMP9 expression, and gastric cancer cell migration. Moreover, silencing of MRTF-A inhibited the CDC42 activation induced by overexpression of MICAL2. In addition, MICAL2-induced ROS generation contributed to the effect exerted by MICAL2 on MRTF-A nuclear translocation. Conclusion: Together, these results provide evidence that MICAL2 facilitates gastric cancer cell migration via positive regulation of nuclear translocation of MRTF-A and subsequent CDC42 activation and MMP9 expression.

MICAL-L2 Is Essential for c-Myc Deubiquitination and Stability in Non-small Cell Lung Cancer Cells.

Objectives: MICAL-L2, a member of the molecules interacting with the CasL (MICAL) family, was reported to be highly expressed in several types of cancers, however, the roles of MICAL-L2 in NSCLC pathogenesis remain to be explored. This study is designed to clarify the mechanisms by which MICAL-L2 participates in NSCLC cell proliferation. Materials and Methods: The expression levels of MICAL-L2 in human lung cancer samples were assessed by immunohistochemical staining. Cells were transfected with siRNA or plasmids to regulate MICAL-L2 expression. Cell proliferation was measured by EdU staining and CCK-8 assays. MICAL-L2 and phosphorylated/total c-Myc expression were examined by Western blotting analysis. Interaction between MICAL-L2 and c-Myc was assessed by immunofluorescence staining, Western blotting and co-immunoprecipitation assays. Western blotting, polyubiquitylation detection and protein stability assays were used to assess whether MICAL-L2 exerts its oncogenic effect via c-Myc. Results: We found that MICAL-L2 was highly expressed in human NSCLC. While overexpressing MICAL-L2 increased NSCLC cell proliferation, MICAL-L2 depletion decreased the proliferation of NSCLC cells, an effect that was linked to cell cycle arrest. MICAL-L2 physically interacted with the c-Myc protein and functioned to maintain nuclear c-Myc levels and prolonged its half-life. Knockdown of MICAL-L2 expression led to decreased c-Myc protein stability through accelerating polyubiquitylation of c-Myc and gave rise to c-Myc degradation. We further found that MICAL-L2 deubiquitinated c-Myc and blocked its degradation, presumably by inhibiting c-Myc phosphorylation at threonine residue 58. Conclusions: These results indicate that MICAL-L2 is a key regulator of c-Myc deubiquitination and stability in the nucleus, and this activity may be involved in promoting NSCLC cell proliferation.

Merely inoculating anammox sludge to achieve the start-up of anammox and autotrophic desulfurization-denitrification process.

Anammox and autotrophic desulfurization-denitrification (AADD) process is feasible for the nitrogen and sulfide removal in the same reactor, and the influence of excess nitrate produced by anammox could also be alleviated simultaneously. This study firstly proposed a novel strategy with inoculating single anammox sludge to start up the AADD process. Results demonstrated that the 90% nitrogen removal efficiency (NRE), 2.55kgm-3 d-1 nitrogen removal rate (NRR), and 95% sulfide removal efficiency (SRE) were obtained at the influent total nitrogen of 280mgL-1 and sulfide of 221.5mgL-1, and the final effluent nitrate concentration was as low as 8mgL-1 under the appropriate operation conditions. Tryptophan-like and protein-like substances were characterized as the main components in bound EPS. Thiobacillus (35.68%) and Pseudoxanthomonas (11.61%) were identified as the predominant genera. This study will pave a potential avenue to promote the treatment of high concentration nitrogen and sulfide in wastewater in the future.