Randomized trial of oral versus enteral feeding for patients with postoperative pancreatic fistula after pancreatoduodenectomy.

BACKGROUND: Whether continued oral feeding may have a negative impact on healing of postoperative pancreatic fistula (POPF) is unclear. The aim was to test the hypothesis that oral feeding is non-inferior to enteral feeding in closure of POPF after pancreatoduodenectomy, and to clarify the effects of oral feeding on the duration and grade of POPF. METHODS: This multicentre, non-inferiority randomized trial of oral or enteral feeding of patients with POPF after pancreatoduodenectomy recruited patients between August 2013 and September 2016. The primary efficacy outcome was the 30-day fistula closure rate. The prespecified non-inferiority margin was 15 per cent. Other efficacy outcomes included grade of fistula, and hospital stay and costs. RESULTS: A total of 114 patients were included, and received oral (57) or enteral (57) feeding. The two groups were balanced in baseline characteristics and no patient was lost to follow-up. In intention-to-treat analysis, oral feeding was non-inferior to enteral feeding in terms of 30-day fistula closure rate (88 versus 89 per cent respectively; difference -1·8 per cent, lower limit of 95 per cent c.i. -14·4 per cent; P = 0·020 for non-inferiority). Compared with enteral feeding, oral feeding significantly reduced hospital costs and duration of stay. No significant differences were noted in the number of patients whose POPF evolved into grade B/C, or other outcomes. CONCLUSION: Oral feeding in patients with POPF after pancreatoduodenectomy did not increase the duration or grade of POPF, and was associated with reduced duration of stay and hospital costs. Registration number: NCT01755260 (http://www.clinicaltrials.gov).

Identification of the chitinase genes from the diamondback moth, Plutella xylostella.

Chitinases have an indispensable function in chitin metabolism and are well characterized in numerous insect species. Although the diamondback moth (DBM) Plutella xylostella, which has a high reproductive potential, short generation time, and characteristic adaptation to adverse environments, has become one of the most serious pests of cruciferous plants worldwide, the information on the chitinases of the moth is presently limited. In the present study, using degenerated polymerase chain reaction (PCR) and rapid amplification of cDNA ends-PCR strategies, four chitinase genes of P. xylostella were cloned, and an exhaustive search was conducted for chitinase-like sequences from the P. xylostella genome and transcriptomic database. Based on the domain analysis of the deduced amino acid sequences and the phylogenetic analysis of the catalytic domain sequences, we identified 15 chitinase genes from P. xylostella. Two of the gut-specific chitinases did not cluster with any of the known phylogenetic groups of chitinases and might be in a new group of the chitinase family. Moreover, in our study, group VIII chitinase was not identified. The structures, classifications and expression patterns of the chitinases of P. xylostella were further delineated, and with this information, further investigations on the functions of chitinase genes in DBM could be facilitated.

The metabolome profiling and pathway analysis in metabolic healthy and abnormal obesity.

OBJECTIVES: Mechanisms of the development of abnormal metabolic phenotypes among obese population are not yet clear. In this study, we aimed to screen metabolomes of both healthy and subjects with abnormal obesity to identify potential metabolic pathways that may regulate the different metabolic characteristics of obesity. METHODS: We recruited subjects with body mass index (BMI) over 25 from the weight-loss clinic of a central hospital in Taiwan. Metabolic healthy obesity (MHO) is defined as without having any form of hyperglycemia, hypertension and dyslipidemia, while metabolic abnormal obesity (MAO) is defined as having one or more abnormal metabolic indexes. Serum-based metabolomic profiling using both liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry of 34 MHO and MAO individuals with matching age, sex and BMI was performed. Conditional logistic regression and partial least squares discriminant analysis were applied to identify significant metabolites between the two groups. Pathway enrichment and topology analyses were conducted to evaluate the regulated pathways. RESULTS: A differential metabolite panel was identified to be significantly differed in MHO and MAO groups, including L-kynurenine, glycerophosphocholine (GPC), glycerol 1-phosphate, glycolic acid, tagatose, methyl palmitate and uric acid. Moreover, several metabolic pathways were relevant in distinguishing MHO from MAO groups, including fatty acid biosynthesis, phenylalanine metabolism, propanoate metabolism, and valine, leucine and isoleucine degradation. CONCLUSION: Different metabolomic profiles and metabolic pathways are important for distinguishing between MHO and MAO groups. We have identified and discussed the key metabolites and pathways that may prove important in the regulation of metabolic traits among the obese, which could provide useful clues to study the underlying mechanisms of the development of abnormal metabolic phenotypes.

Discovery of genes related to formothion resistance in oriental fruit fly (Bactrocera dorsalis) by a constrained functional genomics analysis.

Artificial selection can provide insights into how insecticide resistance mechanisms evolve in populations. The underlying basis of such phenomena can involve complex interactions of multiple genes, and the resolution of this complexity first necessitates confirmation that specific genes are involved in resistance mechanisms. Here, we used a novel approach invoking a constrained RNA sequencing analysis to refine the discovery of specific genes involved in insecticide resistance. Specifically, for gene discovery, an additional constraint was added to the traditional comparisons of susceptible vs. resistant flies by the incorporation of a line in which insecticide susceptibility was 'recovered' within a resistant line by the removal of insecticide stress. In our analysis, the criterion for the classification of any gene as related to insecticide resistance was based on evidence for differential expression in the resistant line as compared with both the susceptible and recovered lines. The incorporation of this additional constraint reduced the number of differentially expressed genes putatively involved in resistance to 464, compared with more than 1000 that had been identified previously using this same species. In addition, our analysis identified several key genes involved in metabolic detoxification processes that showed up-regulated expression. Furthermore, the involvement of acetylcholinesterase, a known target for modification in insecticide resistance, was associated with three key nonsynonymous amino acid substitutions within our data. In conclusion, the incorporation of an additional constraint using a 'recovered' line for gene discovery provides a higher degree of confidence in genes identified to be involved in insecticide resistance phenomena.

Direct duplication of the Y chromosome with normal phenotype - incidental finding in two cases.

Structural rearrangement in the Y chromosome is closely involved in spermatogenesis. However, several Y chromosome variants may have no deleterious effects on male reproduction. Here, we report two cases of Y chromosomal duplication from incidental findings. Their FISH analysis revealed direct duplication of large segments of short and long arms of the Y chromosome. Nearly two intact Y chromosomes were carried in these two cases with normal phenotype.

Gefitinib-induced epidermal growth factor receptor-independent keratinocyte apoptosis is mediated by the JNK activation pathway.

BACKGROUND: Gefitinib (ZD1839) is a selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor with a significant antitumour effect on various cancers. Skin toxicity induced by gefitinib is common, and has been shown to be related to the inhibition of EGFR signalling pathways. However, other mechanisms may be involved in gefitinib-induced skin toxicity. OBJECTIVES: To study the possible EGFR-independent mechanisms of gefitinib-induced skin toxicity. METHODS: The human immortalized keratinocyte cell line HaCaT and human lung adenocarcinoma cell lines (A549 and PC9) were treated with different concentrations of gefitinib for 24, 48 and 72 h. Cell viability was measured by MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] after EGFR gene silencing. The signalling pathways were investigated by immunoblot analysis. Keratinocyte apoptosis was evaluated by nuclear condensation and flow cytometric analysis. RESULTS: Gefitinib maintained its cytotoxicity to HaCaT cells after EGFR gene silencing, indicating that an EGFR-independent mechanism exists. Increased phosphorylation of p38 mitogen-activated protein kinase and JNK by gefitinib was observed in a dose-dependent manner in HaCaT cells. The JNK inhibitor, SP600125, attenuated the gefitinib-induced cytotoxicity and apoptosis of HaCaT cells. Immunohistochemical examination of patient specimens showed an increased expression of phosphorylated JNK in lesional epidermis compared with nonlesional epidermis. CONCLUSIONS: Gefitinib can induce keratinocyte apoptosis through an EGFR-independent JNK activation pathway.

Does the Internet assist clients to carry out contact tracing? A randomized controlled trial using web-based information.

The primary outcome was to determine the acceptability of the Internet, specifically a website for use in standard partner notification. A secondary objective was to determine if a website would enhance the outcomes of currently used partner notification methods. In a randomized control trial, 105 participants diagnosed with chlamydia and non-gonoccocal urethritis (NGU) were randomized and 97 completed the study. About 30% of participants were provided a standard partner letter and 70% were provided the standard partner letter and website. All participants reported that their partners had no objections to the website (0%, 95% confidence interval [CI] 0-5%). There were no complaints received from any partner. The odds ratio for contacting partners was not significantly different between the two groups of participants. The results of this study indicate that providing a website with specific information about the infection to which a partner has been exposed is not harmful.

Quantitative studies of Mn(2+)-promoted specific and non-specific cleavages of a large RNA: Mn(2+)-GAAA ribozymes and the evolution of small ribozymes.

Manganese (Mn(2+)) promotes specific cleavage at two major (I and III) and four minor (II, IV, V and VI) sites, in addition to slow non-specific cleavage, in a 659-nucleotide RNA containing the Cr.LSU group I intron. The specific cleavages occurred between G and AAA sequences and thus can be considered Mn(2+)-GAAA ribozymes. We have estimated rates of specific and non-specific cleavages under different conditions. Comparisons of the rates of major-specific and background cleavages gave a maximal specificity of approximately 900 for GAAA cleavage. Both specific and non-specific cleavages showed hyperbolic kinetics and there was no evidence of cooperativity with Mn(2+) concentration. Interestingly, at site III, Mg(2+) alone promoted weak, but the same specific cleavage as Mn(2+). When added with Mn(2+), Mg(2+) had a synergistic effect on cleavage at site III, but inhibited cleavage at the other sites. Mn(2+) cleavage at site III also exhibited lower values of K (Mn(2+) requirement), pH-dependency and activation energy than did cleavage at the other sites. In contrast, the pH-dependency and activation energy for cleavage at site I was similar to non-specific cleavage. These results increase our understanding of the Mn(2+)-GAAA ribozyme. The implications for evolution of small ribozymes are also discussed.

Epidermal growth factor inhibits follicular response to human chorionic gonadotropin: possible role of cell to cell communication in the response to gonadotropin.

Epidermal growth factor (EGF) affects follicular steroidogenesis and expression of gonadotropin receptors. The effects of EGF on hCG-induced estradiol and progesterone secretion and ovulation were examined in the in vitro perfused rabbit ovary. We also examined the effects of EGF on hCG-induced progesterone secretion by isolated granulosa cells. In addition, distribution of hCG within the follicle was probed by immunohistochemical means 30 min after its administration to the in vitro perfused ovary. EGF significantly (P less than 0.05) reduced hCG-induced secretion of estradiol (control, 117 +/- 12 pg/min.follicle; 10 ng/ml EGF, 55 +/- 10) and progesterone (control, 18.2 +/- 1.2 ng/min.follicle; 10 ng/ml EGF, 11.9 +/- 0.8) by the perfused ovary. In contrast, EGF did not inhibit hCG-induced progesterone secretion by isolated granulosa cells. Ovulatory efficiency (number of ovulated ova per number of mature follicles x 100) when EGF was given 30 min before hCG was reduced dose-dependently from 58.2% with no EGF to 8.3% with 10 ng/ml EGF (P less than 0.001). Ovulation was not inhibited by EGF when it was given 30 min after hCG. Distribution of hCG in the preovulatory follicle was confined to the basement membrane, thecal cell layer, and a small fraction of the outer granulosa cell layer. These observations suggest that gonadotropin stimulates the follicle through the release of a secondary signal(s) from ligand-bound granulosa cells near the follicle wall to unexposed cells of the inner avascular area. EGF may inhibit the follicular response to hCG by attenuation of this cell to cell communication.

Extracellular metalloprotease gene of Streptomyces cacaoi: structure, nucleotide sequence and characterization of the cloned gene product.

The gene (npr) encoding an extracellular neutral metalloprotease (Npr) from Streptomyces cacaoi YM15 was cloned in Streptomyces lividans using pIJ702 as a vector. The nucleotide (nt) sequence of npr was determined. The deduced open reading frame encoded 550 amino acids (aa) (60 kDa) with a putative signal sequence of 34 aa at the N terminus. High-resolution S1 mapping identified the transcriptional start point at about 132-134 nt upstream from the start codon. The nt sequences at both -10 and -35 regions resemble the consensus sequence of typical Escherichia coli promoters and a fragment containing the promoter was functional in an E. coli promoter probe plasmid. In vitro transcription and translation of the cloned npr sequence revealed a 60-kDa protein product, correlated with the sequence data but not with the size (35 kDa) of the extracellular Npr. The N-terminal aa sequence in conjunction with the aa composition analyses on the purified mature Npr led to the conclusion that it was processed from the 60-kDa pre-proenzyme form encoded by npr. The Npr protease contained putative zinc ligand-binding regions and two repeated motifs, Asp-Ser-Gly, similar to the active site residues of the aspartic acid and retroviral proteases.

Are acoustic neuromas encapsulated tumors?

In articles and chapters on the subject of acoustic neuroma, it is almost invariably stated that they are well-encapsulated tumors. During surgical procedures, blunt mechanical dissection defines a natural subsurface cleavage plane that leaves intact a several millimeter thick rind of tumor surface. Occasionally, as a concession to neural integrity, less than complete resection is elected, leaving behind this "capsular" remnant. To clarify the nature of the surface of acoustic neuromas and to test whether this long held description is indeed correct, a microscopic analysis of 10 surgical specimens was performed. A wedge was harvested from the free surface of the tumor in the mid cerebellopontine angle that included a large, undisturbed section of the tumor surface. Histologic analysis showed that for most of the tumor surface only an extremely thin (3 to 5 microm) layer of connective tissue envelops the tumor. Neoplastic Schwann cells, which extend essentially to the margin of the tumor, were found to be somewhat flattened and compressed in the vicinity of the surface. Although acoustic neuromas are surrounded by a continuous layer of connective tissue, it is so exceptionally thin (on average less than the diameter of a red blood cell) that its edge cannot be visualized intraoperatively by a surgeon. Because the pathologic definition of a capsule is a thick, enveloping layer of connective tissue that is both micro- and macroscopically evident, it must be concluded that acoustic neuromas are nonencapsulated, at least in the conventional sense of the term. The surface peel observed intraoperatively is surgically produced during tumor debulking by cleaving of the looser central component from the more compressed portion of neoplastic cells that lies immediately beneath the free margin of the lesion.

Percutaneous epididymal sperm aspiration versus microsurgical epididymal sperm aspiration for irreparable obstructive azoospermia--experience with 100 cases.

PURPOSE: This study investigated the sperm retrieval success rates, fertilization rates, pregnancy rates, and complications of percutaneous epididymal sperm aspiration (PESA) and microsurgical epididymal sperm aspiration (MESA) in cases of irreparable obstructive azoospermia. METHODS: During a period of 36 months, 100 men with irreparable obstructive azoospermia underwent 109 cycles of sperm retrieval procedures and intracytoplasmic sperm injection (ICSI). We routinely performed PESA first in each retrieval cycle; MESA and/or testicular sperm extraction (TESE) were performed if PESA failed. The sperm retrieval success rates, mean fertilization rates, and pregnancy rates of PESA and MESA were evaluated. RESULTS: PESA was performed in all 109 retrieval cycles with a successful sperm retrieval rate of 61%. When PESA failed to retrieve a sufficient number of viable sperm, MESA was subsequently performed with a sperm retrieval rate of 93%. Three cases, which had failed retrieval with both the PESA and MESA procedures, received TESE successfully. The rates of fertilization and pregnancy were 56% and 39% in the 66 PESA-ICSI cycles, respectively, and 47% and 45% in the 40 MESA-ICSI cycles. No significant differences were found in fertilization rates or pregnancy rates among the various sperm retrieval methods and obstruction etiologies. The overall mean fertilization rate and pregnancy rate were 51% and 41%, respectively. CONCLUSION: Both PESA and MESA can be used successfully to obtain sufficient sperm for ICSI. PESA cannot replace MESA in some cases as some epididymal pathologies prevent its success. The results of this study indicate that PESA should be the treatment of choice for patients with ductal obstruction distal to the epididymis, owing to its higher initial success rate. In contrast, patients with irreparable epididymal obstruction might achieve better success rates with MESA.

Coamplification of the ZFX and ZFY genes for sex identification in preimplantation embryos.

Knowledge of the sex of an embryo may be particularly useful for couples who have a high risk of producing offspring with inherited genetic disorders. We present a rapid and reliable nested polymerase chain reaction strategy to simultaneously amplify the ZFX and ZFY genes at the single cell level. Forty single blastomeres isolated from six triploid preembryos were subjected to coamplification of ZFX and ZFY genes. The results obtained from the preembryo were consistent with the assigned genotype. The amplification rate was 80% for ZFX and 84% for ZFY. Our strategy can be applied to preimplantation diagnosis of single gene disorders, and is especially useful for preimplantation diagnosis and prevention of X-linked diseases in in vitro fertilization programs.

Activation of functionally protective K(+) channels by methylmercury in rat alveolar macrophages.

Methylmercury (MeHg) is generally known as a neurotoxic heavy metal while its effect on alveolar macrophages is still rarely studied. In this paper, we attempted to use whole cell and cell-attached patch-clamp recording technique and fura-2 fluorescence measurement to elucidate the effects of MeHg on rat alveolar macrophages. The results showed that extracellular application of MeHg induced a transient outward current I(O)(MeHg), 10-20 s in duration, 100-1000 pA in amplitude at -40 mV associated with a marked increase in conductance. The reversal potential depended distinctly on the external K(+) concentration. Removal of external Ca(2+) as well as bath applied verapamil caused a depression of I(O)(MeHg), and intracellular dialysis with 5 mM EGTA completely abolished I(O)(MeHg). Heparin (5 mg/ml) applied by intracellular dialysis greatly accelerated a run-down of I(O)(MeHg) induced by pressure ejection of MeHg. K(+) channel blockers such as quinine, and 4-aminopyridine especially low concentrations of dequalinium and apamin, but not tetraethylammonium inhibited I(O)(MeHg). Cell-attached single-channel recordings with the pipette solution containing 145 mM KCl revealed that the activation of single-channel currents with a conductance of 12 pS could be induced by application of MeHg outside the patch. Since MeHg increased [Ca(2+)](i), in a concentration-dependent manner which was partially blocked by either verapamil or Ca(2+)-free medium containing 1 mM EGTA, it is concluded that MeHg activates a Ca(2+)-dependent K(+) conductance by an increase of [Ca(2+)](i) through an influx from outside the cells as well as mobilization from intracellular store. A possibility that this membrane hyperpolarizing K(+) current may exhibit a functioning modulator in response to the harmful cytotoxic increase in [Ca(2+)](i) caused by MeHg was tested. Accordingly, this working hypothesis is verified by an increase of MeHg-induced cytotoxicity of cultured rat alveolar macrophages through a blockade of this Ca(2+)-activated K(+) channel by dequalinium.

[Pre- and intra-operative administration of epidural morphine provides good postoperative pain relief after laminectomy].

BACKGROUND: To evaluate the postoperative analgesic effect of epidural morphine administered at different timing in lumbar spine surgery. METHODS: Eighty-four patients who were scheduled for elective lumbar spine surgery were randomized in three groups. Seventeen patients in group I who received non-steroid analgesics postoperatively (diclophenac sodium 50 mg, iv, q4h) served as control while thirty-six patients in group II who received single dose epidural morphine 3 mg in combination with 10 ml 2% lidocaine given at the lesion site (L4-5 or L5-S1) just before general anesthesia and thirty-one patients in group III who received 3 mg morphine in combination with 3 ml 2% lidocaine administered to the targeted epidural space by means of slow drippings just before wound closure were studied subjects. RESULTS: During the first 24 h postoperatively, the patients in group II and group III suffered a pain which was significantly less in intensity as compared with those in group I (p < 0.05). We used the 10 cm visual analog pain score (VAS) to scale post-operative pain with "no pain" and "worst pain" respectively anchored at 0 and 10 cm. The incidence of side effects such as pruritus, nausea and vomiting was higher in group II and III than in group I. We did not evaluate the occurrence of urinary retention because routine retention urinary catheterization in all patients hampered us to do so. There were no significant differences in the quality and duration of analgesia between group II and III. Respiratory depression of clinical significance was not observed. Neither decrease in oxygen saturation below 92% registered on pulse oximetry nor decrease in respiratory rate below 12 cycles/min was found in the PACU. CONCLUSIONS: Preoperative or intraoperative administration of epidural morphine could provide satisfactory analgesia in lumbar spine surgery during the first 24 h postoperatively.

The influence of methylmercury on the nitric oxide production of alveolar macrophages.

Mercury is a global pollutant considered to be a persistent bioaccumulative and toxic chemical. Humans may be exposed to organic forms of mercury by either inhalation, oral, or dermal routes. Methylmercury is more toxic to living organisms than the inorganic forms. In this study, we attempted to elucidate the altered functions of alveolar macrophage including nitric oxide production after methylmercury exposure. Treatment of 7 muM methylmercury for 24 h inhibited lipopolysaccharide-induced nitric oxide and nitric oxide synthase production of alveolar macrophages. The addition of H-89 (PKA inhibitor) significantly decreased the methylmercury inhibition of lipopolysaccharide-mediated nitric oxide production. We found the cell had a calcium-dependent adenylate cyclase, and MeHg could inhibit the phosphorylation of extracellular-signal regulated kinase (ERK). Because methylmercury could increase the intracellular calcium ion concentration, it might activate the adenylate cyclase by increasing [Ca(2+)](i). Though the interaction of methylmercury with the immune system has been studied by several investigators, the actual mechanisms underlying these interactions are still poorly understood. We discovered that methylmercury could activate protein kinase A, which in turn would inhibit the activation of Raf-1-ERK and so inhibit the release of nitric oxide.

A kinetically efficient form of the Chlamydomonas self-splicing ribosomal RNA precursor.

The 23S rRNA gene of Chlamydomonas reinhardtii contains a group IA3 intron, Cr.LSU, whose splicing is essential for cell growth. To better understand Cr.LSU splicing, kinetic analyses were undertaken with 23S.3, a preRNA previously shown to self-splice. Self-splicing of 23S.3 showed biphasic kinetics, with only approximately 33% reacting efficiently. Removal of a region of the 5' exon that could potentially interfere with the intron core (i.e., P3) increased the size (53%) of the active fraction. Replacement of the large P6a-extension by a 20-nt stem-loop further increased the active fraction to approximately 80%. The k(cat) and K(G)(M) for self-splicing (first step) by these latter RNAs were approximately 1 min(-1) and approximately 20 microM, respectively. These results indicate that Cr.LSU is a highly efficient ribozyme whose folding in vitro is impeded by exonic and/or intronic sequences. The implications for in vivo splicing of Cr.LSU are discussed.

Evolution and assembly of an extremely scrambled gene.

The process of gene unscrambling in hypotrichous ciliates represents one of nature's ingenious solutions to the problem of gene assembly. With some essential genes scrambled in as many as 51 pieces, these ciliates rely on sequence and structural cues to rebuild their fragmented genes and genomes. Here we report the complex pattern of scrambling in the DNA polymerase alpha gene of Stylonychia lemnae. The germline (micronuclear) copy of this gene is broken into 48 pieces with 47 dispersed over two loci, with no asymmetry in the placement of coding segments on either strand. Direct repeats present at the boundaries between coding and noncoding sequences provide pointers to help guide assembly of the functional (macronuclear) gene. We investigate the evolution of this complex gene in three hypotrichous species.

A comparison of umbilical venous blood levels of neuropeptide Y and catecholamines between cesarean section and normal spontaneous delivery.

This study was designed to determine the presence and different levels of placental neuropeptide Y (NPY) in women at parturition between normal spontaneous delivery (NSD) and cesarean section (C/S) under spinal anesthesia and to compare it with the level of catecholamines. Umbilical vein plasma levels of neuropeptide Y and catecholamines of the placental cord were measured at delivery in 40 parturient women who were divided into two groups, with 20 patients in each. NSD group underwent vaginal delivery without the presence of intrapartum fetal distress. C/S group received elective cesarean section (C/S) under spinal anesthesia. The results showed that umbilical vein level of NPY in NSD patients was significantly less than C/S patients (160.32 pg/ml vs 346.25 pg/ml, p less than 0.01). But for catecholamines levels, C/S group were significantly less than that of NSD group.