CRISPR/Cas9-Based Genome Editing in the Filamentous Fungus Glarea lozoyensis and Its Application in Manipulating gloF.

Glarea lozoyensis is an important industrial fungus that produces the pneumocandin B0, which is used for the synthesis of antifungal drug caspofungin. However, because of the limitations and complications of traditional genetic tools, G. lozoyensis strain engineering has been hindered. In this study, we established an efficient CRISPR/Cas9-based gene editing tool in G. lozoyensis SIPI1208. With this method, gene mutagenesis efficiency in the target locus can be up to 80%, which enables the rapid gene knockout. According to the reports, GloF and Ap-HtyE, proline hydroxylases involved in pneumocandin and Echinocandin B biosynthesis, respectively, can catalyze the proline to generate different ratios of trans-3-hydroxy-l-proline to trans-4-hydroxy-l-proline. Heterologous expression of Ap-HtyE in G. lozoyensis decreased the ratio of pneumocandin C0 to (pneumocandin B0 + pneumocandin C0) from 33.5% to 11% without the addition of proline to the fermentation medium. Furthermore, the gloF was replaced by ap-htyE to study the production of pneumocandin C0. However, the gene replacement has been hampered by traditional gene tools since gloF and gloG, two contiguous genes indispensable in the biosynthesis of pneumocandins, are cotranscribed into one mRNA. With the CRISPR/Cas9 strategy, ap-htyE was knocked in and successfully replaced gloF, and results showed that the knock-in strain retained the ability to produce pneumocandin B0, but the production of pneumocandin C0 was abolished. Thus, this strain displayed a competitive advantage in the industrial production of pneumocandin B0. In summary, this study showed that the CRISPR/Cas9-based gene editing tool is efficient for manipulating genes in G. lozoyensis.

The patterns and treatment of postoperative hemorrhage and hematoma in total endoscopic thyroidectomy via breast approach: experience of 1932 cases.

PURPOSE: Postoperative hemorrhage and hematoma formation is a potentially lethal complication in thyroid surgery, although the patterns and treatment of hemorrhage after total endoscopic thyroidectomy (TET) via breast approach has not been reported previously. We aim to share our experience about postoperative bleeding. METHODS: A retrospective analysis of 1932 patients who underwent TET from April 2008 to May 2018 in our institution was carried out. The patterns of postoperative hemorrhage and hematoma formation that need surgical treatment were summarized and focused on the relation to the source of bleeding and the time interval between first surgery and hemorrhage. Related risk factors were analyzed by univariate or multivariate analysis processes. RESULTS: The overall rate of hemorrhage and hematoma occurrence was only 0.724% (14 in 1932 patients). Of them, 12 occurred in the first 24 h after surgery, and the other two occurred after withdrawal of the drainage tube. The principle independent risk factors for postoperative hemorrhage and hematoma were age (older than 35 years old) and lateral compartment dissection (LCD) revealed by multivariate regression. During re-exploration, obvious bleeding points were detected in 13 patients. Among them, 12 bled from the vessels in the main trocar cavity and another 1 bled from a broken vein located between the two heads of the sternocleidomastoid (SCM) muscle with LCD. CONCLUSIONS: Hemorrhage after TET usually occurs within 24 h, and the main video trocar cavity was the area most likely to bleed. Age and LCD may increase the bleeding risk. Appropriate dissection level is the main solution to prevent postoperative hemorrhage.

Protection of mouse pancreatic islet function by co­culture with hypoxia pre­treated mesenchymal stromal cells.

Ectogenic pancreatic islet transplantation has long been discussed as having the potential to reverse diabetes. The aim of the present study was to evaluate the therapeutic efficacy of co­transplantation with hypoxia pretreated mesenchymal stem cells (MSCs) and islets in a diabetic mouse model. MSCs were isolated from femoral and tibial bone marrow aspirates from female BALB/c donor mice. MSC proliferation rates and the expression levels of vascular endothelial growth factor A (VEGFA), interleukin (IL)­6, monocyte chemoattractant protein (MCP)­1 and matrix metalloproteinase (MMP)­9 were measured in hypoxic conditions. Subsequently, a streptozotocin­induced diabetic model was established in BALB/c mice. Glucose tolerance and diabetes reversal rate following co­transplantation of hypoxia pre­cultured MSCs and islets were demonstrated at different conditions during transplantation. The present study results demonstrated that MSCs increased their proliferation rate and the secretion of growth­related cytokines, including VEGFA, IL­6, MCP­1 and MMP­9 in a hypoxic environment. In the diabetes animal model, fewer islets (~250) were required to reverse the impaired glucose tolerance condition in Islets + Hypoxia cultured MSCs transplant group compared with the Islets­only group (~400 islets) and the Islets + Normal cultured MSCs group (~300 islets). Hypoxia­cultured MSC co­transplantation accelerated glycemic utilization following glucose intake. In subjects with hyperglycemia control for islet only transplantation group, MSCs pre­cultured in hypoxic condition prior to co­transplantation may potentially improve islet tissue regeneration.

LncRNA-SLC6A9-5:2: A potent sensitizer in 131I-resistant papillary thyroid carcinoma with PARP-1 induction.

Recent studies have indicated that long non-coding RNAs play crucial roles in numerous cancers, including thyroid cancer, while their function in the mechanism of thyroid cancer 131I resistance has not been elucidated to date. The present study identified a functional long non-coding RNA, SLC6A9-5:2, which was involved in the radioactive therapy resistance of thyroid cancer. We demonstrated that SLC6A9-5:2 was remarkably downregulated in 131I-resistant thyroid cancer cell lines and 131I-insensitive patients and was positively correlated with Poly (ADP-ribose) polymerase 1 (PARP-1) expression and its activation. After downregulating SLC6A9 or blocking PARP-1 artificially, the sensitive thyroid cancer cells mostly displayed a tolerant phenotype under 131I exposure. Furthermore, SLC6A9-5:2 overexpression was positively correlated with PARP-1 mRNA and protein levels, which restored the sensitivity of resistant thyroid cancer cells. The present study further revealed that cancer cell death was primarily caused by ATP exhaustion in excessive DNA repair with high PARP-1 activity. In patients with thyroid cancer, a positive correlation between SLC6A9-5:2 and PARP-1 was identified, and low SLC6A9-5:2 expression was associated with a worse prognosis of papillary thyroid carcinoma. Hence, our data provide a new lncRNA-mediated regulatory mechanism implying that SLC6A9-5:2 can be used as a novel therapeutic target for 131I-resistant thyroid cancer.

Beneficial effect of corticosteroids for patients with severe drug-induced liver injury.

OBJECTIVE: The efficacy of corticosteroids in drug-induced liver injury (DILI) remains controversial. We aimed to determine whether corticosteroids were beneficial for severe DILI. METHODS: This was a single-center retrospective study of patients with DILI enrolled between January 2010 and May 2015. RESULTS: Of the 203 patients enrolled, 53 were treated with corticosteroids. The baseline characteristics of patients received corticosteroids were more severe than that of the non-corticosteroid group. Subgroup analyses indicated that almost all patients who died had the higher 50% quartile of total bilirubin (TB) levels. Among the 50-75% quartile of TB level, no patient in the corticosteroids group but 3 (15.0%) of 20 patients in the non-corticosteroid group died (P = 0.261). With the highest 25% quartile of TB level, four patients in the corticosteroids group and four in the non-corticosteroids group died (P = 0.405). Corticosteroid therapy improved the recovery rate from 77.4% to 87.9% in the higher 50% quartile of TB values (P = 0.331). More interestingly, corticosteroid administration hastened the resolution of liver injury by shortening the duration of peak TB to 50% reduction from 17 to 12 days (P < 0.05). Additionally, multivariate analysis revealed that the TB levels and cholestatic injury type were the two independent factors associated with a poor outcome of DILI. CONCLUSIONS: Corticosteroids are not detrimental to DILI, but instead ameliorate liver injury and improve patient survival. Short-time use of corticosteroids is strongly recommended for severe DILI patients with hyperbilirubinemia.

MACC1 upregulation promotes gastric cancer tumor cell metastasis and predicts a poor prognosis.

In various studies, metastasis associated with colon cancer 1 (MACC1) has been frequently reported to be abnormally highly expressed in human lung cancer, colon cancer, and hepatocellular carcinoma. Our study focuses on the association of MACC1 expression with gastric cancer (GC). During our experiment, the MACC1 expression was tested in 105 GC samples using an immunohistochemical (IHC) method. The clinical characteristics and prognosis of these patients were summarized. During analysis, MACC1 distribution in GC samples with distant metastasis was higher than that in normal samples and in tumors with no dissemination. Subsequently, a lower 5-year survival rate had a strong correlation with high MACC1 expression. As a consequence, the present results suggest that MACC1 is more frequently expressed in a poor prognosis phenotype of GC and acts as a promising prognostic prediction parameter for GC.

Phloretin promotes adipocyte differentiation in vitro and improves glucose homeostasis in vivo.

Adipocyte dysfunction is associated with many metabolic diseases such as obesity, insulin resistance and diabetes. Previous studies found that phloretin promotes 3T3-L1 cells differentiation, but the underlying mechanisms for phloretin's effects on adipogenesis remain unclear. In this study, we demonstrated that phloretin enhanced the lipid accumulation in porcine primary adipocytes in a time-dependent manner. Furthermore, phloretin increased the utilization of glucose and nonesterified fatty acid, while it decreased the lactate output. Microarray analysis revealed that genes associated with peroxisome proliferator-activated receptor-γ (PPARγ), mitogen-activated protein kinase and insulin signaling pathways were altered in response to phloretin. We further confirmed that phloretin enhanced expression of PPARγ, CAAT enhancer binding protein-α (C/EBPα) and adipose-related genes, such as fatty acids translocase and fatty acid synthase. In addition, phloretin activated the Akt (Thr308) and extracellular signal-regulated kinase, and therefore, inactivated Akt targets protein. Wortmannin effectively blocked the effect of phloretin on Akt activity and the protein levels of PPARγ, C/EBPα and fatty acid binding protein-4 (FABP4/aP2). Oral administration of 5 or 10 mg/kg phloretin to C57BL BKS-DB mice significantly decreased the serum glucose level and improved glucose tolerance. In conclusion, phloretin promotes the adipogenesis of porcine primary preadipocytes through Akt-associated signaling pathway. These findings suggested that phloretin might be able to increase insulin sensitivity and alleviate the metabolic diseases.

Acute pancreatitis associated left-sided portal hypertension with severe gastrointestinal bleeding treated by transcatheter splenic artery embolization: a case report and literature review.

Left-sided portal hypertension (LSPH) followed by acute pancreatitis is a rare condition with most patients being asymptomatic. In cases where gastrointestinal (GI) bleeding is present, however, the condition is more complicated and the mortality is very high because of the difficulty in diagnosing and selecting optimal treatment. A successfully treated case with severe GI bleeding by transcatheter splenic artery embolization is reported in this article. The patient exhibited severe uncontrollable GI bleeding and was confirmed as gastric varices secondary to LSPH by enhanced computed tomography (CT) scan and CT-angiography. After embolization, the bleeding stopped and stabilized for the entire follow-up period without any severe complications. In conclusion, embolization of the splenic artery is a simple, safe, and effective method of controlling gastric variceal bleeding caused by LSPH in acute pancreatitis.

Novel blood biomarkers of pancreatic cancer-associated diabetes mellitus identified by peripheral blood-based gene expression profiles.

OBJECTIVES: The prognosis of pancreatic cancer is still very poor, and the ability to detect pancreatic cancer in high-risk groups at an early stage is therefore essential for improving its long-term survival. The purpose of this study was to explore specific biomarkers that can differentiate pancreatic cancer-associated diabetes from type 2 diabetes, for the early detection of pancreatic cancer. METHODS: From January 2006 to July 2008, 102 peripheral blood samples were collected from 25 patients diagnosed with pancreatic cancer and diabetes, 27 patients with pancreatic cancer without diabetes, 25 patients with diabetes mellitus >5 years, and 25 healthy controls. Thirty-two samples were used in microarray experiments to find differentially expressed genes specific for pancreatic cancer-associated diabetes. The results were further validated by quantitative real-time PCR for 101 blood samples. Protein expression of selected genes in serum and tissues was also detected. RESULTS: Using microarray analysis, we found 58 genes to be unique in patients with pancreatic cancer-associated diabetes, including 23 upregulated genes and 35 downregulated genes. Eleven upregulated genes were further validated by RT-PCR, and two of these genes-vanin-1 (VNN1) and matrix metalloproteinase 9 (MMP9)-were selected for logistic regression analysis. The combination of VNN1 and MMP9 showed the best discrimination of pancreatic cancer-associated diabetes from type 2 diabetes. The protein expression of MMP9 and VNN1 was in accordance with the gene expression. CONCLUSIONS: Our results indicate that the combination of VNN1 and MMP9 may be used as a novel blood biomarker panel for the discrimination of pancreatic cancer-associated diabetes from type 2 diabetes.

Pancreatic somatostatinoma with obscure inhibitory syndrome and mixed pathological pattern.

Somatostatinoma is a very rare neuroendocrine tumor that originates from D cells and accounts for less than 1% of all gastrointestinal endocrine tumors. The duodenum is the most frequent site for this tumor, followed by the pancreas. We here describe a 46-year-old Chinese woman who developed pancreatic somatostatinoma presenting with the characteristic "inhibitory" syndrome, but the symptoms were obscure and seemingly uncorrelated. This case is also unique for its large tumor size and mixed pathological pattern. Distal pancreatectomy was performed, and the patient has remained well since operation. As the syndromes of somatostatinoma may be obscure and atypical, clinicians should review all clinical findings to obtain an accurate diagnosis. Aggressive surgery is preferred to improve the survival.

The potential effect of proteasome inhibitor PS-341 on severe acute pancreatitis detected by positron emission tomography in ICR mice.

INTRODUCTION: Severe acute pancreatitis is a life threatening disease with a high rate of mortality, and its treatments are still controversial. The purpose of this study is to investigate the potential effects of proteasome inhibitor PS-341 on severe acute pancreatitis induced by cerulein and lipopolysaccharide in mice. MATERIALS AND METHODS: Severe acute pancreatitis was induced by seven intraperitoneal injections of 50 ug/kg cerulein at hourly intervals and one injection of 10mg/kg lipopolysaccharide in mice. Thirty min before the administration of lipopolysaccharide, mice were treated either with PS-341 or vehicle. The severity of acute pancreatitis was then evaluated by serum and pancreatic biochemical assays as well as histologic examination. Positron emission tomography (PET) was used for the first time to determine the therapeutic effects of interventions in situ. RESULTS: PS-341 significantly inhibited NF-kappaB activation, while the pancreatic cell apoptosis was significantly enhanced, resulting in the improved parameters such as serum amylase, C-reactive protein, lactate dehydrogenase, interleukin-1beta, interleukin-6, and pancreatic myeloperoxidase activity. Accordingly, pancreatic damage, including inflammatory cell infiltration, hemorrhage, and necrosis, was markedly reduced. (18)F-fluorodeoxyglucose-positron emission tomography demonstrated that PS-341 significantly reduced the uptake of (18)F-fluorodeoxyglucose within the pancreas. CONCLUSIONS: These observations demonstrate that PS-341 was able to significantly reduce the severity of acute pancreatitis induced by cerulein and lipopolysaccharide in mice. The potential effect is associated with the inhibition of NF-kappaB activation and increased pancreatic cell apoptosis within the pancreas. (18)F-fluorodeoxyglucose-positron emission tomography could be a sensitive and promising means in evaluating the therapeutic effect and adjusting medical interventions for pancreatitis.

Human bone marrow mesenchymal stem cells differentiate into insulin-producing cells upon microenvironmental manipulation in vitro.

It was recently reported that pluripotent mesenchymal stem cells (MSCs) in rodent bone marrow (BM) have the capacity to generate insulin-producing cells (IPCs) in vitro. However, little is known about this capacity in human BM-MSCs. We developed a nongenetic method to induce human BM-MSCs to transdifferentiate into IPCs both phenotypically and functionally. BM-MSCs from 12 human donors were sequentially cultured in specially defined conditions. Their differentiation extent toward beta-cell phenotype was evaluated systemically. Specifically, after induction human BM-MSCs formed spheroid islet-like clusters containing IPCs, which was further confirmed by dithizone (DTZ) staining and electron microscopy. These IPCs expressed multiple genes related to the development or function of pancreatic beta cells (including NKX6.1, ISL-1, Beta2/Neurod, Glut2, Pax6, nestin, PDX-1, ngn3, insulin and glucagon). The coexpression of insulin and c-peptide was observed in IPCs by immunofluorescence. Moreover, they were able to release insulin in a glucose-dependent manner and ameliorate the diabetic conditions of streptozotocin (STZ)-treated nude mice. These results indicate that human BM-MSCs might be an available candidate to overcome limitations of islet transplantation.

In situ high-volume modified continuous closed and/or open lavage for infected necrotizing pancreatitis.

OBJECTIVES: To evaluate the clinical significance of high-volume modified continuous closed and/or open lavage for the treatment of infected necrotizing pancreatitis. METHODS: From August 1997 to December 2006, 53 patients with infected necrotizing pancreatitis who underwent in situ high-volume (>20 L/d) continuous closed lavage using a single-lumen rubber catheter and/or open lavage were retrospectively studied in our hospital, and the advantages of this new technique were analyzed. RESULTS: Modified continuous closed lavage was the initial treatment for all patients; in 6 patients with secondary retroperitoneal sepsis or abscess, continuous open lavage was performed. Impaired tube patency and lavage fluid retention did not occur in any of these patients. The overall mortality was 17.0% (9/53). Twelve patients underwent early surgery, and 5 (41.7%) died; 41 patients underwent delayed surgery, and 4 (9.8%) died. Significant local complications occurred in 14 patients (26.4%); the incidence of bleeding, abscess, and fistula was 13.2% (7/53), 9.4% (5/53), and 9.4% (5/53), respectively. CONCLUSIONS: Our technique of in situ high-volume modified continuous closed and/or open lavage has produced a better control of infected necrotizing pancreatitis.

Risk factors for infectious morbidity in gallbladder cancer patients treated surgically.

BACKGROUND/AIMS: To explore risk factors associated with postoperative infectious morbidity in gallbladder cancer patients. METHODOLOGY: We investigated 58 consecutive patients undergoing surgery for primary gallbladder cancer between January 2000 and June 2005 in our hospital. Records of all patients were retrospectively reviewed. Twenty-two independent tumor-, patient-, and treatment-related variables were analyzed. The dependent variable was clinical infectious complications. A binary logistic regression analysis was used to assess the independent association of variables with the dependent variable. RESULTS: Overall surgical morbidity was 33% (19/58), and 14 (24%) of the 58 patients developed infectious complications postoperatively. On univariate analysis, presence of jaundice, hypoalbuminemia and intraoperative blood transfusion were found to be significantly associated with infectious morbidity. The multivariate analysis of logistic regression disclosed that presence of jaundice and intraoperative blood transfusion of 4 units or more only showed marginally significant impacts on infectious complications (odds ratio, 8.004, 7.782; 95% confidence interval, 0.886-72.278, 0.914-66.283, respectively), while weight loss and hypoalbuminemia were significantly associated with infectious complications postoperatively (odds ratio, 9.609, 40.257; 95% confidence interval, 1.269-72.253, 3.901-415.438, respectively). CONCLUSIONS: Hypoalbuminemia and weight loss are significantly associated with postoperative infectious morbidity independently. While presence of jaundice and intraoperative blood transfusion of 4 units or more appear to be marginally significant factors, modality of operation or liver resection, blood loss, and additional gastrointestinal operation are not risk factors.

[Clinical staging of ankylosing spondylitis].

OBJECTIVE: To compare the chronic changes between the sacroiliac joints (SIJ) and the spine or the hip joints in conventional radiography of patients with ankylosing spondylitis (AS) and discuss the clinical staging of this disease. METHODS: All images of the joints of the AS patients were evaluated twice in blinded manner by two doctors. The cervical spine, lumbar spine, sacroiliac joints and hip joints were separately evaluated by BASRI, and the results were averaged and analyzed. Definite involvement was defined as a score > or=2. RESULTS: Thirty-seven AS patients (81.0% male with mean age of 28.49 years) were examined, and 81.0% of them had definite involvement of the spine and 40.5% of hip involvement. Pearson product-moment correlation coefficient was 0.459 between BASRI-SIJ and BASRI-s, and 0.465 between BASRI-SIJ and BASRI-h. CONCLUSION: Separately, the severity of SIJ changes can not represent the severity of changes in the spine and hip, etc, therefore SIJ changes may not be sufficient evidence for AS staging.