Histopathological staging and differential diagnosis of marginal zone lymphoma of gastric mucosa-associated lymphoid tissue.

The purpose of this study was to explore the histopathological staging and differential diagnosis of marginal zone lymphoma in gastric mucosa-associated lymphoid tissue (MALT lymphoma). We performed detailed histomorphology and immunohistochemistry investigations as well as genetic testing on endoscopic biopsy and endoscopic mucosal resection specimens from 18 patients with gastric MALT lymphoma. We found that gastric MALT lymphoma typically begins as a small, isolated area outside the lymphoid follicular mantle zone or proliferates in a multifocal, patchy manner, gradually spreads to the interfollicular zone, forming diffuse proliferation, invades the gastric mucosal glands, and infiltrates or proliferates into the center of peripheral reactive lymphoid follicles. Abnormally proliferating lymphocytes invade the surrounding lymphoid follicles, resulting in damage, atrophy, and disappearance of their normal follicles as well as of the gastric mucosa glands, forming diffuse proliferation. Redifferentiation and proliferation lead to the transformation of lymphocytes; that is, MALT transitions into highly invasive lymphoma. Based on our findings in this study, we propose the following five stages in the process of development and progression of gastric MALT lymphoma: the stage of cell proliferation outside the lymphoid follicular mantle zone; the stage of heterogeneous proliferative lymphoepithelial lesion; the stage of reactive lymphoid follicular implantation; the stage of lymphoid follicular clonal proliferation; and the stage of MALT transforming into highly invasive lymphoma. We examined the differential diagnosis of histopathological features at each stage. The clinicopathological staging of gastric MALT lymphoma can help clinicians provide accurate treatment and track malignant cell transformation, thus playing a significant role in controlling its development and progression.

Correlation between abnormal energy metabolism of ovarian granulosa cells and in vitro fertilization-embryo transfer outcomes in patients with polycystic ovary syndrome and obesity.

CONTEXT: Granulosa cells (GCs) that surround oocytes in mammalian reproduction play an active role in oocyte differentiation through proliferation and energy production. AIMS: This study aimed to investigate the characteristics of the energy metabolism of ovarian GCs and the influence of GCs on the early embryonic development in polycystic ovary syndrome (PCOS). METHODS: The clinical characteristics and in vitro fertilization-embryo transfer treatment outcomes of 39 patients with PCOS and 68 patients with simple tubal factor infertility who underwent controlled ovarian hyperstimulation were analyzed and summarized. The mitochondrial function and glucose metabolism level of the GCs were determined, as well as the content of oxidative stress markers in the follicular fluid (FF) of patients with and without PCOS. KEY RESULTS: When compared to the non-PCOS group, patients with PCOS had a significantly increased number of retrieved oocytes but a significantly decreased number of high-quality embryos, available embryos, and high-quality blastocyst formation (P < 0.05). Furthermore, the mitochondrial membrane potential, adenosine triphosphate level, and mitochondrial DNA (mtDNA) copy number decreased in the GCs, whereas the levels of reactive oxygen species increased (P < 0.01). The levels of malondialdehyde and 8-oxo-deoxyguanosine (8-OHdG) in the follicular fluid (FF) of the patients with PCOS were higher than those of the control group (P < 0.05), and superoxide dismutase was increased by compensation (P < 0.05). In the PCOS group, the expressions of GLUT1, LDHA, and PFKP were lower than those in the non-PCOS group, and glucose levels were higher. CONCLUSIONS: The low oocyte competence of PCOS may be associated with mitochondrial dysfunction and abnormal glycolysis. IMPLICATIONS: This research offers explanations for the possible connections influencing human ovarian folliculogenesis.

Nickel-Catalyzed Selective C(sp2 )-F Bond Alkylation of Industrially Relevant Hydrofluoroolefin HFO-1234yf.

The selective transition-metal catalyzed C-F bond functionalization of inexpensive industrial fluorochemicals represents one of the most attractive approaches to valuable fluorinated compounds. However, the selective C(sp2 )-F bond carbofunctionalization of refrigerant hydrofluoroolefins (HFOs) remains challenging. Here, we report a nickel-catalyzed selective C(sp2 )-F bond alkylation of HFO-1234yf with alkylzinc reagents. The resulting 2-trifluoromethylalkenes can serve as a versatile synthon for diversified transformations, including the anti-Markovnikov type hydroalkylation and the synthesis of bioactive molecule analogues. Mechanistic studies reveal that lithium salt is essential to promote the oxidative addition of Ni0 (Ln ) to the C-F bond; the less electron-rich N-based ligands, such as bipyridine and pyridine-oxazoline, feature comparable or even higher oxidative addition rates than the electron-rich phosphine ligands; the strong σ-donating phosphine ligands, such as PMe3 , are detrimental to transmetallation, but the less electron-rich and bulky N-based ligands, such as pyridine-oxazoline, facilitate transmetallation and reductive elimination to form the final product.

Cas12a/Guide RNA-Based Platforms for Rapidly and Accurately Identifying Staphylococcus aureus and Methicillin-Resistant S. aureus.

In order to ensure the prevention and control of methicillin-resistant Staphylococcus aureus (MRSA) infection, rapid and accurate detection of pathogens and their resistance phenotypes is a must. Therefore, this study aimed to develop a fast and precise nucleic acid detection platform for identifying S. aureus and MRSA. We initially constructed a CRISPR-Cas12a detection system by designing single guide RNAs (sgRNAs) specifically targeting the thermonuclease (nuc) and mecA genes. To increase the sensitivity of the CRISPR-Cas12a system, we incorporated PCR, loop-mediated isothermal amplification (LAMP), and recombinase polymerase amplification (RPA). Subsequently, we compared the sensitivity and specificity of the three amplification methods paired with the CRISPR-Cas12a system. Finally, the clinical performance of the methods was tested by analyzing the fluorescence readout of 111 clinical isolates. In order to visualize the results, lateral-flow test strip technology, which enables point-of-care testing, was also utilized. After comparing the sensitivity and specificity of three different methods, we determined that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a methods were the optimal detection methods. The nuc-LAMP-Cas12a platform showed a limit of detection (LOD) of 10 aM (~6 copies µL-1), while the mecA-LAMP-Cas12a platform demonstrated a LOD of 1 aM (~1 copy µL-1). The LOD of both platforms reached 4 × 103 fg/µL of genomic DNA. Critical evaluation of their efficiencies on 111 clinical bacterial isolates showed that they were 100% specific and 100% sensitive with both the fluorescence readout and the lateral-flow readout. Total detection time for the present assay was approximately 80 min (based on fluorescence readout) or 85 min (based on strip readout). These results indicated that the nuc-LAMP-Cas12a and mecA-LAMP-Cas12a platforms are promising tools for the rapid and accurate identification of S. aureus and MRSA. IMPORTANCE The spread of methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to global health. Isothermal amplification combined with the trans-cleavage activity of Cas12a has been exploited to generate diagnostic platforms for pathogen detection. Here, we describe the design and clinical evaluation of two highly sensitive and specific platforms, nuc-LAMP-Cas12a and mecA-LAMP-Cas12a, for the detection of S. aureus and MRSA in 111 clinical bacterial isolates. With a limit of detection (LOD) of 4 × 103 fg/µL of genomic DNA and a turnaround time of 80 to 85 min, the present assay was 100% specific and 100% sensitive using either fluorescence or the lateral-flow readout. The present assay promises clinical application for rapid and accurate identification of S. aureus and MRSA in limited-resource settings or at the point of care. Beyond S. aureus and MRSA, similar CRISPR diagnostic platforms will find widespread use in the detection of various infectious diseases, malignancies, pharmacogenetics, food contamination, and gene mutations.

Gene expression and methylation profiles show the involvement of POMC in primary hyperparathyroidsm.

Primary hyperparathyroidism (PHPT) is mainly caused by parathyroid adenoma, which produces excess parathyroid hormones. Its pathogenic mechanisms have not yet been fully understood. To investigate the mechanism in the pathogenesis of PHPT, the transcriptome and genome-wide DNA methylation profiles of parathyroid adenoma were analyzed. The candidate genes that may be involved in the PHPT were verified via qRT-PCR, immunohistochemistry, western blot, and methylation-specific PCR. A total of 1650 differentially expressed genes and 2373 differentially methylated regions were identified. After the integration of its transcriptome and DNA methylation data, IL6, SYP, GNA01, and pro-opiomelanocortin (POMC) were the candidate genes that demonstrated a similar pattern between their mRNA expression and DNA methylation status. Of the 4 candidate genes, POMC, a pro-peptide which is processed to a range of bioactive peptide products like ACTH, was further confirmed to be expressed at low levels at both the mRNA and protein levels, which may be due to POMC promoter hypermethylation. Hypermethylation of the POMC promoter may contribute to its low expression, which may be involved in the pathogenesis of PHPT.

Mutational Profile and Potential Molecular Therapeutic Targets of Pheochromocytoma.

Purpose: Pheochromocytoma/paraganglioma (PCC/PGL; collectively known as PPGL) can be driven by germline and somatic mutations in susceptibility genes. We aimed to investigate the mutation profile and clinical features of pathogenic genes in highly genetically heterogeneous PPGL and to preliminary explore molecular therapeutic targets in PPGL. Methods: We established a panel of 260 genes, including susceptibility genes of PPGL and other important tumorigenic genes to sequence 107 PPGL tissues. Results: Overall, 608 genomic mutations were identified in 107 PPGL tissues. Almost 57% of PPGL tissue samples exhibited pathogenic mutations, and the most frequently mutated gene was SDHB (15/107, 14%). SDHB and HRAS were the most commonly mutated genes in germline-mutated PPGL (25/107, 23%) and nongermline-mutated PPGL (36/107, 34%), respectively. In addition, novel pathogenic mutations were detected in sporadic PPGL. PPGL with mutations in the hypoxia pathway had an earlier onset and higher norepinephrine level than those in the kinase pathway. Receptor tyrosine kinase (RTK; 22%, 24/107), mitogen-activated protein kinase (MAPK; 14%, 15/107), and tyrosine kinase (TK; 2%, 2/107) pathways were the most frequently mutated pathways in PPGL. Conclusion: Our results provided the genetic mutation profile in PPGL tissues. Genetic mutations in PPGL were mainly concentrated in the RTK, TK, and MAPK pathways, suggesting potential molecular therapeutic targets for PPGL.

Convergence between germline and somatic mutations in pancreatic neuroendocrine tumors.

Objectives: The pancreatic neuroendocrine tumors (PanNETs) are a group of clinically heterogeneous neoplasms. Although previous studies illustrated the somatic mutation pattern for PanNETs, the germline mutation pattern is still unclear. Here, we comprehensively screened the underlying germline mutations in a cohort of multiple endocrine neoplasia type 1 (MEN1)-related and sporadic PanNETs to reveal the characteristics of germline mutation in PanNET patients. Methods: Patients diagnosed with PanNETs by biopsy or surgical pathology were enrolled in this study. Peripheral blood samples were used for genomic DNA purification and subsequent sequencing. The following sequencing techniques were used and compared for validation: (1) targeted gene capture with a customized panel; (2) whole exome sequencing data from previous study. Results: A total of 184 PanNET patients were enrolled, including 20 MEN1-related and 164 sporadic cases. In this study, MEN1 mutation rate in MEN1-related PanNETs was 60% (12/20), of which 50% were novel mutation sites. For sporadic PanNETs, the overall germline mutation rate was very low. Besides the rare MEN1 mutation, previously unreported germline variant in DAXX was found in one non-functional PanNET. Conclusions: This study revealed distinctive germline mutation rates between MEN1-related and sporadic PanNETs. The novel MEN1 mutations contribute to revealing the spectrum of MEN1 mutations in PanNETs. The newly discovered germline variant of DAXX in sporadic PanNET implies a tendency of convergence between germline and somatic mutation genes.

[Application of ultrasound-guided endoscopic retrograde appendicitis therapy in children with appendix-related chronic abdominal pain]. / è¶ å£°å¼•å¯¼å† é•œä¸‹é€†è¡Œé˜‘å°¾ç‚Žæ²»ç–—æœ¯åœ¨é˜‘å°¾ç›¸å ³æ ¢æ€§è ¹ç—›æ‚£å„¿ä¸­çš„åº”ç”¨ä»·å€¼.

OBJECTIVES: To study the clinical efficacy of ultrasound-guided endoscopic retrograde appendicitis therapy in children with appendix-related chronic abdominal pain. METHODS: A retrospective analysis was performed on the medical data of 30 children with the chief complaint of chronic abdominal pain who were admitted from August 2019 to May 2021. All the children were found to have inflammation of the appendix or intracavitary stool and fecalith by ultrasound and underwent ultrasound-guided endoscopic retrograde appendicitis therapy. The medical data for analysis included clinical manifestations, endoscopic findings, white blood cell count, neutrophil percentage, length of hospital stay, and cure rate. RESULTS: Among the 30 children with chronic abdominal pain, there were 13 boys (43%) and 17 girls (57%), with a mean age of (9±3) years (range 3-15 years) at diagnosis. The median duration of the disease was 12 months, and the median length of hospital stay was 3 days. The children had a median white blood cell count of 6.7×109/L and a neutrophil percentage of 50%±13%. Fecalith and a large amount of feces were flushed out of the appendix cavity for 21 children (70%) during surgery. The follow-up rate was 97% (29/30), and the median follow-up time was 11 months (range 5-26 months). Of the 29 children, abdominal pain completely disappeared in 27 children (93%). CONCLUSIONS: Ultrasound-guided endoscopic retrograde appendicitis therapy is effective in children with chronic abdominal pain caused by feces or fecalith in the appendix cavity.

Recurrent Germline Mutations of CHEK2 as a New Susceptibility Gene in Patients with Pheochromocytomas and Paragangliomas.

PURPOSE: Recently, pheochromocytomas and paragangliomas (PPGLs) have been strongly suspected as hereditary tumors, as approximately 40% of patients carry germline mutations. In the cancers where defects occur to corrupt DNA repair and facilitate tumorigenesis, a CHEK2 strong association has been observed. Therefore, the purpose of this study was to investigate the effect of CHEK2 mutations for its possible pathogenicity in PPGLs. METHODS: Four patients with CHEK2 mutations were recruited, as previously detected by the whole exome sequencing. Sanger sequencing was used to verify the germline mutations as well as the loss of heterozygosities (LOHs) in their somatic DNAs. Immunohistochemistry was used to analyze the expression of CHEK2 and its downstream target p53 Ser20 (phosphorylated p53). RESULTS: The average age of studied patients was 44.25 ± 11.18 years, at the time diagnosis. One patient had multiple tumors which recurred quickly, while two patients had distant metastasis. None of the patient had any relevant family history. Four germline CHEK2 mutations were identified (c.246_260del; c.715G > A; c.1008+3A > T; and c.1111C > T). All the patients were predicted to have either pathogenic or suspected pathogenic mutations. There was no LOH of CHEK2 gene in somatic DNAs found. Additionally, neither CHEK2 proteins nor its downstream target p53 Ser20 were expressed in the tumor tissues. The inactivation of CHEK2 leads to the decrease in the p53 phosphorylation, which might promote tumorigenesis. CONCLUSIONS: For the first time, CHEK2 was identified as a susceptibility gene for PPGLs. However, the penetrance of CHEK2 gene with genotype-phenotype correlation needs to be investigated.

Sequence and Structure Characteristics of 22 Deletion Breakpoints in Intron 44 of the DMD Gene Based on Long-Read Sequencing.

Purpose: Exon deletions make up to 80% of mutations in the DMD gene, which cause Duchenne and Becker muscular dystrophy. Exon 45-55 regions were reported as deletion hotspots and intron 44 harbored more than 25% of deletion start points. We aimed to investigate the fine structures of breakpoints in intron 44 to find potential mechanisms of large deletions in intron 44. Methods: Twenty-two dystrophinopathy patients whose deletion started in intron 44 were sequenced using long-read sequencing of a DMD gene capture panel. Sequence homology, palindromic sequences, and polypyrimidine sequences were searched at the breakpoint junctions. RepeatMasker was used to analyze repetitive elements and Mfold was applied to predict secondary DNA structure. Results: With a designed DMD capture panel, 22 samples achieved 2.25 gigabases and 1.28 million reads on average. Average depth was 308× and 99.98% bases were covered at least 1×. The deletion breakpoints in intron 44 were scattered and no breakpoints clustered in any region less than 500 bp. A total of 72.7% of breakpoints located in distal 100 kb of intron 44 and more repetitive elements were found in this region. Microhomologies of 0-1 bp were found in 36.4% (8/22) of patients, which corresponded with non-homologous end-joining. Microhomologies of 2-20 bp were found in 59.1% (13/22) of patients, which corresponded with microhomology-mediated end-joining. Moreover, a 7 bp insertion was found in one patient, which might be evidence of aberrant replication origin firing. Palindromic sequences, polypyrimidine sequences, and small hairpin loops were found near several breakpoint junctions. No evidence of large hairpin loop formation in deletion root sequences was observed. Conclusion: This study was the first to explore possible mechanisms underlying exon deletions starting from intron 44 of the DMD gene based on long-read sequencing. Diverse mechanisms might be associated with deletions in the DMD gene.

Adjustment Disorder in Female Breast Cancer Patients: Prevalence and Its Accessory Symptoms.

Women diagnosed with breast cancer may have serious psychological problems and will suffer from adjustment disorder (AjD). We investigated the prevalence of AjD in female breast cancer patients who were diagnosed within 1 year and examined the severe life events they experienced, and the most common symptoms of AjD. 342 newly diagnosed (<1 year) female breast cancer patients were recruited from Tongji Hospital and Hubei Cancer Hospital in Hubei, China, from July 2018 to May 2019. The patients completed the self-report questionnaire including demographic characteristics and the scale ADNM-20 for the diagnosis of AjD. SPSS20.0 was used for data analysis. As a result, the prevalence of AjD in breast cancer patients was 38.6%. Patients from rural areas and lacking of exercise were more likely to suffer from AjD (P<0.05). The prevalence of AjD in patients who did not regard breast cancer as the most severe life event was higher than that in patients who took breast cancer as the most severe life event (44.4% vs. 33.9%, P<0.05; OR=1.728, 95% CI=1.072-2.787). The symptom that scored highest was preoccupation (3.15). We found that the prevalence of AjD in women with breast cancer in this study is very high and warrants more attention. Patients from rural areas, lacking of exercise and subject to multiple stressors are more likely to suffer from AjD. The commonest and severe symptom is preoccupation.

Whole exome sequencing identified a novel truncation mutation in the NHS gene associated with Nance-Horan syndrome.

BACKGROUND: Nance-Horan syndrome (NHS) is an X-linked inheritance disorder characterized by bilateral congenital cataracts, and facial and dental dysmorphism. This disorder is caused by mutations in the NHS gene. However, NHS may be difficult to detect in individuals with subtle facial dysmorphism and dental abnormalities in whom congenital cataracts are the primary clinical manifestations. METHODS: In this study, we present a three-generation family with NHS. Whole exome sequencing was performed to determine the potential pathogenic variant in the proband. Further validation was explored with Sanger sequencing in 9 of the available individuals of the family and additional 200 controls. RESULTS: A novel truncation mutation in gene NHS (c.C4449G, p.Tyr1483Ter) was found in the proband, who presented with a long-narrow face, prominent nose and large anteverted pinnae ear, screw-driver like incisors, mild mulberry like molars, one missing maxillary second molar and malocclusion. We found this mutation was detected in 2 male patients and 4 female carriers in the family. However, the mutation was never detected in the control subjects. CONCLUSIONS: In conclusion, we identified a novel truncation mutation in the NHS gene, which might associate with NHS. Our review on the NHS studies illustrated that NHS has significantly clinical heterogeneity. And NHS mutations in the NHS-affected individuals typically result in premature truncation of the protein. And the new mutation revealed in this study would highlight the understanding of the causative mutations of NHS.

Rare compound heterozygous mutations in gene MSH6 cause constitutive mismatch repair deficiency syndrome.

Few studies reported patients who harbored three kinds of primary tumors simultaneously. Here, we present a 9-year-old boy with colon carcinoma, brain medulloblastoma, and lymphoma. Genetic mutation detection was explored with next-generation sequencing, and compound heterozygous mutations in gene MSH6 c.3103C>T p.Arg1035Ter and c.3261dupC p.Phe1088LeufsTer were discovered.

Next-generation sequencing for identifying genetic mutations in adults with bronchiectasis.

BACKGROUND: Defective airway host-defense (e.g., altered mucus properties, ciliary defects) contributes to the pathogenesis of bronchiectasis. This study aims to determine whether genetic mutations associated with defective airway host-defense are implicated in the pathogenesis of bronchiectasis. METHODS: Based on the systematic screening of 32 frequently reported bronchiectasis-associated genes, we performed next-generation sequencing (NGS) on peripheral blood samples from 192 bronchiectasis patients and 100 healthy subjects. The variant distribution frequency and pathogenicity of mutations were analyzed. RESULTS: We identified 162 rare variants in 192 bronchiectasis patients, and 85 rare variants among 100 healthy subjects. Among bronchiectasis patients, 25 (15.4%), 117 (72.2%) and 18 (11.1%) rare variants were associated with cystic fibrosis transmembrane receptor (CFTR), epithelial sodium channel, and primary ciliary dyskinesia genes, respectively. Biallelic CFTR variants were detected in four bronchiectasis patients but none of the healthy subjects. Carriers of homozygous p.M470 plus at least one CFTR rare variant were detected in 6.3% of bronchiectasis patients (n=12) and in 1.0% of healthy subjects (n=1, P=0.039). Twenty-six patients (16 with idiopathic and 6 with post-infectious bronchiectasis) harbored biallelic variants. Bronchiectasis patients with biallelic DNAH5 variants, or biallelic CFTR variants plus an epithelial sodium channel variant, tended to have greater disease severity. CONCLUSIONS: Genetic mutations leading to impaired host-defense might have implicated in the pathogenesis of bronchiectasis. Genetic screening may be a useful tool for unraveling the underlying causes of bronchiectasis, and offers molecular information which is complementary to conventional etiologic assessment for bronchiectasis.

Overexpression of MPC1 inhibits the proliferation, migration, invasion, and stem cell-like properties of gastric cancer cells.

Invasion and metastasis are major malignant characteristics of human gastric cancer (GC), but the molecular mechanisms underlying the invasion and metastasis of GC cells remain elusive. MPC1, a key factor that controls pyruvate transportation through the inner mitochondrial membrane, was reported to be downregulated and correlated with poor prognosis in several cancers. However, the effects of MPC1 on human GC have not been illustrated. In this study, we investigated the potential role of MPC1 in the proliferation, migration, invasion, and stem cell-like properties of human GC cells and evaluated its prognostic significance for patients with GC. We found that MPC1 protein and mRNA levels were significantly decreased in GC tissues and cell lines. Low MPC1 expression was associated with tumor T stage, N stage, and advanced tumor node metastasis stage. Decreased MPC1 expression was an independent prognostic marker and correlated with poor overall survival of patients with GC. Furthermore, overexpression of MPC1 inhibited the proliferation, migration, invasion, and stem cell-like properties of GC cells. These findings suggest that MPC1 may be a novel prognostic marker and a potential therapeutic target in human GC.

A novel heterozygous germline deletion in MSH2 gene in a five generation Chinese family with Lynch syndrome.

Lynch syndrome (LS) is one of the most common familial forms of colorectal cancer predisposing syndrome with an autosomal dominant mode of inheritance. LS is caused by the germline mutations in DNA mismatch repair (MMR) genes including MSH2, MLH1, MSH6 and PMS2. Clinically, LS is characterized by high incidence of early-onset colorectal cancer as well as endometrial, small intestinal and urinary tract cancers, usually occur in the third to fourth decade of the life. Here we describe a five generation Chinese family with LS clinically diagnosed according to the Amsterdam II criteria. Immuno-histochemical staining of MSH2 and MSH6 shows only foci nuclear positive on the surface of the tumor with strong expression of MLH1 and PMS2 with diffuse immunoreactivity. In order to dig into the molecular basis of this LS pedigree, we collected the proband's blood sample, extracted the genomic DNA and applied the genetic screening. As a result, we identified a novel heterozygous deletion in MSH2 gene by targeted next generation sequencing, which is also proved to be co-segregated among other affected family members by following validation. To our knowledge, this novel heterozygous deletion (c.1676_1679 delTAAA) in MSH2 gene causes frameshift mutation (p.Asn560Lysfs*29) and leads to the formation of a truncated MSH2 protein which is confirmed to be a deleterious mutation according to the variant interpretation guidelines of American College of Medical Genetics and Genomics (ACMG). Identification of novel DNA mismatch repair (MMR) gene mutations can definitely benefit to the clinical diagnosis and management.

[Effect of eicosapentaenoic acid on mRNA expression of tight junction protein ZO-1 in intestinal epithelial cells after Escherichia coli LF82 infection].

OBJECTIVE: To investigate the change in the expression of tight junction protein ZO-1 in intestinal epithelial cells (Caco-2 cells) and the protective effect of eicosapentaenoic acid (EPA) after adherent-invasive Escherichia coli (E.coli) LF82 infection. METHODS: The Caco-2 cell line was used to establish an in vitro model of tight junction of intestinal epithelial cells. Caco-2 cells were divided into EPA treatment groups (0, 25, 50, 100, and 200 µmol/L EPA) and EPA (0, 25, 50, 100, and 200 µmol/L EPA)+E.coli LF82 treatment (0, 6, and 12 hours) groups. A microscope was used to observe the morphological characteristics of the cells. MTT assay was used to determine the cell growth curve. The activity of alkaline phosphatase (ALP) at both sides of the cell membrane was compared to evaluate the Caco-2 cell model. MTT assay and flow cytometry were used to investigate the effects of different concentrations of EPA on the survival rate and apoptosis rate of Caco-2 cells. RT-qPCR was used to measure the mRNA expression of ZO-1 in Caco-2 cells after EPA and/or E.coli LF82 treatment. ELISA was used to measure the change in the level of tumor necrosis factor-α (TNF-α) in culture supernatant. RESULTS: After EPA treatment (25 and 50 µmol/L), the proliferation of Caco-2 cells was induced in a dose-dependent manner. The survival rates of the cells were significantly higher than those in the control group (P<0.05). The EPA treatment (100 and 200 µmol/L) groups had a significant inhibitory effect on the proliferation of Caco-2 cells in a dose-dependent manner. The survival rates of the cells were significantly lower than those in the control group (P<0.05). The EPA treatment (100 and 200 µmol/L) groups had a significant increase in cell apoptosis rate compared with the control group (P<0.05). The 6- and 12-hour E.coli LF82 treatment groups had decreasing mRNA expression of ZO-1 in Caco-2 cells over the time of treatment and had significantly lower mRNA expression of ZO-1 than the untreated group (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 µmol/L EPA for 6 or 12 hours showed an increase in the mRNA expression of ZO-1 with the increasing concentration of EPA, as well as significantly higher mRNA expression of ZO-1 than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). The Caco-2 cells treated with E.coli LF82 alone for 6 or 12 hours had increasing secretion of TNF-α over the time of treatment and had significantly higher secretion than the untreated Caco-2 cells (P<0.05). The Caco-2 cells treated with E.coli LF82 and 25 or 50 µmol/L EPA for 6 or 12 hours showed a reduction in the secretion of TNF-α with the increasing concentration of EPA and had significantly lower secretion than the Caco-2 cells treated with E.coli LF82 alone (P<0.05). CONCLUSIONS: EPA can effectively prevent the destruction of tight junction of intestinal epithelial cells induced by E.coli LF82 infection and inhibit the secretion of inflammatory factors. Therefore, it has a certain protective effect on intestinal mucosal barrier.

Early VEGF inhibition attenuates blood-brain barrier disruption in ischemic rat brains by regulating the expression of MMPs.

Vascular endothelial growth factor (VEGF) inhibition has been demonstrated to be an effective strategy in preserving the integrity of the blood-brain barrier (BBB) in patients with acute ischemic stroke. Loss of the BBB is the key event associated with morbidity and mortality in these patients. However, the underlying mechanisms remain poorly understood. In the present study, the effects of VEGF inhibition and the possible mechanism that underlies acute cerebral ischemia in rats was investigated. Following the induction of transient middle cerebral artery occlusion for a 90­min period, either an anti­VEGF neutralizing antibody (RB­222; 5 or 10 µg), or IgG (control), was administered by intracerebroventricular injection at 1 h following reperfusion. Functional outcomes, BBB leakage, brain edema, microvessel numbers and the relative protein levels of VEGF, matrix metalloproteinase (MMP)-2, MMP-9, occludin and collagen-IV were then determined using neurological assessments, Evans Blue staining, brain water content, CD31 staining and western blotting. Treatment with RB­222 at a dose of 5 and 10 µg significantly improved neurological functional outcomes and diminished infarct size, BBB leakage and brain edema compared with the MCAO and IgG groups at 24 h following reperfusion; 10 µg RB­222 was more effective than a 5 µg dose of the antibody. In addition, RB­222 reduced the number of immature microvessels, which subsequently attenuated BBB permeability. RB­222 significantly repressed VEGF expression as well as decreased MMP­2 and MMP­9 expression. However, it enhanced occludin and collagen­IV levels in the ischemic rat brain compared with the MCAO and IgG groups. Taken together, the results indicate that early inhibition of VEGF may have significant potential against cerebral ischemia, partly by regulating the expression of MMPs.

Tandem Difluoroalkylation-Arylation of Enamides Catalyzed by Nickel.

A nickel-catalyzed three-component reaction for the synthesis of difluoroalkylated compounds through tandem difluoroalkylation-arylation of enamides has been developed. The reaction tolerates a variety of arylboronic acids and widely available difluoroalkyl bromides, and even the relatively inert substrate chlorodifluoroacetate. The significant advantages of this protocol are the low-cost nickel catalyst, synthetic convenience, excellent functional-group compatibility and high reaction efficiency.

Can S-1 replace fluorouracil for advanced gastric cancer? A PRISMA-compliant systematic review and meta-analysis.

It remains to be seen whether S-1 can be a replacement for infusional fluorouracil (5-FU) for advanced gastric cancer (AGC). The aim of this study was to compare the efficacy and safety of S-1 with 5-FU in AGC.PubMed and Cochrane Library were searched. Randomized controlled trials and meta-analyses comparing S-1 with 5-FU for AGC were eligible. Meta-analysis was performed using RevMan 5.2.Seven trials involving 2443 patients were included. Compared with 5-FU, S-1 showed no significant prolongation of overall survival (OS) (hazard ratio [HR] = 0.91, 95% confidence interval [CI] [0.83-1.01], P = 0.07) and progression-free survival (HR = 0.89, 95% CI [0.70-1.13], P = 0.35), but longer time to treatment failure (HR = 0.74, 95% CI [0.56-0.97], P = 0.03). The objective response rates were comparable (risk ratio [RR] = 1.36, 95% CI [0.95, 1.96], P = 0.10). Regarding treatment-related deaths and hematological toxicities, there was significant heterogeneity between Asian and non-Asian trials, and subgroup analysis was applied. In Asian patients, there was a significant increase in hematological toxicities such as leukopenia (grade 1-4: RR = 1.22, 95% CI [1.08, 1.37], P = 0.001; grade 3-4: RR = 2.21, 95% CI [1.52, 3.21], P < 0.0001), neutropenia (grade 1-4: RR = 1.29, 95% CI [1.11, 1.48], P = 0.0005; grade 3-4: RR = 1.87, 95% CI [1.11, 3.17], P = 0.02), and thrombocytopenia (grade 1-4: RR = 1.71, 95% CI [1.22, 2.41], P = 0.002) in S-1-containing regimens compared with 5-FU-containing regimens, but without significant difference in treatment-related mortality rate (risk difference [RD] = 0.00, 95% CI [-0.01, 0.01], P = 0.68). In non-Asian patients, S-1-containing regimens were, however, associated with significantly fewer treatment-related deaths (RD = -0.02, 95% CI [-0.05, -0.00], P = 0.04), as well as less all grade 1-4 and grade 3-4 hematological toxicities except anemia. There was no significant heterogeneity in nonhematologic toxicities between Asian and non-Asian trials. Lower incidence of grade 1-4 nausea, diarrhea, mucositis, grade 3-4 mucositis, increased creatinine, and decreased calculated creatinine clearance was observed in S-1-containing regimens.S-1 could not improve OS, but increase some hematological toxicities in Asian patients. Therefore, special attention on hematological toxicities should be paid to Asian patients because S-1 is administered on an outpatient basis.