Chemoproteomics reveals arctigenin as a phagophore-closure blocker via targeting ESCRT-I subunit VPS28.

Arctigenin is the active ingredient of the traditional medicines Arctium lappa and Fructus Arctii and has been extensively investigated for its diverse pharmacological functions, including its novel anti-austerity activity. Although several mechanisms have been proposed, the direct target of arctigenin to induce anti-austerity activity remains unclear. In this study, we designed and synthesized photo-crosslinkable arctigenin probes and utilized them in the chemoproteomic profiling of potential target proteins directly in living cells. Vacuolar protein sorting-associated protein 28 (VPS28), a key subunit of the ESCRT-I complex implicated in phagophore closure, was successfully identified. Unexpectedly, we found that arctigenin degraded VPS28 via the ubiquitin-proteasome pathway. We also demonstrated that arctigenin induces a prominent phagophore closure-blockade phenotype in PANC-1 cells. To the best of our knowledge, this is the first report of a small molecule acting as a phagophore-closure blocker and a VPS28 degrader. The arctigenin-modulating phagophore closure provides a new druggable target for cancers that rely heavily on autophagy activation and may also be used for other diseases associated with the ESCRT system.

Sequential butylphthalide therapy combined with dual antiplatelet therapy in the treatment of acute cerebral infarction.

OBJECTIVE: To observe the clinical efficacy of sequential butylphthalide therapy combined with dual antiplatelet therapy in the treatment of elderly patients with acute cerebral infarction (ACI). METHODS: One hundred and twenty-two elderly patients with ACI who were admitted to the department of neurology of our hospital at May 2016-August 2018 were selected grouped into a control group and an observation group by random number table method, 61 in each group. On the basis of conventional treatment, the patients in the control group were given dual antiplatelet therapy (aspirin enteric-coated tablets + clopidogrel bisulfate tablets), while the patients in the observation group were given sequential butylphthalide therapy on the basis of the control group. The clinical effects of the two groups were compared after four weeks of treatment, and the changes of National Institutes of Health Stroke Scale (NIHSS), ADL score, plasma 3-mercaptopyruvate sulphurtransferase (3-MST) and Amyloid ß42 (Aß42) levels and the occurrence of adverse reactions during treatment were recorded. RESULTS: The clinical efficacy of the observation group was better than that of the control group (P<0.05). There was no significant difference in NIHSS and ADL scores between the two groups before treatment (P>0.05). After treatment, the NIHSS and ADL scores of the observation group were better than those of the control group (P<0.05). There was no significant difference in plasma levels of 3-MST and AB42 between the two groups before treatment (P>0.05). The level of plasma 3-MST in the observation group was higher than that in the control group, and the level of plasma Aß42 was lower than that in the control group (P<0.05). No serious adverse reactions occurred during the treatment period in both groups. CONCLUSION: Butylphthalide sequential therapy combined with dual antiplatelet therapy is effective in the treatment of elderly ACI. It can effectively improve the plasma level of 3-MST and decrease the plasma level of Aß42, which is conducive to improving the living ability and neurological function of patients and has high safety.

DEP domain-containing mTOR-interacting protein suppresses lipogenesis and ameliorates hepatic steatosis and acute-on-chronic liver injury in alcoholic liver disease.

Alcoholic liver disease (ALD) is characterized by lipid accumulation and liver injury. However, how chronic alcohol consumption causes hepatic lipid accumulation remains elusive. The present study demonstrates that activation of the mechanistic target of rapamycin complex 1 (mTORC1) plays a causal role in alcoholic steatosis, inflammation, and liver injury. Chronic-plus-binge ethanol feeding led to hyperactivation of mTORC1, as evidenced by increased phosphorylation of mTOR and its downstream kinase S6 kinase 1 (S6K1) in hepatocytes. Aberrant activation of mTORC1 was likely attributed to the defects of the DEP domain-containing mTOR-interacting protein (DEPTOR) and the nicotinamide adenine dinucleotide-dependent deacetylase sirtuin 1 (SIRT1) in the liver of chronic-plus-binge ethanol-fed mice and in the liver of patients with ALD. Conversely, adenoviral overexpression of hepatic DEPTOR suppressed mTORC1 signaling and ameliorated alcoholic hepatosteatosis, inflammation, and acute-on-chronic liver injury. Mechanistically, the lipid-lowering effect of hepatic DEPTOR was attributable to decreased proteolytic processing, nuclear translocation, and transcriptional activity of the lipogenic transcription factor sterol regulatory element-binding protein-1 (SREBP-1). DEPTOR-dependent inhibition of mTORC1 also attenuated alcohol-induced cytoplasmic accumulation of the lipogenic regulator lipin 1 and prevented alcohol-mediated inhibition of fatty acid oxidation. Pharmacological intervention with rapamycin alleviated the ability of alcohol to up-regulate lipogenesis, to down-regulate fatty acid oxidation, and to induce steatogenic phenotypes. Chronic-plus-binge ethanol feeding led to activation of SREBP-1 and lipin 1 through S6K1-dependent and independent mechanisms. Furthermore, hepatocyte-specific deletion of SIRT1 disrupted DEPTOR function, enhanced mTORC1 activity, and exacerbated alcoholic fatty liver, inflammation, and liver injury in mice. CONCLUSION: The dysregulation of SIRT1-DEPTOR-mTORC1 signaling is a critical determinant of ALD pathology; targeting SIRT1 and DEPTOR and selectively inhibiting mTORC1-S6K1 signaling may have therapeutic potential for treating ALD in humans. (Hepatology 2018).

Distinct pituitary hormone levels of 184 Chinese children and adolescents with multiple pituitary hormone deficiency: a single-centre study.

BACKGROUND: Pituitary tumors and/or their treatment are associated with multiple pituitary hormone deficiency (MPHD) in adults, but the distinct pituitary hormone profile of MPHD in Chinese children and adolescents remains unclear. METHODS: Patients with MPHD were divided into four groups according to their MRI results: 1) pituitary stalk interruption syndrome (PSIS); 2) hypoplasia; 3) normal; and 4) tumor survivor. RESULTS: Among the 184 patients, 93 patients (50.5%) were with PSIS, 24 (13.0%) had hypoplastic pituitary gland, 10 (5.4%) patients were normal, and 57 (31.0%) were tumor survivors. There was an association between abnormal fetal position and PSIS (P ≤ 0.001). The CA/BA in PSIS, hypoplasia, normal, tumor survivor groups were 2.27 ± 1.05, 1.48 ± 0.39, 1.38 ± 0.57, 1.49 ± 0.33, and HtSDS were - 3.94 ± 1.39, - 2.89 ± 1.09, - 2.50 ± 1.05, - 1.38 ± 1.63. Patients in PSIS group had the largest CA/BA (P ≤ 0.001 vs. hypoplasia group, P = 0.009 vs. normal group, P ≤ 0.001 vs. tumor survivors) and lowest HtSDS (P ≤ 0.001 vs. hypoplasia group, P = 0.003 vs. normal group, P ≤ 0.001 vs. tumor survivors). The levels of TSH in the PSIS, hypoplasia, normal, and tumor survivor groups were 1.03 ± 1.08 (P = 0.149 vs. tumor survivors), 1.38 ± 1.47 (P = 0.045 vs. tumor survivors), 2.49 ± 1.53 (P < 0.001 vs. tumor survivors), and 0.76 ± 1.15 µIU/ml. The levels of GH peak in PSIS, hypoplasia, normal, tumor survivor groups were 1.37 ± 1.78, 1.27 ± 1.52, 3.36 ± 1.79, 0.53 ± 0.52 ng/ml and ACTH were 27.50 ± 20.72, 25.05 ± 14.64, 34.61 ± 59.35, 7.19 ± 8.63 ng/ml. Tumor survivors had the lowest levels of GH peak (P ≤ 0.001 vs. PSIS group, P = 0.002 vs. hypoplasia group, P ≤ 0.001 vs. normal group) and ACTH (all the P ≤ 0.001 vs. the other three groups). CONCLUSION: The frequency of PSIS is high among children and adolescents with MPHD. The severity of hormone deficiencies in patients with MPHD was more important in the tumor survivor group compared with the other groups.

Transumbilical single-incision laparoscopic cholecystectomy: long-term review from a single center.

BACKGROUND: Currently, researches about single-incision laparoscopic cholecystectomy (SILC) are various, but long-term reviews assessing relevant complications after SILC with considerable amount of case series are rare. STUDY DESIGN: We retrospectively reviewed a large series of 529 patients undergoing SILC to assess the long-term postoperative recovery, including postoperative complications, retained symptoms, and quality of life. Finally, we assessed its associated risk factors related to SILC patients' recovery in the long term. RESULTS: During a mean follow-up period of 36.8 ± 8.8 months after SILC, 402 (76.0 %) patients underwent complete resolution. Frequent diarrhea (12.1 %) and recurrent omphalitis (5.9 %) were most commonly seen among other complications and retained symptoms within overall the patients. We identified 1 (0.3 %) incision hernia and 1 (0.3 %) intra-abdominal abscess among overall the patients, while 3 (0.8 %) common bile duct stones and 1 (0.3 %) biliary pancreatitis among the patients with symptomatic cholelithiasis during long-term review period. No significant differences were identified between patients with symptomatic cholelithiasis and gallbladder polyps when considering other incidences (all p > 0.05). Patients undergoing SILC with older age (p = 0.023) or female gender (p = 0.020) contributed to complete resolution. CONCLUSIONS: SILC via traditional devices is feasible and safe with acceptable postoperative incidence rate in the long run. Patients with older age or female gender, who have no severe systemic diseases, tend to benefit more from the surgical intervention.

Identification of mitochondria-targeting anticancer compounds by an in vitro strategy.

Mitochondria play a pivotal role in determining the point-of-no-return of the apoptotic process. Therefore, anticancer drugs that directly target mitochondria hold great potential to evade resistance mechanisms that have developed toward conventional chemotherapeutics. In this study, we report the development of an in vitro strategy to quickly identify the therapeutic agents that induce apoptosis via directly affecting mitochondria. This result is achieved by treating isolated mitochondria with potential anticancer compounds, followed by simultaneously measuring the side scatter and mitochondrial membrane potential (Δψ(m)) fluorescence of individual mitochondria using a laboratory-built high-sensitivity flow cytometer. The feasibility of this method was tested with eight widely used anticarcinogens. Dose-dependent Δψ(m) losses were observed for paclitaxel, antimycin A, betulinic acid, curcumin, ABT-737, and triptolide, but not for cisplatin or actinomycin D, which agrees well with their mechanisms of apoptosis induction reported in the literature. The as-developed method offers an effective approach to identify mitochondria-targeting anticancer compounds.

Transumbilical single-incision laparoscopic distal pancreatectomy: preliminary experience and comparison to conventional multi-port laparoscopic surgery.

BACKGROUND: Single-incision laparoscopic surgery (SILS), which has been demonstrated to be safely applied on kinds of surgeries, may represent an improvement over conventional multi-port laparoscopic surgery. However, there are still few clinical experiences of SILS in pancreatic surgery until now. In this study, we will summarize our experience of transumbilical single-incision laparoscopic distal pancreatectomy (TUSI-LDP), and compare its related parameters with conventional multi-port laparoscopic distal pancreatectomy (C-LDP). METHODS: A retrospective analysis was conducted for the patients who underwent C-LDP or TUSI-LDP in our department. The demographic data, operative parameters, and postoperative complications in the two groups were summarized and compared. RESULTS: Laparoscopic distal pancreatectomy was performed in a total of 21 cases, among which TUSI-LDP was performed in 14 cases. As far as the demographical results concerned, there were no significant differences between the two groups. The conversion to open surgery was conducted in one case in the TUSI-LDP group because of severe adhesion between pancreatic cyst and surrounding tissues, while in the C-LDP group the only one conversion was for the difficult detection of small lesion. The mean operating time and intraoperative blood loss in TUSI-LDP group was a little shorter (166.4 ± 57.4 versus 202.1 ± 122.5 minutes, p > 0.05, and 157.1 ± 162.4 versus 168.6 ± 157.4 ml, p > 0.05). The postoperative pain and post-operation lengths of hospital stay in the TUSI-LDP group were also less, though there was no significant statistical difference between the two groups. For the post-operation complications, in TUSI-LDP group the pancreatic leakage occurred in only one case, and ceased spontaneously with only a drain for 61 days. There were no other complications including postoperative hemorrhage, venous thrombosis, infections and so on in both groups. CONCLUSION: For the experienced laparoscopic surgeons, in selected patients, TUSI-LDP is a feasible technique, with excellent cosmetic effect, less postoperative pain and post-operation lengths of hospital stay. With the experience accumulated, the operating time and intraoperative blood loss of TUSI-LDP could also gradually reduce.

Asymptomatic Rosai-Dorfman-Destombes disease presenting as isolated bilateral perinephric infiltration: a case report and review of the literature.

Background: Rosai-Dorfman-Destombes disease (RDD) is a rare histioproliferative disease with unknown etiology. It commonly occurs in lymph nodes and can affect extra-nodal tissues and organs. Renal RDD is extremely rare, only a few cases have been reported, and its clinical symptoms and imaging findings are non-specific. To date, no literature has summarized its imaging manifestations in a large number of cases. Due to the involvement of different tissues and organs, there is no standard treatment for RDD. It has been reported that RDD patients with kidney involvement have a poor prognosis. Thus, understanding of renal RDD need to be extended. Case Description: We present a rare case of renal RDD in an asymptomatic 67-year-old male. The results of an ultrasound examination indicated that both kidneys were surrounded by hypoechoic soft tissue lesions, and there was a huge mass in the left kidney, which had a clear boundary with the renal capsule. The results of contrast-enhanced ultrasound (CEUS) showed hypo-enhancement in the bilateral perinephric lesions and mass. However, the computed tomography urography (CTU) findings revealed no obvious enhancement. The patient then underwent a series of laboratory tests, but no relevant information was found. To make a clear diagnosis, the urologist then removed the left perirenal mass and some perirenal tissues, and the patient was finally pathologically diagnosed with extra-nodal RDD. The patient remains asymptomatic, and no treatment has been administered to date. Conclusions: This case may be the first reported case in which CEUS was performed and the second reported case of asymptomatic renal RDD. Based on the previous literature reports, we found that some specific characteristics of renal RDD include bilateral perirenal lesions with a "hairy kidney" appearance. CEUS and/or CTU can be used to help differentiate a solitary mass of RDD from common tumors, to avoid misdiagnosis leading to unnecessary nephrectomy.

Biliary Microbiota in Choledocholithiasis and Correlation With Duodenal Microbiota.

Background: The pathogenesis of choledocholithiasis is closely related to the role of bacteria. However, little is known about the predictive role of bile bacteria in clinical conditions of patients and the compositional and functional characteristics of biliary microbiota in choledocholithiasis. Methods: To investigate the predictive value of biliary bacteria, clinical data of 488 patients with choledocholithiasis were collected. The predictive value of common bile bacteria to patients' clinical conditions was analyzed by logistic regression. Samples of bile and corresponding duodenal juice from 10 selected patients with choledocholithiasis were obtained, and the composition and function of microbial communities were analyzed based on 16S rRNA sequencing and Tax4Fun. Results: The clinical conditions of patients with choledocholithiasis, such as recurrence, the severity of acute cholangitis, and duration of hospital stay were closely related to different species of bile bacteria as well as antimicrobial-resistant bacteria. Employing 16S rRNA sequencing, the dominant phyla of biliary and duodenal microbiota were Proteobacteria and Firmicutes. The top three core microbiota at the genus level were Escherichia-Shigella, Fusobacterium, and Enterococcus. Escherichia coli accounted for the most abundant annotated species in both. Differences in composition between biliary and duodenal microbiota were not significant according to the alpha and beta diversities. Differential abundant features were not found in biliary microbiota indicated by A linear discriminant analysis effective size algorithm. The major pathways identified in biliary and duodenal microbiota were related to membrane transport, translation, replication and repair, carbohydrate and amino acid metabolism. However, no significant difference in those major pathways, as well as antimicrobial-resistance patterns, was observed between biliary and duodenal microbiota. Conclusion: Our study first demonstrates the predictive contribution of biliary bacteria to the clinical conditions of patients with choledocholithiasis, and then it offers new insights into the compositional and functional features of biliary and duodenal microbiota. Similarities between biliary and duodenal microbiota support the theory of bacterial duodenal-biliary reflux in patients with choledocholithiasis. Meanwhile, when it is impracticable to obtain a bile sample, duodenal juice may be used as an alternative for bacterial culture and susceptibility tests.

Molecular mechanism underlying miR­130b­Sp1 transcriptional regulation in LPS­induced upregulation of MUC5AC in the bile duct epithelium.

Hepatolithiasis is a common disease that represents a serious health threat to the Chinese population. The pathological mechanism underlying hepatolithiasis is closely related to bacterial infections of the intrahepatic bile duct, followed by chronic inflammation and the overexpression of mucin 5AC (MUC5AC). However, the exact mechanism responsible for the lipopolysaccharide (LPS)­induced upregulation of MUC5AC has yet to be elucidated. Specificity protein 1 (Sp1) is a ubiquitous transcription factor that plays a vital role in the regulation of a number of genes that are responsible for normal cellular function. microRNA (miR/miRNA)­130b is a member of the miRNA family. miRNAs can bind to the 3'­untralsated region (3'­UTR) of a target gene and influence its expression levels. The present study found that LPS increases the expression of MUC5AC by influencing Sp1 secretion. Chromatin immunoprecipitation­quantitative PCR experiments further verified three Sp1 binding sites in the MUC5AC promoter sequence that can regulate the expression of MUC5AC. Further analysis demonstrated that Sp1 expression was regulated by miR­130b. Luciferase experiments identified one miR­130b binding site in the Sp1 3'­UTR region. In vivo experiments also confirmed the role of the miR­130b­Sp1­MUC5AC signaling pathway in the formation of biliary stones and indicated that this pathway may provide targeted therapeutic strategies for the treatment of intrahepatic bile duct stones.

Rapid and quantitative in vitro analysis of mitochondrial fusion and its interplay with apoptosis.

Mitochondrial fusion is essential to maintain genomic stability and physiological functions of mitochondria. Since mitochondrial fusion and fission work in concert to regulate mitochondrial morphology and functions, it has been challenging to quantitatively measure the direct roles of mitochondrial fusion in apoptosis and cancer progression. Here, we report the development of a high-throughput in vitro method to quantify mitochondrial fusion through single mitochondria analysis by a laboratory-built nano-flow cytometer (nFCM). Isolated mitochondria expressing green fluorescent protein (GFP-mito) or discosoma red fluorescent protein (DsRed-mito) were mixed together, induced to fuse, and analyzed by nFCM. A particle exhibiting both green and red fluorescence was identified as an event of heterotypic fusion, and the efficiency of heterotypic fusion was used as a surrogate of overall fusion efficiency. The as-developed method was applied to reveal the interplay between mitochondrial fusion and apoptosis without the interference of fission. We show that cytosolic components promoted mitochondrial fusion, and this upregulation was diminished during apoptosis. Combined with the translocation of Bid and Bax from cytosol to mitochondria, these findings suggest that cytosolic pro-apoptotic Bcl-2 family proteins could be the positive mediators of mitochondrial fusion. On the other hand, fusion also renders mitochondria more resistant to membrane potential collapse upon apoptosis induction. Our data suggest that disruption of mitochondrial fusion could be a potent strategy for cancer therapy. Furthermore, the as-developed method offers an effective approach to identify fusion inhibitors, including betulinic acid and antimycin A, giving reasons for their powerful utility in cancer treatment.

Growth and Adult Height during Human Growth Hormone Treatment in Chinese Children with Multiple Pituitary Hormone Deficiency Caused by Pituitary Stalk Interruption Syndrome: A Single Centre Study

Objective: The aim was to assess growth velocity (GV) during human recombinant growth hormone (hGH) treatment of children with multiple pituitary hormone deficiency (MPHD) caused by pituitary stalk interruption syndrome (PSIS) and to analyze the characteristics of patients that attained normal adult heights. Methods: Data from 74 (16 female) children with MPHD caused by PSIS with GH, thyroid stimulating hormone, gonadotropin and adrenocorticotropic hormone deficiencies were collected. Subjects were divided into groups: 12 pre-pubescent females (Female-Group) and 36 pre-pubescent males (Male-Group 1). The remaining 22 males were further sub-divided into two groups (Male-Group 2 and Male-Group 3) according to the initiation of gonadotropin replacement treatment, based on bone age and height. Results: No differences in change in height standard deviation score (△HtSDS) and GV were observed at different time points of hGH treatment between the Female-Group and Male-Group 1 (p>0.05). GV was significantly greater in the first year of hGH therapy than in subsequent years: Female-Group p=0.011; Male-Group 1 p<0.001; Male-Group 2 p=0.005; and Male-Group 3 p=0.046. Adult height was achieved by 23 (19 males and 4 females) patients. The total gain in height positively correlated with the GV during the first year (r=0.626, p<0.001). Conclusion: GV during hGH treatment were similar amongst pre-pubescent males and females with MPHD caused by PSIS. GV during the first year of hGH treatment appears to be an effective predictor of final height in patients with MPHD caused by PSIS.

Use of PEGylated Recombinant Human Growth Hormone in Chinese Children with Growth Hormone Deficiency: A 24-Month Follow-Up Study.

OBJECTIVE: Once-weekly PEGylated recombinant human growth hormone (rhGH) is the sole long-acting GH formulation available currently for pediatric patients with GH deficiency (GHD). The aim of this study was to evaluate the efficacy and safety of PEGylated rhGH therapy compared to daily rhGH therapy in GHD children treated for two years. METHODS: A total of 98 children (49 children for the PEGylated rhGH group and 49 children for the daily rhGH group) with GHD were enrolled in this single-center, prospective, nonrandomized cohort study. PEGylated rhGH or daily rhGH was administered for 2 years. Height, height SDS, height velocity (HV), IGF-1, bone age (BA), and adverse events were determined throughout the treatment. RESULTS: HV significantly increased over the baseline and was similar in both groups. In the PEGylated rhGH cohort, the mean ± SD HV was improved from 3.78 ± 0.78 cm/y at the baseline to 12.44 ± 3.80 cm/y at month 3, to 11.50 ± 3.01 cm/y at month 6, to 11.00 ± 2.32 cm/y at month 12, and finally 10.08 ± 2.12 cm/y at month 24 in the PEGylated rhGH group. In the daily rhGH group, HV was 3.36 ± 1.00 cm/y at baseline, increasing to 12.56 ± 3.71 cm/y at month 3, to 11.82 ± 2.63 cm/y at month 6, to 10.46 ± 1.78 cm/y at month 12, and to 9.28 ± 1.22 cm/y at month 24. No serious adverse event related to PEGylated rhGH or daily rhGH occurred during the 24-month study. CONCLUSION: PEGylated rhGH replacement therapy is effective and safe in pediatric patients with GHD. The adherence to once-weekly PEGylated rhGH therapy is superior to daily rhGH in children with GHD.

T-tube-free single-incision laparoscopic common bile duct exploration plus cholecystectomy: a single centre experience.

BACKGROUND: The present study aimed to explore the indications and feasibility of T-tube-free trans-umbilical single-incision laparoscopic common bile duct exploration (SILCBDE) plus laparoscopic cholecystectomy (LC) for treating choledocholithiasis. METHODS: Patients hospitalized in the Second Affiliated Hospital (Shengjing Hospital) of China Medical University from January 2010 to January 2017 with the diagnosis of common bile duct stones and treated with T-tube-free trans-umbilical single-incision LC plus common bile duct exploration were retrospectively analysed. RESULTS: A total of 37 male/female choledocholithiasis patients (mean age 65 years, range 29-86) were treated with T-tube-free trans-umbilical SILCBDE plus LC. No intraoperative complication or conversion to open surgery occurred in any of the cases. The mean operative time was 99.8 min (range 84-125) for endoscopic nasobiliary drainage group (n = 6), 113.8 min (range 70-150) for endoscopic retrogradebiliary drainage group (n = 2), 131.1 min (range 75-161) for pigtail J-tube group (n = 24), 113.7 min (range 100-150) for primary closure group (n = 5). The mean post-operative hospital stay length was 5.5 days (range 4-7) for endoscopic nasobiliary drainage group, 12.5 days (range 10-15) for endoscopic retrogradebiliary drainage group, 6.5 days (range 4-10) for J-tube group, 5.8 days (range 4-9) for primary closure group. Pancreatitis, bile leakage and peritonitis were not presented in any of the group. After 17-101 months follow-up, three patients presented recurrent common bile duct stones. CONCLUSION: In selected cases, T-tube-free trans-umbilical SILCBDE plus LC is feasible and safe for experienced surgeons, and can achieve similar therapeutic effects as common LC plus common bile duct exploration procedures.

Physcion 8-O-ß-glucopyranoside exhibits anti-leukemic activity through targeting sphingolipid rheostat.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common fatal cancer in people younger than 20 years of age. This study was designed to explore the anti-leukemia activity of physcion 8-O-ß-glucopyranoside (PG) in B-cell ALL. METHODS: NALM6 and SupB15 cells were used as model cell lines. Cell viability, cell apoptosis, cell cycle distribution were determined by CCK-8 assay, DNA fragmentation assay and flow cytometry, and flow cytometry, respectively. Expression of proteins involved in cell apoptosis and cell cycle regulation was determined by western blot and the levels of ceramide and sphingosine 1-phosphate (S1P) were determined by ELISA. Activity of sphingosine kinase 1 (SphK1) was also determined with a Sphingosine Kinase Assay Kit. In the present study, both model cell lines were transfected with siRNA targeting SphK1 or an overexpression plasmid to examine the role of SphK1 in the anti-leukemia activity of PG. Moreover, the efficacy of PG was examined in vivo in a mouse model by measuring survival and spleen weight. RESULTS: Our results provided experimental evidence that PG could significantly induce apoptosis and cell cycle arrest in vitro. Mechanistically, the anti-leukemia activity of PG was mediated by its ability to repress SphK1 and thus modulate ceramide-S1P rheostat. Moreover, the anti-leukemia activity of PG was also verified in a murine model. CONCLUSION: Collectively, our results indicate that PG may be a promising agent for the treatment of B-cell leukemia.

Single-incision laparoscopic biliary bypass for malignant obstructive jaundice.

Biliary bypass is a major management of resolution to malignant obstructive jaundice. Laparoscopic approach is an ideal alternative to open surgery with the less recurrence compared with endoscopic stenting. Single incision surgery approach has not been applied to biliary bypass due to technical challenge. The aim of this study is to evaluate the safety and feasibility of single-incision laparoscopic biliary bypass. Eighteen patients with periampulla tumor underwent single-incision laparoscopic cholecystojejunostomy. The preoperation and postoperation data were retrospectively analyzed. All the cases underwent surgery successfully without conversion to open or traditional laparoscopic surgery. The operation time and blood loss were 172.8 min and 101.1 ml, respectively. The postoperative hospital stay was 9.9 days. The jaundice was released, and the liver function was improved after the surgery. The mean survival of the patients was 9.5 months. The single-incision laparoscopic cholecystojejunostomy is safe and feasible with acceptable short-term outcomes in selected patients. The benefits still need to be evaluated in comparative study.

Quantification of protein copy number in single mitochondria: The Bcl-2 family proteins.

Bcl-2 family proteins, represented by antiapoptotic protein Bcl-2 and proapoptotic protein Bax, are key regulators of mitochondria-mediated apoptosis pathway. To build a quantitative model of how Bcl-2 family protein interactions control mitochondrial outer membrane permeabilization and subsequent cytochrome c release, it is essential to know the number of proteins in individual mitochondria. Here, we report an effective method to quantify the copy number and distribution of proteins in single mitochondria via immunofluorescent labeling and sensitive detection by a laboratory-built high sensitivity flow cytometer (HSFCM). Mitochondria isolated from HeLa cells were stained with Alexa Fluor 488 (AF488)-labeled monoclonal antibodies specifically targeting Bcl-2 or Bax and with nucleic acid dye. A series of fluorescent nanospheres with fluorescence intensity calibrated in the unit of molecules of equivalent soluble fluorochrome (MESF)-AF488 were used to construct a calibration curve for converting the immunofluorescence of a single mitochondrion to the number of antibodies bound to it and then to the number of proteins per mitochondrion. Under the normal condition, the measured mean copy numbers were 1300 and 220 per mitochondrion for Bcl-2 and Bax, respectively. A significant variation in protein copy number was identified, which ranged from 130 to 6000 (2.5-97.5%) for Bcl-2 and from 65 to 700 (2.5-97.5%) for Bax, respectively. We observed an approximately 4.4 fold increase of Bax copy number per mitochondrion upon 9h of apoptosis stimulation while the abundance of Bcl-2 remained almost unchanged. To the best of our knowledge, this is the first report of Bcl-2 family protein copy number and variance in single mitochondria. Collectively, we demonstrate that the HSFCM-based immunoassay provides a rapid and sensitive method for determining protein copy number distribution in single mitochondria.