Curtis J. Wray

Sepsis upregulates the gene expression of multiple ubiquitin ligases in skeletal muscle.

Muscle wasting during sepsis reflects increased expression and activity of the ubiquitin-proteasome proteolytic pathway and is at least in part mediated by glucocorticoids. The ubiquitination of proteins destined to be degraded by the proteasome is regulated by multiple enzymes, including ubiquitin ligases. We tested the hypothesis that sepsis upregulates the gene expression of the newly described ubiquitin ligases, MuRF1 and atrogin-1/MAFbx. Sepsis was induced in rats by cecal ligation and puncture. Control rats were sham-operated. In some experiments, rats were treated with the glucocorticoid receptor antagonist RU 38486 before induction of sepsis. At various time points after induction of sepsis, mRNA levels for MuRF1 and atrogin-1/MAFbx were determined in extensor digitorum longus muscles by real-time PCR. Sepsis resulted in a 10-16-fold increase in gene expression of the ubiquitin ligases studied here. These changes were much greater than those observed previously for another ubiquitin ligase, E3alpha, in muscle during sepsis. Treatment of rats with RU 38486 prevented the sepsis-induced increase in mRNA levels for MuRF1 and atrogin-1/MAFbx, suggesting that glucocorticoids participate in the upregulation of these genes in muscle during sepsis. The present results lend further support to the concept that the ubiquitin-proteasome pathway plays an important role in sepsis-induced muscle proteolysis and suggest that multiple ubiquitin ligases may participate in the development of muscle wasting during sepsis.

Superinduction of IL‐6 by cycloheximide is associated with mRNA stabilization and sustained activation of p38 map kinase and NF‐κB in cultured caco‐2 cells

Protein synthesis inhibitors paradoxically increase the expression of early‐gene products, including various cytokines, through a process known as superinduction. Superinduction is cell‐specific and the mechanisms involved are not fully understood but are usually attributed to decreased mRNA degradation. There is, however, increasing evidence that activation of signaling cascades and increased transcriptional activation may be involved as well. Recent studies suggest that IL‐6 production in the intestinal mucosa is particularly important due to its anti‐inflammatory and protective effects. The effect of protein synthesis inhibitors on IL‐6 production in enterocytes, however, is unknown. Treatment of Caco‐2 cells with cycloheximide (10 μg/ml) increased IL‐6 mRNA and protein levels in IL‐1β‐treated cells and this was associated with increased mRNA stabilization. In addition, cycloheximide suppressed IκBα resynthesis and prolonged p38MAP kinase activation and these changes were associated with sustained activation of the transcription factor NF‐κB. NF‐κB activation, in turn, was prevented by the specific p38MAP kinase inhibitor SB208350. Our results suggest that superinduction of IL‐6 by cycloheximide in enterocytes results from both increased mRNA stabilization and upregulated transcriptional activity mediated by prolonged activation of the p38 MAP kinase and NF‐κB.

Failure to Recognize Multiple Endocrine Neoplasia 2B: More Common Than We Think?

BackgroundMultiple endocrine neoplasia 2B (MEN2B) has a classic childhood phenotypic presentation characterized by mucosal neuromas and marfanoid habitus. However, the diagnosis of MEN2B is often delayed beyond childhood, at which time medullary thyroid carcinoma (MTC) may be regionally advanced or metastatic. We examined the extent of this delay and its impact on the treatment of MTC.MethodsPatients in the MEN database were retrospectively analyzed to determine the age at first presentation for a MEN2B-related complaint and the subsequent time to correct diagnosis. Operative and pathology reports were reviewed to determine the extent of thyroidectomy and cervical lymphadenectomy during the initial and subsequent neck operations.ResultsWe identified 22 patients with MEN2B, 20 were de novo cases and a M918T RET gene mutation was confirmed in 18 of the 22 patients. Median age at diagnosis of MTC was 13 years (range 6–25 years). The median delay in diagnosis was 26 months (range 0–18 years). Persistent local-regional MTC was present following the initial cervical operation in 12 of 22 patients (55%); including 4 of 13 with MEN2B diagnosed prior to initial surgery and 8 of 9 with MEN2B diagnosed after initial surgery.ConclusionsMost patients displayed phenotypic characteristics of MEN2B long before the correct diagnosis was made. Half of the patients failed to undergo complete resection of MTC at their initial thyroid surgery. Early recognition of the MEN2B phenotype with a thoughtful approach to preoperative staging and surgery will maximize control of MTC and minimize the need for reoperation.

The significance and clinical factors associated with a subcentimeter resection of colorectal liver metastases.

BackgroundPrognosis after resection of colorectal liver metastases is influenced by various factors. A positive margin of resection (MOR) has been shown to adversely influence prognosis. Although a 1-cm MOR has been accepted as adequate, the data to support this guideline are sparse.MethodsOur hepatobiliary database was queried for patients who underwent liver resection for colorectal metastases between January 1992 and July 2003. All patients were divided into three groups: MOR <.5 cm (group A), .5 to 1 cm (group B), and >1 cm (group C). Operative reports from each hepatic resection were analyzed to determine local factors that may have contributed to a subcentimeter MOR.ResultsA total of 112 patients (67 men and 45 women) underwent liver resection for colorectal metastases with negative margins. Fifty-three patients were in group A, 26 patients were in group B, and 33 patients were in group C. Group C demonstrated decreased local recurrence (LR; P = .003), distant recurrence (DR; P = .008), and disease-free recurrence (P = .002). A significant difference in the overall time to LR (P = .003), time to DR (P = .003), and disease-free survival (P = .002) was also demonstrated. Factors associated with a subcentimeter MOR included nonanatomical resection (P = .043), proximity to a major vessel (P = .003), and central location (P = .002).ConclusionsA <1-cm resection for colorectal liver metastases is associated with increased LR and DR, as well as decreased disease-free survival. When a nonanatomical resection is performed, a MOR >1 cm should be attempted, because an adequate margin is often underestimated. Considerations should be made for extended resections when tumors are centrally located or near major vessels.

Adverse Events after Ventral Hernia Repair: The Vicious Cycle of Complications

BACKGROUND Ventral hernia repairs are one of the most common procedures performed by the general surgeon. They are also among the most complex procedures performed. We hypothesized that with each surgical failure, subsequent ventral hernia repair becomes more complicated and morbid. STUDY DESIGN We assessed a multicenter database of patients who underwent an elective ventral hernia repair from 2000 to 2012 with at least 6 months of follow-up and elective repairs. Patients were evaluated by the number of previous ventral hernia repairs they had: primary ventral hernia repair (PVHR), first time incisional hernia repair (IHR1), second time incisional hernia repair (IHR2), or third time or greater incisional hernia repair (IHR3). The main outcomes measured were abdominal reoperation, operative duration, surgical site infection (SSI), and hernia recurrence. Complications were assessed and compared between the 4 groups. Time to recurrence was estimated using the Kaplan-Meier curve method by study cohort (PVHR, IHR1, IHR2, IHR3). RESULTS A total of 794 patients were assessed; of these, 481 (60.6%) had PVHR, 207 (26.1%) had IHR1, 78 (9.8%) had IHR2, and 28 (3.5%) had IHR3. Patients with multiple repairs were more likely to undergo subsequent reoperation, have a longer operative duration, develop SSI, and have a recurrence. At 140 months of follow-up, 37% of primary ventral hernias and 64% of incisional hernias have recurred. The highest recurrence rates are seen in IHR3, with 73% recurring. CONCLUSIONS Previous ventral hernia repair increases the complication profile of repair, creating a vicious cycle of repair, complications, reoperation, and re-repair. Furthermore, long-term outcomes for ventral hernia repair are poor. Future studies should focus on hernia prevention and improving long-term outcomes after hernia repair.

Genetic Variation in the PNPLA3 Gene and Hepatocellular Carcinoma in USA: Risk and Prognosis Prediction

Nonalcoholic fatty liver disease (NAFLD) is an emerging epidemic with high prevalence in Western countries. Genome‐wide association studies had reported that a variation in the patatin‐like phospholipase domain containing 3 (PNPLA3) gene is associated with high susceptibility to NAFLD. However, the relationship between this variation and hepatocellular carcinoma (HCC) has not been well established. We investigated the impact of PNPLA3 genetic variation (rs738409: C>G) on HCC risk and prognosis in the United States by conducting a case−control study that included 257 newly diagnosed and pathologically confirmed Caucasian patients with HCC (cases) and 494 healthy controls. Multivariate logistics and Cox regression models were used to control for the confounding effects of HCC risk and prognostic factors. We observed higher risk of HCC for subjects with a homozygous GG genotype than for those with CC or CG genotypes, the adjusted odds ratio (OR) was 3.21 (95% confidence interval [CI], 1.68–6.41). We observed risk modification among individuals with diabetes mellitus (OR = 19.11; 95% CI, 5.13–71.20). The PNPLA3 GG genotype was significantly associated with underlying cirrhosis in HCC patients (OR = 2.48; 95% CI, 1.05–5.87). Moreover, GG allele represents an independent risk factor for death. The adjusted hazard ratio of the GG genotype was 2.11 (95% CI, 1.26–3.52) compared with CC and CG genotypes. PNPLA3 genetic variation (rs738409: C>G) may determine individual susceptibility to HCC development and poor prognosis. Further experimental investigations are necessary for thorough assessment of the hepatocarcinogenic role of PNPLA3.

Clinical implications of DNA methylation in hepatocellular carcinoma

BACKGROUND Epigenetics is a rapidly evolving field of genetic study applicable to nearly every aspect of genome-related research. The importance of epigenetics has been recognised in human hepatocellular carcinoma (HCC). Changes in DNA methylation patterns, including global hypomethylation and promoter hypermethylation, are thought to be early events in hepatocarcinogenesis. OBJECTIVES This review aimed to summarise the role of epigenetics in HCC, to describe the mechanisms of epigenetic changes in HCC and to examine the clinical relevance of epigenetics in HCC. METHODS This review examines the role of CpG-rich regions and DNA methylation, and describes an epigenetic model of cancer, tumour type-specific methylation, the relationships among methylation, cirrhosis and hepatocarcinogenesis, and the role of DNA methylation in HCC. The clinical implications of epigenetics in HCC are discussed. RESULTS A multivariate predictor model based on traditional clinical factors and DNA methylation profile may have important applications in the early detection of neoplastic transformation in populations at high risk for HCC. CpG methylation may be valuable in HCC prognostics. DNA methylation profiles may enable clinical prediction in pre-therapy patient biopsies, paraffin-embedded samples or plasma DNA. CONCLUSIONS Epigenetic changes and profiles may correlate to the biological behaviour of tumours and clinical outcome of HCC patients. The use of DNA methylation profiles as a surrogate biomarker remains an active area of clinical cancer research.

Acute Appendicitis: Controversies in Diagnosis and Management

Appendicitis is a common problem; there are more than 300,000 hospital discharges for appendicitis in the United States per year. Although the clinical scenario of periumbilical pain migrating to the right lower quadrant is classically associated with acute appendicitis, the presentation is rarely typical and the diagnosis cannot always be based on history and physical examination alone. Diagnostic errors are common, with over-diagnosis leading to negative appendectomies and with delays in diagnosis leading to perforations. The misdiagnosis of appendicitis has significant economic ramifications; in a nationwide study of administrative data over a 1-year period in the late 1990s, a negative appendectomy rate of 15% resulted in more than

Insulin-Like Growth Factor-I Inhibits Lysosomal and Proteasome-Dependent Proteolysis in Skeletal Muscle After Burn Injury

740 million in hospital charges. Diagnostic strategies for evaluating patients with abdominal pain and for identifying patients with suspected appendicitis should all start with a thorough history and physical examination. The Surgical Infection Society (SIS) and Infectious Diseases Society of America (IDSA) published guidelines that recommend the establishment of local pathways for the diagnosis and management of acute appendicitis. The guidelines note that the combination of clinical and laboratory findings of characteristic abdominal pain, localized tenderness, and laboratory evidence of inflammation will identify most patients with suspected appendicitis. Other diagnostic strategies may include radiologic imaging or the use of scoring systems with or without computer support. Ultimately, the ‘‘gold standard’’ for a positive diagnosis is the histopathologic confirmation of appendicitis, although standard criteria are lacking. A negative diagnosis may be confirmed by intra-operative findings or clinical follow-up or both. There are different measures for evaluating a diagnostic test or strategy (Table 1). Sensitivity refers to the proportion of true positive tests among all patients who have the disease (A/[AþC]). Specificity refers to the proportion of true negatives among all patients who do not have the disease (D/[BþD]). Highly sensitive tests rule disease out, whereas highly specific tests rule disease in. Accuracy refers to the proportion of true positives and negatives among all patients tested ([AþD]/ [AþBþCþD]). The positive predictive value of a test refers to the proportion of true positives among all patients who test positive (A/[AþB]), whereas the negative predictive value refers to the proportion of true negatives among all patients who test negative (D/[CþD]). The predictive values of a test should be applied with caution to local patients as they depend upon the incidence

Original scientific articleAdverse Events after Ventral Hernia Repair: The Vicious Cycle of Complications

Previous studies suggest that insulin-like growth factor-I (IGF-I) inhibits burn-induced muscle wasting mainly by reducing muscle protein degradation. The intracellular mechanisms of this effect of IGF-I are not known. In the present study, we examined the influence of IGF-I on individual proteolytic pathways in muscles from burned rats. Extensor digitorum longus muscles from burned rats were incubated with specific blockers of lysosomal, calcium-calpain-dependent, and ubiquitin-proteasome-dependent proteolytic pathways in the absence or presence of IGF-I. In addition, cathepsin B and L activities and 20S proteasome activity were determined. IGF-I inhibited lysosomal and ubiquitin-proteasome-dependent protein breakdown in skeletal muscle from burned rats by 70 and 90%, respectively, but did not influence calcium-calpain-dependent protein breakdown. The hormone blocked the burn-induced increase in cathepsin B and L activities but did not reduce 20S proteasome activity. Results are important because they provide novel information about intracellular mechanisms by which IGF-I inhibits the catabolic response to burn injury in skeletal muscle.