Translational study identifies XPF and MUS81 as predictive biomarkers for oxaliplatin-based peri-operative chemotherapy in patients with esophageal adenocarcinoma.

Oxaliplatin-based chemotherapy is used to treat patients with esophageal adenocarcinoma (EAC), but no biomarkers are currently available for patient selection. We performed a prospective, clinical trial to identify potential biomarkers associated with clinical outcomes. Tumor tissue was obtained from 38 patients with resectable EAC before and after 2 cycles of oxaliplatin-fluorouracil chemotherapy. Pre-treatment mRNA expression of 280 DNA repair (DNAR) genes was tested for association with histopathological regression at surgery, disease-free survival (DFS) and overall survival (OS). High expression of 13 DNA damage repair genes was associated with DFS less than one year (P < 0.05); expression of 11 DNAR genes were associated with worse OS (P < 0.05). From clinical associations with outcomes, two genes, ERCC1 and EME1, were identified as candidate biomarkers. In cell lines in vitro, we showed the mechanism of action related to repair of oxaliplatin-induced DNA damage by depletion and knockout of protein binding partners of the candidate biomarkers, XPF and MUS81 respectively. In clinical samples from the clinical trial, pre-treatment XPF protein levels were associated with pathological response, and MUS81 protein was associated with 1-year DFS. XPF and MUS81 merit further validation in prospective clinical trials as biomarkers that may predict clinical response of EAC to oxaliplatin-based chemotherapy.

Analgesic efficacy of bilateral superficial cervical plexus block for thyroid surgery: meta-analysis and systematic review.

BACKGROUND: Thyroid surgery is moderately painful, but is increasingly being considered as a day-case procedure. Bilateral superficial cervical plexus block (BSCPB) provides an adjuvant technique to facilitate this approach, but there is great evidential heterogeneity in randomised controlled trials (RCTs) about its use. METHODS: A systematic search, critical appraisal, and analysis of RCTs was performed. Trials investigating preoperative or postoperative BSCPB compared with control in patients undergoing thyroid surgery via neck incision were included. Odds ratio (OR) and 95% confidence interval (95% CI) were calculated for dichotomous data, whilst continuous data were analysed using standard mean difference. Primary outcome was rescue analgesic requirement in the first 24 postoperative hours. Secondary outcomes were visual analogue scale (VAS) scores at 0, 4, and 24 h, time until first analgesic request, intraoperative analgesic requirements, length of hospital stay, and incidence of postoperative nausea and vomiting (PONV). RESULTS: Fourteen RCTs published between 2001 and 2016 including 1154 patients were included. The overall effect of BSCPB compared with control showed a reduction in analgesic requirement (OR 0.30; 95% CI 0.18, 0.51; P<0.00001). There was improvement in VAS scores (P<0.002) and time to first analgesic requirement in the BSCPB group (P<0.00001). Length of hospital stay was reduced by 6 h by use of BSCPB. There was no significant change in the incidence of PONV with its use (OR 0.82; 95% CI 0.49-1.37; P=0.44). CONCLUSIONS: BSCPB offers analgesic efficacy in the early postoperative period for up to 24 h after thyroid surgery, with reduced length of hospital stay, but without any beneficial effect on PONV.

Regulation of c-Myc expression by the histone demethylase JMJD1A is essential for prostate cancer cell growth and survival.

The histone demethylase JMJD1A, which controls gene expression by epigenetic regulation of H3K9 methylation marks, functions in diverse activities, including spermatogenesis, metabolism and stem cell self-renewal and differentiation. Here, we found that JMJD1A knockdown in prostate cancer cells antagonizes their proliferation and survival. Profiling array analyses revealed that JMJD1A-dependent genes function in cellular growth, proliferation and survival, and implicated that the c-Myc transcriptional network is deregulated following JMJD1A inhibition. Biochemical analyses confirmed that JMJD1A enhances c-Myc transcriptional activity by upregulating c-Myc expression levels. Mechanistically, JMJD1A activity promoted recruitment of androgen receptor (AR) to the c-Myc gene enhancer and induced H3K9 demethylation, increasing AR-dependent transcription of c-Myc mRNA. In parallel, we found that JMJD1A regulated c-Myc stability, likely by inhibiting HUWE1, an E3 ubiquitin ligase known to target degradation of several substrates including c-Myc. JMJD1A (wild type or mutant lacking histone demethylase activity) bound to HUWE1, attenuated HUWE1-dependent ubiquitination and subsequent degradation of c-Myc, increasing c-Myc protein levels. Furthermore, c-Myc knockdown in prostate cancer cells phenocopied effects of JMJD1A knockdown, and c-Myc re-expression in JMJD1A-knockdown cells partially rescued prostate cancer cell growth in vitro and in vivo. c-Myc protein levels were positively correlated with those of JMJD1A in a subset of human prostate cancer specimens. Collectively, our findings identify a critical role for JMJD1A in regulating proliferation and survival of prostate cancer cells by controlling c-Myc expression at transcriptional and post-translational levels.

Disruption of SF3B1 results in deregulated expression and splicing of key genes and pathways in myelodysplastic syndrome hematopoietic stem and progenitor cells.

The splicing factor SF3B1 is the most commonly mutated gene in the myelodysplastic syndrome (MDS), particularly in patients with refractory anemia with ring sideroblasts (RARS). We investigated the functional effects of SF3B1 disruption in myeloid cell lines: SF3B1 knockdown resulted in growth inhibition, cell cycle arrest and impaired erythroid differentiation and deregulation of many genes and pathways, including cell cycle regulation and RNA processing. MDS is a disorder of the hematopoietic stem cell and we thus studied the transcriptome of CD34(+) cells from MDS patients with SF3B1 mutations using RNA sequencing. Genes significantly differentially expressed at the transcript and/or exon level in SF3B1 mutant compared with wild-type cases include genes that are involved in MDS pathogenesis (ASXL1 and CBL), iron homeostasis and mitochondrial metabolism (ALAS2, ABCB7 and SLC25A37) and RNA splicing/processing (PRPF8 and HNRNPD). Many genes regulated by a DNA damage-induced BRCA1-BCLAF1-SF3B1 protein complex showed differential expression/splicing in SF3B1 mutant cases. This is the first study to determine the target genes of SF3B1 mutation in MDS CD34(+) cells. Our data indicate that SF3B1 has a critical role in MDS by affecting the expression and splicing of genes involved in specific cellular processes/pathways, many of which are relevant to the known RARS pathophysiology, suggesting a causal link.

Potential for detection and discrimination between mycotoxigenic and non-toxigenic spoilage moulds using volatile production patterns: a review.

There has been interest in the development of techniques for the rapid early detection of mycotoxigenic moulds in the food production chain. The development of sensor arrays that respond to the presence of different volatiles produced by such moulds has been examined as a potential method for the development of such detection systems. Commercial devices based on such sensor arrays, so-called 'electronic noses', have been examined extensively for the potential application of determining the presence of mycotoxigenic moulds in food raw materials. There is also interest in using the qualitative volatile production patterns to discriminate between non-mycotoxigenic and mycotoxigenic strains of specific mycotoxigenic species, e.g. Fusarium section Liseola, Penicillium verrucosum and Aspergillus section Nigri. This paper reviews the technology and available evidence that the non-destructive analysis of the headspace of samples of food raw materials or the discrimination between strains (mycotoxigenic and non-mycotoxigenic) can be determined using volatile fingerprints.

Trichophyton species: use of volatile fingerprints for rapid identification and discrimination.

BACKGROUND: Fungal infection of the skin is a common clinical problem, and laboratory confirmation of the diagnosis is important to ensure appropriate treatment. The identification of the species of fungus is also important, because different fungal species have different modes of transmission, and this may be of importance both in preventing re-infection and in avoidance of infection of others. OBJECTIVES: This study examined the potential of using volatile production patterns for the detection and discrimination between four Trichophyton species (T. mentagrophytes, T. rubrum, T. verrucosum and T. violaceum) in vitro on solid media and in broth culture. METHODS: Two different sensor array systems (conducting polymer and metal oxide sensors) were examined for comparing the qualitative volatile fingerprints produced in the headspace by these species over periods of 24-120 h. The relative sensitivity of detection of two of the species (T. mentagrophytes, T. rubrum) was determined for log 1 to log 7 inoculum levels over the same time period. RESULTS: The conducting polymer-based system was unable to differentiate between species based on volatile fingerprints over the experimental period. However, metal oxide-based sensor arrays were found to be able to differentiate between the four species within 96 h of growth using principal component analysis which accounted for approximately 94% of the data in principal components 1 and 2 based on the qualitative volatile production patterns. This differentiation was confirmed by cluster analysis of the data using Euclidean distance and Ward's linkage. Studies of the sensitivity of detection showed that for T. mentagrophytes and T. rubrum it was possible to differentiate between log 3, log 5 and log 7 inoculum levels within 96 h. CONCLUSIONS: This is the first detailed study of the use of qualitative volatile fingerprints for identification and discrimination of dermatophytes. This approach could have potential for rapid identification of patient samples, reducing significantly the time to treatment.

Effectiveness of gloves in the prevention of hand carriage of vancomycin-resistant enterococcus species by health care workers after patient care.

Gloving reduces acquisition of vancomycin-resistant Enterococcus species (VRE) on the hands, and it should be considered for routine inpatient care, even for contact with the intact skin of patients who may be colonized with VRE. However, gloving does not completely prevent contamination of the hands, and hand washing is necessary after glove removal.

T cell responses to recall antigens, alloantigen, and mitogen of HIV-infected patients receiving long-term combined antiretroviral therapy.

The effect of highly active antiretroviral therapy (HAART) on T cell responses in 30 HIV-infected patients was studied. Lymphocyte proliferation in response to influenza A virus, HIV-1 p24, gp160, allogeneic leukocytes, and mitogen, as well as influenza-specific cytotoxic T lymphocyte (CTL) responses, were measured. AIDS patients had decreased T cell-proliferative responses to influenza and alloantigen compared with asymptomatic patients. Absence of positive proliferative responses of HIV-infected patients to HIV-1 antigens was not associated with increased interleukin 10 production. Correlation was observed between influenza-specific CTL response and T cell proliferation, as well as CD4+ T lymphocyte counts, indicating the importance of CD4+ helper T cells for generating antiviral CTL responses. Finally, these results show that HAART-treated asymptomatic patients, but not AIDS patients, have T cell responses comparable to those of control individuals. It remains to be determined whether immune-based therapy will contribute any additional benefit to patients who received HAART.

Identification of two new nonclassical members of the rat prolactin family.

The prolactin (PRL) family is comprised of a group of hormones/cytokines that are expressed in the anterior pituitary, uterus, and placenta. These proteins participate in the control of maternal and fetal adaptations to pregnancy. In this report, we have identified two new nonclassical members of the rat PRL family through a search of the National Center for Biotechnology Information dbEST database. The cDNAs were sequenced and their corresponding mRNAs characterized. Overall, the rat cDNAs showed considerable structural similarities with mouse proliferin-related protein (PLF-RP) and prolactin-like protein-F (PLP-F), consistent with their classification as rat homologs for PLF-RP and PLP-F. The expression of both cytokines/hormones was restricted to the placenta. The intraplacental sites of PLF-RP and PLP-F synthesis differed in the rat and the mouse. In the mouse, PLF-RP was expressed in the trophoblast giant cell layer of the midgestation chorioallantoic and choriovitelline placentas and, during later gestation, in the trophoblast giant cell and spongiotrophoblast layers within the junctional zone of the mouse chorioallantoic placenta. In contrast, in the rat, PLF-RP was first expressed in the primordium of the chorioallantoic placenta (ectoplacental cone region) and, later, exclusively within the labyrinth zone of the chorioallantoic placenta. In the mouse, PLP-F is an exclusive product of the spongiotrophoblast layer, whereas in the rat, trophoblast giant cells were found to be the major source of PLP-F, with a lesser contribution from spongiotrophoblast cells late in gestation. In summary, we have established the presence of PLF-RP and PLP-F in the rat.

Improved pulmonary distribution of recombinant human Cu/Zn superoxide dismutase, using a modified ultrasonic nebulizer.

Prophylactic, intratracheal instillation of recombinant human Cu/Zn superoxide dismutase (rhSOD) has been shown to lessen lung injury produced by 48 h of hyperoxia and mechanical ventilation in neonatal piglets. However, instillation of small volumes of rhSOD intratracheally would not be expected to result in uniform pulmonary distribution. Aerosolization is a technique that may improve pulmonary distribution of drugs, but is limited by the poor efficiency of most nebulizers. A newly modified ultrasonic nebulizer was tested to assess pulmonary distribution of rhSOD compared to that achieved by intratracheal instillation. rhSOD was dual-labeled with technetium-99m (99mTc) and a fluorescent analog (permitting quantitative and qualitative assessments of pulmonary distribution), and administered to neonatal piglets by intratracheal instillation or by aerosolization. Intratracheal instillation of rhSOD to piglets when supine resulted in nonuniform distribution, with most of the drug being found in the right caudal lobe, and localized in airways. Placing animals in 30 degrees of Trendelenburg and administering half the dose in the left and half in the right lateral decubitus positions improved distribution, but alveolar deposition remained patchy. Aerosolization using a modified ultrasonic nebulizer uniformly delivered 45.8 +/- 3.8% of the rhSOD to the lungs that had been placed in the nebulizer. The rhSOD was still active and present in airways and alveoli in a homogeneous fashion. We conclude that intratracheal instillation of rhSOD in small volumes results in nonuniform pulmonary distribution, while aerosolization enhances rhSOD distribution and alveolar deposition. This has important implications for ongoing clinical trials of rhSOD for the prevention of acute and chronic lung injury in premature neonates.

Intracellular uptake of recombinant superoxide dismutase after intratracheal administration.

We have previously demonstrated that recombinant human copper-zinc superoxide dismutase (rhCu,ZnSOD) is rapidly incorporated into cells of airways, respiratory bronchioles, and alveoli after intratracheal administration. The present study examines whether this cellular uptake is specific for rhCu,ZnSOD or whether other proteins are similarly incorporated into lung cells. Twenty-two newborn piglets (2-3 days old, 1.2-2.0 kg) were intubated and mechanically ventilated. Eight piglets received fluorescently labeled recombinant human manganese superoxide dismutase (rhMnSOD), six received fluorescently labeled albumin, two received free (unbound) fluorescent label intratracheally, and two piglets served as untreated controls. To determine whether endogenous surfactant was important in the process of intracellular uptake, four additional piglets were made surfactant deficient by repeated bronchoalveolar lavage and then given rhCu,ZnSOD intratracheally. All animals were killed after 30-60 min. Lung sections were examined blindly by laser confocal microscopy. Similar to our previous observations with rhCu,ZnSOD, intracellular uptake of rhMnSOD and albumin was noted throughout the lung. The free label did not localize intracellularly. The uptake of proteins did not appear to be affected by surfactant deficiency. rhMnSOD administration was associated with a greater than twofold increase in lung MnSOD activity. Data suggest that the cellular uptake of antioxidants and other proteins in the lung may reflect a nonspecific host defense system for clearing proteins from the lumen of airways and alveoli.

Localization and activity of recombinant human CuZn superoxide dismutase after intratracheal administration.

Hyperoxia and mechanical ventilation cause acute lung injury which may be mitigated by prophylactic intratracheal (IT) administration of recombinant human CuZn superoxide dismutase (rhSOD). However, little is known about the localization, activity, and metabolism of rhSOD after IT administration by instillation or nebulization. Twenty-six newborn piglets were intubated, mechanically ventilated, and given either saline or fluorescently labeled rhSOD (5 mg/kg IT) by instillation or nebulization. Animals were killed 1, 6, or 12 h later. Intact rhSOD (% total fluorescence still associated with macromolecules) and total SOD activity in lung tissue were then determined. Results indicate that, after 1 and 6 h of administration, the majority of rhSOD present in the lung was still associated with the fluorescent label. By 12 h, most of the rhSOD was no longer fluorescently labeled. At 1 h, lung SOD activity increased by 100% compared with untreated control values, with activity remaining elevated at 6 and 12 h. Laser confocal microscopy of lung tissue showed that at 1 h, labeled rhSOD was found throughout the lung, inside a variety of cell types of airways, respiratory bronchioles, and alveoli. Deposition was more homogeneous after nebulization. Negative controls had minimal background fluorescence. These data indicate that after IT administration, rhSOD is rapidly incorporated into cells in the lung and significantly increases lung SOD activity. These observations have important implications for the clinical use of rhSOD in human trials.

Combined effects of nitric oxide and hyperoxia on surfactant function and pulmonary inflammation.

NO and its derivative ONOO- are potent free radicals that can cause cell damage, especially in the presence of O2. To determine the potential pulmonary toxicities of nitric oxide (NO) and peroxynitrite (ONOO-) in vitro, Survanta (2.5 mg/ml) was exposed to ONOO- (0.3-8 mM) in the presence of two different buffering systems (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and phosphate buffer) and minimum surface tension (MST) was determined with an oscillating bubble surfactometer. Significant increases in MST were seen only with exposure to 8 mM ONOO-, indicating that in vitro, high concentrations of ONOO- can inhibit natural surfactant function. The in vivo effects of NO and hyperoxia were then studied in four groups of newborn piglets ventilated for 48 h with 21% O2, 100% O2, 21% O2 and 100 ppm NO, or with 90% O2 and 100 ppm NO. Five animals served as an untreated control group. Bronchoalveolar lavage fluid (BAL) obtained at 48 h was subjected to centrifugation and the surfactant pellet was reconstituted to 5 mg phospholipid/ml. Significant increases in MST were seen in surfactant from piglets ventilated with NO and 90% O2, compared with either untreated controls or piglets ventilated with 21% O2 for 48 h (P < 0.05, analysis of variance). Significant increases in neutrophil chemotactic activity (NCA) of BAL were also found in the NO and O2 group (P < 0.05), with significant positive interaction between NO and O2 found (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of hyperoxia, mechanical ventilation, and dexamethasone on pulmonary antioxidant enzyme activity in the newborn piglet.

It has been previously shown that prophylactic, intravenous dexamethasone (DEX) and intratracheal recombinant human Cu/Zn superoxide dismutase (SOD) ameliorate lung injury in newborn piglets treated with 48 hr of hyperoxia and mechanical ventilation. DEX has many pharmacologic effects, including the possible induction of antioxidant enzyme systems. To investigate whether the effects of DEX are mediated by an increase in endogenous antioxidant enzyme activity, 5 groups of term newborn piglets were studied: Group 1 piglets were ventilated with room air for 48 hr; Group 2 animals were ventilated with 100% O2 for 48 hr; Group 3 animals were ventilated with room air for 48 hr and received DEX (0.7 mg/kg) every 12 h; Group 4 were ventilated with 100% O2 for 48 hr and also received DEX; Group 5 animals were no ventilated and were sacrificed at time 0. At the conclusion of the studies, bronchoalveolar lavage (BAL) was performed and the lungs were removed and homogenized. Lung tissue and BAL were analyzed for SOD, catalase, GPX activities, and total protein concentration. No significant differences in any of these assays were seen in either lung tissue or BAL in the 5 groups. These observations indicate that 48 hr of hyperoxia, mechanical ventilation, or dexamethasone treatment does not induce activity of SOD, catalase, or glutathione peroxidase (GPX) in the lungs of newborn piglets. Thus postnatal DEX appears to minimize neonatal lung injury by mechanisms that are independent of these enzymes.