Patient-reported rates of chronic pain and recurrence after groin hernia repair.

BACKGROUND: The effectiveness of different procedures in routine surgical practice for hernia repair with respect to chronic postoperative pain and reoperation rates is not clear. METHODS: This was prospective cohort study based on a unique combination of patient-reported outcomes and national registry data. Virtually all patients with a groin hernia repair in Sweden between September 2012 and April 2015 were sent a questionnaire 1 year after surgery. Persistent pain, defined as at least 'pain present, cannot be ignored, and interferes with concentration on everyday activities' in the past week was the primary outcome. Reoperation for recurrence recorded in the register was the secondary outcome. RESULTS: In total, 22 917 patients (response rate 75·5 per cent) who had an elective unilateral groin hernia repair were analysed. Persistent pain present 1 year after hernia repair was reported by 15·2 per cent of patients. The risk was least for endoscopic total extraperitoneal (TEP) repair (adjusted odds ratio (OR) 0·84, 95 per cent c.i. 0·74 to 0·96), compared with open anterior mesh repair. TEP repair had an increased risk of reoperation for recurrence (adjusted OR 2·14, 1·52 to 2·98), as did open preperitoneal mesh repair (adjusted OR 2·34, 1·42 to 3·71) at 2·5-year follow-up. No other methods of repair differed significantly from open anterior mesh repair. CONCLUSION: The risk of significant pain 1 year after groin hernia repair in routine surgical practice was 15·2 per cent. This figure was lower in patients who had surgery by an endoscopic technique, but at the price of a significantly higher risk of reoperation for recurrence.

Effects of a large northern European no-take zone on flatfish populations.

In March 2006, a 360 km² no-take zone (NTZ) was established north of Gotland in the central Baltic Sea, with the purpose to scientifically evaluate the effects of a fishing ban on flatfish populations. A monitoring programme was set up to study the populations in the NTZ and in a reference area east of Gotland where the fishing pressure was high. The programme included fishing with multimesh survey nets, modelling of potential larval export and estimation of fish consumption by large marine predators. Overall, the results showed a clear positive effect of the NTZ on turbot Scophthalmus maximus, with higher densities in the closed area compared with the fished area and also higher densities after closure compared with before. The NTZ also had older individuals and a more even sex ratio. This, in combination with a high potential for larval export from the NTZ to Gotland, shows that the marine reserve may be important for maintaining a viable S. maximus stock at Gotland. Also, for flounder Platichthys flesus, the densities were higher in the NTZ compared to the reference area and there was a net larval export to the fished area. For both species, density-dependent growth was evident, with a lower length at age in the closed area. Potential predation by grey seal Halichoerus grypus and great cormorant Phalacrocorax carbo sinesis on flatfishes, that could hamper the evaluation of the marine reserve, was also addressed. Taken together, the results show that there are clear benefits of the fishing ban for both flatfish species within the NTZ, while the net effects on fisheries are difficult to quantify.

A mutation in PRKAG3 associated with excess glycogen content in pig skeletal muscle.

A high proportion of purebred Hampshire pigs carries the dominant RN- mutation, which causes high glycogen content in skeletal muscle. The mutation has beneficial effects on meat content but detrimental effects on processing yield. Here, it is shown that the mutation is a nonconservative substitution (R200Q) in the PRKAG3 gene, which encodes a muscle-specific isoform of the regulatory gamma subunit of adenosine monophosphate-activated protein kinase (AMPK). Loss-of-function mutations in the homologous gene in yeast (SNF4) cause defects in glucose metabolism, including glycogen storage. Further analysis of the PRKAG3 signaling pathway may provide insights into muscle physiology as well as the pathogenesis of noninsulin-dependent diabetes mellitus in humans, a metabolic disorder associated with impaired glycogen synthesis.

Short- and long-term effects of immunization against gonadotropin-releasing hormone, using Improvac, on sexual maturity, reproductive organs and sperm morphology in male pigs.

The objective of this study was to determine the short and long term effects of a gonadotropin-releasing hormone (GnRH) vaccine (Improvac Pfizer Ltd.), on sexual maturity, development of the reproductive organs, and the morphology of caudal epididymal spermatozoa in non-castrated male pigs. The pigs were slaughtered 4, 16 or 22 weeks after the second Improvac vaccination. A total of 80 crossbred non-castrated male pigs were included in this study comprising two experiments, a short-effect (Experiment 1) and a long-effect (Experiment 2). The first experiment included 56 pigs, 24 of them were maintained as controls and 32 were vaccinated twice, and slaughtered 4 weeks after the second vaccination. The second experiment included 24 pigs, 12 controls and 12 vaccinated twice, and slaughtered either 16 weeks (n=6) or 22 weeks (n=6) after the second vaccination. None of the immunized pigs was sexually mature at slaughter, i.e. 4, 16 or 22 weeks after second vaccination. Corresponding results of the control pigs showed that 50% had reached sexual maturity at the age corresponding to 4 weeks after the second vaccination, and 100% at slaughter 16, respectively, 22 weeks after vaccination. At 4, 16 and 22 weeks after second vaccination both testes weight and bulbourethral length were significantly reduced (p<0.001). The percentages of proximal droplets and abnormal heads were significantly lower in the control pigs than in the immunized pigs at slaughter 4 weeks after vaccination, whereas distal droplets were higher. For the other morphological parameters no significant differences were seen, but all mean values except for acrosome defects were numerically lower in the control pigs compared with the immunized pigs. For pigs slaughtered 16 or 22 weeks after vaccination, the vaccination effect was significant for percentages of proximal droplets, distal droplets, acrosome defects, acrosome abnormality and abnormal heads (p=0.017-0.001). The immunization clearly disrupted the number and morphology of the interstitial Leydig cells, lasting throughout the study period (4-22 weeks after vaccination). Spermatogenesis was also clearly affected in the immunized pigs, to various degrees, from mild disruption (spermatocyte loss, decrease of the normal number of layers of germ cells) to severe loss of germ cells including tubuli with Sertoli cells-only (complete disappearance of germ cells), also covering the entire study period. The results indicated that the effect of immunization persisted for at least 22 weeks after the second vaccination.

The effect of a c.-8G>T polymorphism on the expression of cytochrome b5A and boar taint in pigs.

The level of cytochrome b5A (CYB5A) in pig testis is correlated with boar taint from androstenone and an AF016388:c.-8G>T polymorphism in CYB5A has been linked with low androstenone levels in the fat of pigs. In this study, we developed a polymerase chain reaction-based assay to genotype 1242 boars from eight lines for the c.-8G>T SNP. The c.-8T allele was found in all eight lines at a frequency ranging from 1.8% to 20.3% with an overall frequency of 8.6%. Significant deviations from Hardy-Weinberg equilibrium were found in the Hampshire, Landrace and Yorkshire breeds. The homozygous mutant c.-8TT occurred infrequently and was not found in some lines, but was consistently associated with low androstenone levels in fat. Both CYB5A mRNA and CYB5A protein levels were decreased in the c.-8TT genotype in a subset of Yorkshire boars, suggesting that low levels of CYB5A protein in the c.-8TT mutant were not due to inefficient translation of CYB5A mRNA. There were significant but modest marker effects on fat androstenone levels in Landrace, Yorkshire and a Large White/Duroc cross and fat skatole in Duroc and Sire Line breeds. There was no effect of CYB5A genotype on bulbourethral gland length, suggesting that this SNP will not affect reproductive traits. We conclude that the c.-8G>T SNP in the CYB5A gene has a significant but modest effect on boar taint in male pigs and could be useful in some breeds as part of a panel of SNP markers in a marker-assisted selection programme to produce low boar taint pigs.

Effect of a gonadotropin-releasing hormone vaccine (Improvac) on steroid hormones, boar taint compounds and performance in entire male pigs.

The objective of this study, comprising two trials, was to evaluate the effect of a gonadotropin-releasing hormone (GnRH [corrected] vaccine Improvac; Pfizer Ltd) in a sample of the Swedish pig population. The pigs (n = 120) were assigned to three groups: control (entire male pigs), surgical castration and immunization against GnRH. Surgically castrated pigs did not express detectable levels of either testosterone or estrone sulphate (E1S) in plasma, or androstenone in fat and had lower skatole and indole levels in fat than entire male pigs. Immunization significantly reduced testes weight and bulbourethral gland length, plasma levels of the testicular hormones testosterone and E1S, and fat levels of androstenone, skatole and indole. Skatole levels in plasma were significantly lower than in entire male pigs in the second trial, but not in the first due to overall low skatole levels. All immunized pigs and surgically castrated pigs expressed skatole concentrations in fat below the level of 0.2 microg/g, above which meat is regarded as tainted. In contrast, eight entire male pigs exceeded this level. Indole levels in plasma from immunized pigs were lower than those from entire male pigs. Surgical castration caused lower daily weight gain in the suckling period compared with piglets raised intact, whereas in the post-weaning period no difference was observed. Immunization resulted in higher feed intake and daily weight gain after the second injection. The estimated lean meat content was improved in comparison with the castrated pigs, but was lower than for entire male pigs. Dressing percentage was lower in immunized pigs than in surgically castrated and entire male pigs. The frequency of skin damage did not differ between immunized and entire male pigs or between immunized and surgically castrated pigs.

Effect of freezing on sensory quality, shear force and water loss in beef M. longissimus dorsi.

The objective of this study was to determine how sensory quality, shear force and water loss differ between beef stored either chilled or frozen before cooking. Meat tenderness was analysed instrumentally and sensorially using both a consumer panel and a semi-trained panel. Both M. longissimus dorsi (LD) from eight young Holstein bulls were cut into eight samples, weighed, vacuum packed and aged at 4°C for 2, 7 or 14 days. After ageing, the frozen samples were kept at -20°C prior to heat treatment. Water holding capacity was recorded as purge or thawing loss and cooking loss or as combined loss. Sensory analyses were performed on samples aged 7 days. Peak force values declined with ageing time and freezing. Frozen meat aged 2 days had the same peak force values as chilled meat aged 7 days. Total energy was the same for both treatments at day 2 and 7, whereas at day 14 frozen samples showed significantly higher values than chilled samples. The sensory panel experienced the chilled meat to be more tender, juicier and having a more intense meat taste than the frozen meat, whereas the consumers could not find any significant difference in degree of liking. Water holding capacity was lower for the frozen samples. The results indicate that conclusions from studies concerning sensory quality of beef will depend on whether the meat has been kept chilled or frozen before testing.

Review on Medusa:a polymer-based sustained release technology for protein and peptide drugs.

The polymer-based Medusa system (Flamel Technologies) has been designed for slow release of therapeutic proteins and peptides. The Medusa II consists of a poly L-glutamate backbone grafted with hydrophobic alpha-tocopherol molecules, creating a colloidal suspension of nanoparticles (10 - 50 nm) in water. The sustained drug release is based on reversible drug interactions with hydrophobic nanodomains within the nanoparticles. In vivo, it is suggested that the therapeutic protein is displaced by endogenous proteins present in physiological fluids, leading to a slow drug release. The peak concentration is dramatically decreased and the protein release substantially extended. The Medusa technology has been applied to subcutaneous injection for several therapeutic proteins, such as IL-2 and IFN-alpha(2b), in animal models (rats, dogs, monkeys) and clinical trials in renal cancer (IL-2) and hepatitis C (IFN-alpha(2b)) patients.

Effect of testicular steroids on catalytic activities of cytochrome P450 enzymes in porcine liver microsomes.

The testicular steroids androstenone (A), 17beta-oestradiol (E2) and testosterone (T) were tested for their ability to alter CYP2E1 and CYP2A activity in porcine liver microsomes from male and female pigs. This is the first in vitro study indicating that sex steroids have a potential to modify microsomal CYP2E1 activity, the main skatole-metabolising enzyme. A and E2 exerted an inhibitory effect on CYP2E1 mediated hydroxylation of p-nitrophenol to p-nitrocatechol although the mechanism of this inhibition differed for these steroids. The inhibitory effect of A on CYP2E1, as determined by kinetic analysis, might be due to the competitive binding of A and p-nitrophenol to the same site of CYP2E1. Including E2 into the incubations resulted in decreased activities of CYP2E1 in male microsomes through a mixed mode of inhibition. Including pre-incubation steps eliminated this inhibition in male microsomes, and resulted in increased CYP2E1 activities in the microsomes from female pigs. Testosterone was ineffective as an inhibitor of either CYP2E1 or CYP2A activities. Overall, our findings indicate that A and E2 have the potential to modify the catalytic activities of porcine CYP2E1 in vitro. However, the significance of this modification for skatole metabolism in vivo is questionable.

Effects of sex, weight, diet and hCG administration on levels of skatole and indole in the liver and hepatic activities of cytochromes P4502E1 and P4502A6 in pigs.

Cytochromes P4502E1 (CYP2E1) and P4502A6 (CYP2A6) catalyse metabolic reactions of skatole and indole metabolism. The objectives of this study were as follows: to evaluate whether activities of CYP2E1 and CYP2A6 in pigs of two live weights (LW) differ between males and females; to investigate whether activities of CYP2E1 and CYP2A6 are affected by hCG stimulation; and to investigate whether the levels of skatole and indole in the liver and the activities of CYP2E1 and CYP2A6 are affected by raw potato starch (RPS). Female pigs expressed higher CYP2A6 activity at 90kg LW, and higher CYP2E1 activity at 115kg LW compared to male pigs. Skatole levels in the liver were higher in male pigs than in female pigs at both LW, whereas indole levels were higher in males only at 115 kg LW. Neither levels of indolic compounds in the liver nor enzyme activities were affected by hCG stimulation. The inclusion of RPS in the diet reduced skatole levels in the liver in both sexes and increased CYP2A6 activity in female pigs. It was concluded that the incidence of boar taint may depend on both skatole amount, which reach the liver, and the activities of enzymes involved in skatole metabolism, which may vary depending on sex, live weight, and diet.

Effect of hCG administration on the relationship between testicular steroids and indolic compounds in fat and plasma in entire male pigs.

Our objective was to evaluate pigs injected with human chorionic gonadotropin (hCG) as a potential model to study the effect of high testicular steroid levels on the variation in indolic compounds. Pre-selection of the sires based on their plasma skatole levels was used, to evaluate whether the response to hCG would be affected. Out of 34 entire male pigs, 17 pigs were injected with hCG and the remaining pigs were injected with sterile saline four days prior to slaughter. HCG injection increased the levels of testicular steroids (androstenone, testosterone, dehydroepiandrosterone sulphate, oestrone sulphate and oestrone; p<0.001), and indole levels in plasma and fat (p<0.05). Skatole levels in fat increased slightly but not significantly (p=0.107), whereas skatole levels in plasma were not affected. The correlation coefficients between androstenone and skatole levels in fat were 0.54 in the hCG-injected group and non-significant in the control group. Skatole levels in fat were not correlated with testicular steroids in either hCG-injected pigs or controls. Skatole and indole levels in fat were positively correlated in both groups. We concluded that hCG-injected pigs might be useful for studying factors responsible for the role of testicular steroids in the occurrence of high indole levels. Pre-selection of sires based on plasma skatole levels affected both the levels of indolic compounds and testicular steroids, especially in fat, which might have suppressed the response to hCG. Whether hCG affects skatole or not is therefore not fully elucidated.

Skatole metabolites in urine as a biological marker of pigs with enhanced hepatic metabolism.

Boar taint is a quality defect in meat, related to accumulation of skatole and androstenone in male pigs. The levels of skatole and its main metabolites in plasma and urine samples were measured with a validated liquid chromatography-MS method and related to activity of hepatic cytochrome P450 (CYP450) in order to identify 'fast metabolizing' pigs. Urine (n=46), blood (n=12), liver (n=25) and adipose tissue (n=46) were sampled from a total of 46 entire male pigs. Skatole levels in fat were negatively correlated to CYP2E1 activity and positively to 3-hydroxy-3-methyloxindole (HMOI), indole-3-carboxylic acid (ICA) and 2-aminoacetophenone in urine. HMOI and ICA levels in urine were the best predictors of high skatole levels in fat. In summary, the present study provided further evidence for the key role of CYP2E1 in skatole metabolism and suggested that measurement of HMOI and/or ICA in urine might provide information about skatole levels in live pigs.

Expression of enzymatically active rat liver and human placental catechol-O-methyltransferase in Escherichia coli; purification and partial characterization of the enzyme.

To produce sufficient amounts of recombinant catechol-O-methyltransferase (COMT) for structural and functional studies the coding regions of the rat liver and human placental COMT genes have been introduced into a bacterial expression vector pKEX14. Recombinant COMT was produced in Escherichia coli up to 10% of total bacterial protein after the induction of the T7 RNA polymerase gene with isopropyl-beta-D-thiogalactopyranoside. Both the rat and human enzymes were enzymatically active, soluble and reacted with anti-COMT antiserum in Western blotting. Both enzymes were purified from E. coli cells and partially characterized by determining their specific activity, apparent molecular weight and pI.

Multiple marker mapping of quantitative trait loci in a cross between outbred wild boar and large white pigs.

A quantitative trait locus (QTL) analysis of growth and fatness data from a three generation pig experiment is presented. The population of 199 F2 animals was derived from a cross between wild boar and Large White pigs. Animals were typed for 240 markers spanning 23 Morgans of 18 autosomes and the X chromosome. A series of analyses are presented within a least squares framework. First, these identify chromosomes containing loci controlling trait variation and subsequently attempt to map QTLs to locations within chromosomes. This population gives evidence for a large QTL affecting back fat and another for abdominal fat segregating on chromosome 4. The best locations for these QTLs are within 4 cM of each other and, hence, this is likely to be a single QTL affecting both traits. The allele inherited from the wild boar causes an increase in fat deposition. A QTL for intestinal length was also located in the same region on chromosome 4 and could be the same QTL with pleiotropic effects. Significant effects, owing to multiple QTLs, for intestinal length were identified on chromosomes 3 and 5. A single QTL affecting growth rate to 30 kg was located on chromosome 13 such that the Large White allele increased early growth rate, another QTL on chromosome 10 affected growth rate from 30 to 70 kg and another on chromosome 4 affected growth rate to 70 kg.

Alphaviruses as expression vectors.

Alphavirus vectors have been used for efficient high-level expression of a variety of topologically different proteins, allowing studies of protein transport, localization and functional activity in a broad range of host cells. Complex transmembrane proteins have been produced in large quantities through the establishment of scale-up technology. Alphavirus vectors have also shown promising potential in vaccine production and preliminary gene therapy applications.

Chemical synthesis and molecular cloning of a STOP oligonucleotide encoding an UGA translation terminator in all three reading frames.

We have chemically synthesized an oligonucleotide 5'd(TGATTGATTGA)3' 3'd(ACTAACTAACT)5' that encodes the translation termination codon TGA in all three reading frames. After ligation of appropriate restriction endonuclease linkers to the ends, the double-stranded oligonucleotide (STOP-oligonucleotide) was joined to the plasmid pBR322 between the EcoRI and BamHI, or HindIII and BamHI sites, and the hybrid plasmids were transformed into Escherichia coli HB101. Four different constructions were obtained: (i) EcoRI-STOP-BamHI (STOP-oligonucleotide flanked by EcoRI and BamHI linkers; pKTH606), (ii) HindIII-STOP-BamHI (pKTH601), (iii) BamHI-STOP-HindIII (pKTH604), and (iv) HindIII-STOP-POTS-BamHI (two STOP-oligonucleotides in opposite orientation; pKTH605). The inserts in pKTH606 and pKTH601 were excised and transferred to a modified plasmid constructed previously for the expression and secretion of foreign gene products from Bacillus subtilis. The resulting secretion plasmids now contain the promoter/signal sequence region of the alpha-amylase gene from Bacillus amyloliquefaciens joined to the STOP-oligonucleotide by EcoRI or HindIII linkers. Foreign genes can be cloned into these sites. The plasmids can be used to express foreign genes truncated at their C-terminal end and therefore lacking their own translation termination codon. One such plasmid has been successfully used to express the Semliki Forest virus (SFV) membrane protein E1 truncated at its C-terminus.

Molecular cloning and characterization of rat liver catechol-O-methyltransferase.

The coding sequence of rat liver catechol-O-methyl-transferase (COMT; EC 2.1.1.6) was determined from rat cDNA and genomic libraries were screened with DNA probes and specific antiserum. The open reading frame consisted of 663 nucleotides coding for a 221-amino acid (aa) polypeptide with a deduced Mr of 24,747. No obvious hydrophobic signal sequence, membrane-spanning domains, or potential N-glycosylation sites were found in this sequence. The identity of the clone and the accuracy of the sequence was verified by direct aa sequencing of the tryptic peptides derived from the purified rat liver enzyme. Primer extension analysis showed that the transcription start point of the rat liver COMT mRNA was 450 bp upstream from the translation start codon. A putative polyadenylation signal (ATTAAA) was found in the 3'-noncoding region. The predicted size of the COMT transcript was 1.8-2.0 kb, which could be confirmed from Northern hybridization analyses of the isolated rat liver mRNA. One polypeptide of 25 kDa, could be immunoprecipitated with anti-COMT antibody from in vitro translation of rat liver mRNA. Employing the DNA blot analysis only one COMT-encoding gene was found in the rat genome.

Semliki Forest virus vectors: efficient vehicles for in vitro and in vivo gene delivery.

Rapidly generated high-titer Semliki Forest virus (SFV) vectors can infect numerous mammalian cell lines and primary cell cultures, and result in high levels of transgene expression. SFV-based expression of transmembrane receptors has been characterized by specific ligand-binding activity and functional responses. Adaptation of the SFV technology for mammalian suspension cultures has allowed the production of hundreds of milligrams of recombinant receptor for purification and structural studies. The same SFV stock solutions used for the infection of mammalian cells in culture have also been successfully applied for efficient transgene expression in organotypic hippocampal slices, as well as in vivo in rodent brain.

Alphavirus vectors for gene therapy applications.

High-titer alphavirus vectors have been generated for efficient gene delivery both in vitro and in vivo. Studies on CNS infection via intranasal and peripheral injections with virulent and avirulent replication-competent Semliki Forest virus (SFV) strains has demonstrated the potential of gene delivery. Replication-deficient alphavirus particles have shown high local transgene expression of a transient nature in rodent brain. Alphavirus vectors have been demonstrated to induce apoptosis in infected human tumor cell lines and SFV vectors expressing interleukin-12 resulted in tumor regression in a B16 murine melanoma model. Repeated SFV injections led to stronger anti-tumor effects without immunogenic response detected against SFV. It has also been shown that intra-tumoral SFV-injections into nude mice with implanted human lung carcinomas led to tumor regression. Likewise, injection of replicative SFV-LacZ RNA resulted in tumor response as well as prophylactic protection against tumor formation. Alphaviruses have also showed potential in vaccine production. Additionally, modifications in the envelope structure of Sindbis virus resulted in substantial change in host range and demonstrated the feasibility of targeting alphavirus vectors. Moreover, SFV has been used as an expression vector for the generation of high-titer retrovirus-like particles. Recent alphavirus vector development has introduced novel non-cytopathogenic vectors, tightly temperature-regulated vectors as well as replication-persistent forms that should prolong the duration of expression. Alphavirus vectors can therefore be considered as highly potential gene delivery vehicles for future gene therapy applications, especially where only short-term expression is required, or even preferred.