A Closed-Tube Nested Quantitative PCR Assay for Rapid Detection of Intron 22 Inversions in the Factor VIII Gene.

BACKGROUND: An inversion of intron 22 in the Factor VIII gene (Inv22) is the causative mutation for 45% of severe hemophilia A cases. Available methods for molecular diagnosis of Inv22 are generally tedious and not ideal for routine clinical use. METHODS: We report here a new method using a single closed-tube nested quantitative PCR (CN-qPCR) for rapid detection of Inv22. This method combines a 12-cycle long-distance PCR (LD-PCR) amplifying the int22h regions, followed by a duplex qPCR targeting two specific regions close to the int22h regions. All reagents were added to a single PCR mixture for the closed-tube assay. Sequential LD-PCR and qPCR was achieved by designing primers at substantially different melting temperatures and optimizing PCR conditions. RESULTS: Seventy-nine male hemophilia A patients of different disease severity were tested by both the CN-qPCR assay and the standard LD-PCR assay. CN-qPCR successfully made calls for all samples, whereas LD-PCR failed in eight samples. For the 71 samples where both methods made calls, the concordance was 100%. Inv22 was detected in 17 out of the 79 samples. Additionally, CN-qPCR achieved clear separation for 10 female carriers and 10 non-Inv22 females, suggesting the assay may also be useful for molecular diagnosis of female carriers. CONCLUSIONS: This new CN-qPCR method may provide a convenient and accurate F8 Inv22 test suitable for clinical use.

Highly multiplexed quantifications of 299 somatic mutations in colorectal cancer patients by automated MALDI-TOF mass spectrometry.

BACKGROUND: Detection of somatic mutations in tumor tissues helps to understand tumor biology and guide treatment selection. Methods such as quantitative PCR can analyze a few mutations with high efficiency, while next generation sequencing (NGS) based methods can analyze hundreds to thousands of mutations. However, there is a lack of cost-effective method for quantitatively analyzing tens to a few hundred mutations of potential biological and clinical significance. METHODS: Through a comprehensive database and literature review we selected 299 mutations associated with colorectal cancer. We then designed a highly multiplexed assay panel (8-wells covering 299 mutations in 109 genes) based on an automated MADLI-TOF mass spectrometry (MS) platform. The multiplex panel was tested with a total of 319 freshly frozen tissues and 92 FFPE samples from 229 colorectal cancer patients, with 13 samples also analyzed by a targeted NGS method covering 532 genes. RESULTS: Multiplex somatic mutation panel based on MALDI-TOF MS detected and quantified at least one somatic mutation in 142 patients, with KRAS, TP53 and APC being the most frequently mutated genes. Extensive validation by both capillary sequencing and targeted NGS demonstrated high accuracy of the multiplex MS assay. Out of 35 mutations tested with plasmid constructs, sensitivities of 5 and 10% mutant allele frequency were achieved for 19 and 16 mutations, respectively. CONCLUSIONS: Automated MALDI-TOF MS offers an efficient and cost-effective platform for highly multiplexed quantitation of 299 somatic mutations, which may be useful in studying the biological and clinical significance of somatic mutations with large numbers of cancer tissues.

Dynamics of Plasma EGFR T790M Mutation in Advanced NSCLC: A Multicenter Study.

BACKGROUND: Droplet digital polymerase chain reaction (ddPCR) is an emerging technology for quantitative cell-free DNA oncology applications. However, a ddPCR assay for the epidermal growth factor receptor (EGFR) p.Thr790Met (T790M) mutation suitable for clinical use remains to be established with analytical and clinical validations. OBJECTIVE: We aimed to develop and validate a new ddPCR assay to quantify the T790M mutation in plasma for monitoring and predicting the progression of advanced non-small-cell lung cancer (NSCLC). METHODS: Specificity of the ddPCR assay was evaluated with genomic DNA samples from healthy individuals. The inter- and intraday variations of the assay were evaluated using mixtures of plasmid DNA containing wild-type EGFR and T790M mutation sequences. We assessed the clinical utility of the T790M assay in a multicenter prospective study in patients with advanced NSCLC receiving tyrosine kinase inhibitor (TKI) treatment by analyzing longitudinal plasma DNA samples. RESULTS: We set the criteria for a positive call when the following conditions were satisfied: (1) T790M mutation frequency > 0.098% (3 standard deviations above the background signal); (2) at least two positive droplets in duplicate ddPCR reactions. Among the 62 patients with advanced NSCLC exhibiting resistance to TKI treatment, 15 had one or more serial plasma samples that tested positive for T790M. T790M mutation was detected in the plasma as early as 205 days (median 95 days) before disease progression, determined by imaging analysis. Plasma T790M concentrations also correlated with intervention after disease progression. CONCLUSIONS: We developed a ddPCR assay to quantify the T790M mutation in plasma. Quantification of longitudinal plasma T790M mutation may allow noninvasive assessment of drug resistance and guide follow-up treatment in TKI-treated patients with NSCLC. TRIAL REGISTRATION: Clinical Trials.gov identifier: NCT02804100.

The impact of early enteral nutrition on pediatric patients undergoing gastrointestinal anastomosis a propensity score matching analysis.

This study was conducted to assess the clinical advantages of early enteral nutrition (EEN) in pediatric patients who underwent surgery with gastrointestinal (GI) anastomosis.EEN has been associated with clinical benefits in various aspect of surgical intervention, including GI function recovery and postoperative complications reduction. Evaluable data documenting clinical advantages with EEN for pediatric patients after surgery with GI anastomosis are limited.We retrospectively reviewed the medical records of 575 pediatric patients undergoing surgical intervention with GI anastomosis. Among them, 278 cases were managed with EEN and the remaining cases were set as late enteral nutrition (LEN) group. Propensity score (PS) matching was conducted to adjust biases in patient selection. Enteral feeding related complications were evaluated with symptoms, including serum electrolyte abnormalities, abdominal distention, abdominal cramps, and diarrhea. Clinical outcomes, including GI function recovery, postoperative complications, length of hospital stay, and postoperative follow-up, were assessed according to EEN or LEN.Following PS matching, the baseline variables of the 2 groups were more comparable. There were no differences in the incidence of enteral feeding-related complications. EEN was associated with postoperative GI function recovery, including time to first defecation (3.1 ±â€Š1.4 days for EEN vs 3.8 ±â€Š1.0 days for LEN, risk ratio [RR], 0.62; 95% confidence interval [CI] 0.43-1.08, P = .042). A lower total episodes of complication, including infectious complications and major complications were noted in patients with EEN than in patients with LEN (117 [45.9%] vs 137 [53.7%]; OR, 0.73, 95% CI 0.52-1.03, P = .046). Mean postoperative length of stay in the EEN group was 7.4 ±â€Š1.8 days versus 9.2 ±â€Š1.4 days in the LEN group (P = .007). Furthermore, the incidence of adhesive small bowel obstruction was lower for patients with laxative administration compared with control, but no significant difference was attained (P = .092)EEN was safe and associated with clinical benefits, including shorten hospital stay, and reduced overall postoperative complications on pediatric patients undergoing GI anastomosis.

The efficacy of combined therapy with metronidazole and broad-spectrum antibiotics on postoperative outcomes for pediatric patients with perforated appendicitis.

The aim of this study was to evaluate the efficacy of combined therapy with metronidazole and broad-spectrum antibiotics for patients with perforated appendicitis who underwent surgical intervention.Broad-spectrum antibiotic therapy is warranted in the treatment of perforated appendicitis. Metronidazole has been used as anaerobic antimicrobial therapy. However, few studies about the use of metronidazole in perforated appendicitis have been reported.The medical records of 249 patients treated with metronidazole combined with broad-spectrum antibiotics following perforated appendicitis surgery were reviewed retrospectively and compared with the medical records of 149 patients treated only with broad-spectrum antibiotics. Propensity score matching was performed to adjust for selected baseline variables. Clinical outcomes, including postoperative complications and length of hospital stay, were compared between the 2 groups.No differences were found between the use of combined therapy with metronidazole and the use of solely broad-spectrum antibiotic agents with regard to postoperative duration of intravenous antibiotic treatment (6.8 ±â€Š1.3 vs 7.9 ±â€Š2.1 days, respectively, P = .18), inflammation variables at POD 5 (white blood cell [WBC] [risk ratio [RR], 1.06; 95% confidence interval [CI], 0.67-1.93, P = .15] and C-reactive protein [CRP] [RR, 1.18; 95% CI, 0.73-2.25, P = .36]) (Table 2), and the mean postoperative length of hospital stay (LOS) (RR, 0.68, 95% CI, 0.41-0.94, P = .41). There were also no differences in the incidence of postoperative complications, including the intra-abdominal or pelvic abscess rate (7[7.1%] vs 9[9.2%], respectively, P = .40), the incidence of wound infection (14[14.3%] vs 15[15.3%], respectively, P = .50), and the 30-day readmission rate (9[9.2%] vs 12[12.2%], respectively, P = .32).Regarding overall postoperative outcomes and complications, our study demonstrated no beneficial clinical effects of metronidazole administration in patients with perforated appendicitis who underwent surgical intervention. Therefore, metronidazole is not indicated when broad-spectrum antibiotics such as aminopenicillins with ß-lactam inhibitors or carbapenems and select cephalosporins are used.

Prophylactic vacuum sealing drainage (VSD) in the prevention of postoperative surgical site infections in pediatric patients with contaminated laparotomy incisions.

Surgical site infection (SSI) continues to be an issue in abdominal surgery, especially for contaminated (class III) and dirty-infected (class IV) wounds. Vacuum sealing drainage (VSD) was reported effective in the management of various types of wounds or skin grafts. Our goal was to investigate the efficacy of prophylactic VSD to better orient their medicosurgical care of high-risk incisions following laparotomy in a pediatric population.A total of 331 pediatric patients with contaminated (class III) and dirty-infected (class IV) wounds following emergency laparotomy were retrospectively reviewed between January 2005 and January 2013. Among them, 111 cases were placed with prophylactic VSD when incisions were closed. Clinical outcomes, including, overall surgical site complication, device effectiveness, and mean postoperative LOS were evaluated based on VSD usage or not.VSD was applied for an average of 5.8 days (range, 5-7 days), with 3 to 15 mL sucked fluid. The overall SSIs rate was 3% for patients with prophylactic VSD and 17% for patients with convention dressing (OR, 0.27; 95% CI, 0.10-0.71, P = 0.004). In patients with prophylactic VSD, only 1 of 96 wound developed postoperative incision dehiscence, which is significant reduced compared with patients for conventional dressings (OR, 0.12; 95% CI, 0.01-0.95; P = 0.017) (Table 2). It also exhibited a decreased mean postoperative LOS (P < 0.001) for prophylactic VSD over conventional dressings.Our study demonstrated beneficial postoperative clinical effects of prophylactic VSD for high-risk laparotomy incisions following emergency laparotomy, such as shorter length of hospitalization, which may be attributed to the reduced overall SSIs rate.

Impact of hypertonic saline on postoperative complications for patients undergoing upper gastrointestinal surgery.

The aim of this study was to explore the impact of 3% hypertonic saline (HS) intragastric administration for patients who underwent upper gastrointestinal surgery.During the postoperative period, 3% HS has been suggested as a means to improve the intestinal edema and reduce gastrointestinal complications.The medical records of 111 patients with HS intragastric administration following upper gastrointestinal surgery and 268 patients, served as control, were reviewed retrospectively. Propensity score matching was performed to adjust for selected baseline variables. Clinical outcomes, including early gastrointestinal function recovery, postoperative complications, and length of hospital stay, were compared according to the HS intragastric administration or not.HS intragastric administration was associated with prompt postoperative gastrointestinal function recovery, including first flatus (risk ratio [RR], 1.32; 95% confidence interval [CI], 0.89-1.65; P = 0.048) and feeding within 3 postoperative days (RR (95% CI), 0.57 (0.49-0.77); P = 0.036). Early ileus occurred in 25 of 108 patients with HS treatment versus 36 of 108 patients without HS treatment (RR (95% CI), 1.43 (0.63-2.15); P = 0.065). The patients with HS experienced a lower overall postoperative complication (odds ratio [OD] 0.57; 95% CI, 0.33-1.09; P = 0.063), including trend toward a decrease for infectious complications (15[13.9] vs 23[21.3]; P = 0.11; OD, 0.59; 95% CI, 0.29-1.22). There was a decreased incidence of anastomotic leakage (1[0.9] vs 7[6.5]; P = 0.033) and postoperative ileuas (5[4.6%] vs 11[10.2%]; P = 0.096) in the HS administration patients.Our study demonstrated beneficial postoperative clinical effects of HS intragastric administration in patients who had undergone upper gastrointestinal surgery, such as prompt postoperative gastrointestinal function recovery and reduced overall postoperative complications, which may be attributed to a reduced intestinal edema.

Contribution of glutaredoxin-1 to S-glutathionylation of endothelial nitric oxide synthase for mesenteric nitric oxide generation in experimental necrotizing enterocolitis.

Endothelial nitric oxide synthase (eNOS) is critical for intestinal microcirculatory perfusion and therefore plays a key role in the development of necrotizing enterocolitis (NEC). eNOS-derived nitric oxide (NO) is inhibited by S-glutathionylation of eNOS (eNOS-SSG), which can be reversed by glutaredoxin-1 (Grx1). Therefore, the objective of this study was to investigate the interplay between Grx1 and eNOS in regulating the following inflammation signal during the development of NEC. Primary mouse intestinal microvascular endothelial cells (MIMECs) and peritoneal macrophages were subjected to lipopolysaccharide treatment, and Grx1-/- mice were subjected to an NEC-inducing regimen of formula feeding in combination with hypoxia and hypothermia. The eNOS-SSG level and its activity were assessed using immunoprecipitated assay and NO production evaluation. NO-mediated Toll-like receptor 4 (TLR4) signaling and inflammation injury were further defined. NEC severity was significantly increased in Grx1-/- mice. Grx1-/- mice with NEC showed significantly decreased NO and increased O2•- production with increases in eNOS-SSG. Furthermore, TLR4 signaling, which is required for the development of NEC, was enhanced in the Grx1-deficient mice. These results suggest that eNOS-SSG within the MIMECs inhibited NO production and enhanced TLR4 activity, which were implicated in the pathogenesis of NEC. Grx1 deficiency increases the severity of NEC in association with eNOS-SSG.

Fecal microbiota transplantation (FMT) could reverse the severity of experimental necrotizing enterocolitis (NEC) via oxidative stress modulation.

Fecal microbiota transplantation (FMT) has been used successfully to treat a variety of gastroenterological diseases. The alterations of microbiota in mouse models of necrotizing enterocolitis (NEC) as well as in patients suggested the possibility of treating NEC with FMT. Here we show that FMT caused an improvement in the histopathology and symptoms of NEC in WT mice, but not Grx1-/- mice. FMT eliminated O2•- production and promoted NO production in experimental NEC mice though the modulation of S-glutathionylation of eNOS (eNOS-SSG). FMT decreased the extent of TLR4-mediated proinflammatory signaling though TLR9 in the intestinal mucosa tissue. FMT also suppressed intestinal apoptosis and bacterial translocation across the intestinal barrier, which was accompanied by decreased inflammatory cytokine levels, altered bacterial microbiota, and regulated lymphocyte proportions. FMT is effective in a mouse model of NEC through the modulation of oxidative stress and reduced colon inflammation.

[The criminological characteristics of mental retardation].

OBJECTIVE: To explore the criminological characteristics of mental retardation (MR) in forensic psychiatry. METHODS: The record scale of forensic psychiatric assessment designed by ourselves was used to analyse the criminological characteristics of 83 offenders with MR, and to compare the criminological characteristics of mild MR with that of moderate and severe MR. RESULTS: The mild MR accounted for 62.7%, moderate and severe MR was 22.9%. The percentage of sex offenders in MR was 37.3%, manslaughter 34.7%, property offences 28.0%, respectively. Additionally, 96.1% cases with MR have definite criminal motives, and the criminal history was established in 34.7% cases. Significant differences of criminal premeditation (X2chi-squared l11,P=0.001), criminal aim(x2chi-squared 7.531, P=0.006), criminal motive(X 2chi-squared . 920, P= 0.019) and criminal types(s 2chi-squared .855, P=0.02) were found between the mild MR and the moderate, severe MR. CONCLUSIONS: The criminal offenders were mostly found in mild MR. The sex offenders and manslaughter were in outright majority, and most of them had definite criminal motives. The proportion of offenders in mild MR who had criminal premeditation and criminal aim was higher significantly than which in the moderate, severe MR. The proportion of offenders in moderate, severe MR whose criminal motive was for sex was higher than that in mild MR.

Mitochondrial ATF2 translocation contributes to apoptosis induction and BRAF inhibitor resistance in melanoma through the interaction of Bim with VDAC1.

BACKGROUND: The mitochondrial accumulation of ATF2 is involved in tumor suppressor activities via cytochrome c release in melanoma cells. However, the signaling pathways that connect mitochondrial ATF2 accumulation and cytochrome c release are not well documented. METHODS: Several melanoma cell lines, B16F10, K1735M2, A375 and A375-R1, were treated with paclitaxel and vemurafenib to test the function of mitochondrial ATF2 and its connection to Bim and voltage-dependent anion channel 1 (VDAC1). Immunoprecipitation analysis was performed to investigate the functional interaction between the involved proteins. VDAC1 oligomerization was evaluated using an EGS-based crosslinking assay. RESULTS: The expression and migration of ATF2 to the mitochondria accounted for paclitaxel stimuli and acquired resistance to BRAF inhibitors. Mitochondrial ATF2 facilitated Bim stabilization through the inhibition of its degradation by the proteasome, thereby promoting cytochrome c release and inducing apoptosis in B16F10 and A375 cells. Studies using B16F10 and A375 cells genetically modified for ATF2 indicated that mitochondrial ATF2 was able to dissociate Bim from the Mcl-1/Bim complex to trigger VDAC1 oligomerization. Immunoprecipitation analysis revealed that Bim interacts with VDAC1, and this interaction was remarkably enhanced during apoptosis. CONCLUSION: These results reveal that mitochondrial ATF2 is associated with the induction of apoptosis and BRAF inhibitor resistance through Bim activation, which might suggest potential novel therapies for the targeted induction of apoptosis in melanoma therapy.

Potential synergism of Bim with p53 in mice with Myc­induced lymphoma in a mouse lymphoma model.

We report that Bim has an apoptotic function through Bax/mitochondrial apoptosis signaling in the p53-independent mode, which is somewhat additive to the effects of p53. Bim-deficient mouse embryonic fibroblast (MEF) cells were resistant to the apoptotic effects of Myc, while loss of Bim accelerated lymphoma development. Furthermore, Bim was overexpressed at the same frequency in Myc-initiated lymphomas, irrespective of p53 status, suggesting that Bim resides in a pathway separate from p53. Loss of Bim further augmented resistance to apoptosis in p53-/- MEFs. Mice with p53 knockdown exhibited exacerbated malignancies in the absence of Bim. The combined loss of these proteins promoted more severe spontaneous tumorigenesis. Thus, Myc-induced apoptotic signals through Bim and p53 must bifurcate to activate Bax, suggesting that the activation of Bim and p53 significantly contribute to apoptosis. Our results, therefore, establish that p53 and Bim are effective key initiators of apoptosis in lymphoma cells, particularly when combined.