Scalable methanol-free production of recombinant glucuronoyl esterase in Pichia pastoris.

OBJECTIVE: Glucuronoyl esterase (GE) is an emerging enzyme that improves fractionation of lignin-carbohydrate complexes. However, the commercial availability of GE is limited, which hinders the research of GE-based bioprocesses for its industrial application in lignocellulose biorefineries. This study evaluated a workable, cost-effective, and commercially scalable production strategy to improve the ease of GE-based research. This strategy consisted of a constitutive and methanol-free enzyme production step coupled with a two-step filtration process. The aim was to determine if this strategy can yield copious amounts of GE, by secretion into the extracellular medium with an acceptable purity that could allow its direct application. This approach was further validated for cellobiose dehydrogenase, another emerging lignocellulose degrading enzyme which is scarcely available at high cost. RESULTS: The secreted recombinant enzymes were functionally produced in excess of levels previously reported for constitutive production (1489-2780 mg L-1), and were secreted at moderate to high percentages of the total extracellular protein (51-94%). The constant glycerol feed, implemented during fed-batch fermentation, lead to a decline in growth rate and plateaued productivity. Tangential flow ultrafiltration was used to concentrate cell-free enzyme extracts 5-6-fold, reaching enzyme activity levels (1020-202 U L-1) that could allow their direct application.

Spinal cord stimulation for the management of pain: Recommendations for best clinical practice.

Spinal cord stimulation (SCS) is an accepted method of pain control. SCS has been used for many years and is supported by a substantial evidence base. A multidisciplinary consensus group has been convened to create a guideline for the implementation and execution of an SCS programme for South Africa (SA). This article discusses the evidence and appropriate context of SCS delivery, and makes recommendations for patient selection and appropriate use. The consensus group has also described the possible complications following SCS. This guideline includes a literature review and a summary of controlled clinical trials of SCS. The group notes that, in SA, SCS is performed mainly for painful neuropathies, failed back surgery, and chronic regional pain syndrome. It was noted that SCS is used to treat other conditions such as angina pectoris and ischaemic conditions, which have therefore been included in this guideline. These recommendations give guidance to practitioners delivering this treatment, to those who may wish to refer patients for SCS, and to those who care for patients with stimulators in situ. The recommendations also provide a resource for organisations that fund SCS. This guideline has drawn on the guidelines recently published by the British Pain Society, and parts of which have been reproduced with the society's permission. These recommendations have been produced by a consensus group of relevant healthcare professionals. Opinion from outside the consensus group has been incorporated through consultation with representatives of all groups for whom these recommendations have relevance. The recommendations refer to the current body of evidence relating to SCS. The consensus group wishes to acknowledge and thank the task team of the British Pain Society for their help and input into this document.

The diagnostic performance of the GenoType MTBDRplus version 2 line probe assay is equivalent to that of the Xpert MTB/RIF assay.

Molecular diagnostics for Mycobacterium tuberculosis have recently been endorsed by the World Health Organization. The Xpert MTB/RIF assay was endorsed for use on patient material, regardless of smear gradation, while the GenoType MTBDRplus (version 1) has been limited for use on smear-positive patient material. In this study, we evaluated the diagnostic performance of the Xpert MTB/RIF and GenoType MTBDRplus (version 2) assays on smear-positive and smear-negative patient specimens submitted to a high-throughput diagnostic laboratory. A total of 282 consecutive specimens were subjected to the two new molecular assays, and their performance characteristics were assessed relative to the routine diagnostic standard. Both assays showed similar diagnostic performance characteristics. The sensitivities of the GenoType MTBDRplus (v2.0) and Xpert MTB/RIF assays for the detection of culture-positive M. tuberculosis were 73.1% and 71.2%, respectively, while the specificities of both assays were 100%. Both assays were able to diagnose the presence of M. tuberculosis in 57 to 58% of smear-negative cases, suggesting that the performance characteristics were dependent on bacillary load. The detection of M. tuberculosis in culture-negative specimens confirmed that molecular assays should not be used for treatment monitoring. The sensitivity and specificity for rifampin resistance detection were 100% in both assays; however, the GenoType MTBDRplus (v2.0) assay provided additional information on isoniazid susceptibility. The GenoType MTBDRplus (v2.0) assay will complement the Xpert MTB/RIF screening assay by validating rifampin susceptibility and providing information on isoniazid susceptibility. In addition, the GenoType MTBDRplus (v2.0) assay will provide pharmacogenetic information that may be critical in guiding appropriate treatment.

Mycobacterium tuberculosis population structure determines the outcome of genetics-based second-line drug resistance testing.

The global emergence of multidrug-resistant tuberculosis has highlighted the need for the development of rapid tests to identify resistance to second-line antituberculosis drugs. Resistance to fluoroquinolones and aminoglycosides develops through nonsynonymous single nucleotide polymorphisms in the gyrA and gyrB genes and the rrs gene, respectively. Using DNA sequencing as the gold standard for the detection of mutations conferring resistance, in conjunction with spoligotyping, we demonstrated heteroresistance in 25% and 16.3% of Mycobacterium tuberculosis isolates resistant to ofloxacin and amikacin, respectively. Characterization of follow-up isolates from the same patients showed that the population structure of clones may change during treatment, suggesting different phases in the emergence of resistance. The presence of underlying mutant clones was identified in isolates which failed to show a correlation between phenotypic resistance and mutation in the gyrA or rrs gene. These clones harbored previously described mutations in either the gyrA or rrs gene, suggesting that rare mutations conferring resistance to ofloxacin or amikacin may not be as important as was previously thought. We concluded that the absence of a correlation between genotypic and phenotypic resistance implies an early phase in the emergence of resistance within the patient. Thus, the diagnostic utility of genetics-based drug susceptibility tests will depend on the proportion of patients whose bacilli are in the process of acquiring resistance in the study setting. These data have implications for the interpretation of molecular and microbiological diagnostic tests for patients with drug-susceptible and drug-resistant tuberculosis who fail to respond to treatment and for those with discordant results.

Rise in rifampicin-monoresistant tuberculosis in Western Cape, South Africa.

SETTING: Brewelskloof Hospital, Western Cape, South Africa. OBJECTIVES: To verify the perceived increase in rifampicin monoresistant tuberculosis (RMR-TB) in the Cape Winelands-Overberg region and to identify potential risk factors. DESIGN: A retrospective descriptive study of trends in RMR-TB over a 5-year period (2004-2008), followed by a case-control study of RMR and isoniazid (INH) monoresistant TB cases, diagnosed from April 2007 to March 2009, to assess for risk factors. RESULTS: The total number of RMR-TB cases more than tripled, from 31 in 2004 to 98 in 2008. The calculated doubling time was 1.63 years (95%CI 1.18-2.66). For the assessment of risk factors, 95 RMR-TB cases were objectively verified on genotypic and phenotypic analysis. Of 108 specimens genotypically identified as RMR cases, 13 (12%) were misidentified, multidrug-resistant TB. On multivariate analysis, previous use of antiretroviral therapy (OR 6.4, 95%CI 1.3-31.8), alcohol use (OR 4.8, 95%CI 2.0-11.3) and age ≥ 40 years (OR 5.8, 95%CI 2.4-13.6) were significantly associated with RMR-TB. CONCLUSION: RMR-TB is rapidly increasing in the study setting, particularly among patients with advanced human immunodeficiency virus (HIV) disease. Routine drug susceptibility testing should be considered in all TB-HIV co-infected patients, and absence of INH resistance should be confirmed phenotypically if genotypic RMR-TB is detected.

Dried culture spots for Xpert MTB/RIF external quality assessment: results of a phase 1 pilot study in South Africa.

Implementation of Xpert MTB/RIF requires quality assessment. A pilot program using dried culture spots (DCSs) of inactivated Mycobacterium tuberculosis is described. Of 274 DCS results received, 2.19% generated errors; the remainder yielded 100% correct Mycobacterium tuberculosis detection. The probe A cycle threshold (C(T)) variability of three DCS batches was ≤ 3.47. The study of longer-term DCS stability is ongoing.

inhA promoter mutations: a gateway to extensively drug-resistant tuberculosis in South Africa?

SETTING: Western Cape and Eastern Cape Provinces, South Africa. OBJECTIVE: To assess the potential association between the evolution of extensively drug-resistant (XDR) strains of Mycobacterium tuberculosis and mutations in the inhA promoter or the katG gene. DESIGN: Analysis of the frequency distribution of isoniazid (INH) resistance conferring mutations in a population sample of drug-resistant isolates of M. tuberculosis. RESULTS: In the Western Cape and Eastern Cape Provinces, the percentage of isolates exhibiting inhA promoter mutations increased significantly from respectively 48.4% and 62.4% in multidrug-resistant tuberculosis (MDR-TB) isolates to 85.5% and 91.9% in XDR isolates. Data from the Western Cape revealed that significantly more XDR-TB isolates showed mutations in the inhA promoter than in katG (85.5% vs. 60.9%, P < 0.01), while the respective proportions were equal for INH-resistant non-MDR-TB isolates (∼30%). CONCLUSIONS: inhA promoter mutations are strongly associated with XDR-TB in South Africa. We suggest that this is due to the dual resistance to ethionamide and (low-dose) INH conferred by inhA promoter mutations. The use of molecular probe assays such as the GenoType® MTBDRplus assay, which allows the detection of inhA promoter mutations, could enable treatment regimens to be adjusted depending on the pharmacogenetic properties of the mutations detected.

Drug-resistant tuberculosis epidemic in the Western Cape driven by a virulent Beijing genotype strain.

Temporal analysis of drug-resistant tuberculosis (TB) cases in the Western Cape, South Africa, showed a 1.5-fold increase over a 2-year period, suggesting a doubling time of 8.2 years. This increase was strongly associated with multidrug resistance and the Beijing genotype. Forty-two per cent of the overall increase was due to the Beijing genotype strain R220, suggesting that this strain had evolved unique properties that allowed for both acquisition and transmission of drug resistance. To curb the drug-resistant TB epidemic in this setting, it will be essential to implement rapid diagnostics and efficient infection control measures, improve contact screening and ensure treatment adherence.

Validation of a Rapid Tuberculosis PCR Assay for Detection of MDR-TB Patients in Gauteng; South Africa

Nucleic acid amplification tests offer shorter turnaround times for diagnosis of tuberculosis (TB) and drug resistance of isolates compared to conventional culture methods. The rapid molecular-based multidrug-resistant (MDR)-TB assay; GenoTyper MTBDRplus (Hain Lifescience) was evaluated in Gauteng; South Africa; as a pilot investigation to assess its performance for detection of MDR-TB in patients who were at high risk of drug-resistant TB. A total of 945 sputum specimens sequentially received within a period of six weeks from seven hospitals were assessed by MTBDRplus and compared to liquid culture drug susceptibility tests (DST) using the MGIT 960 system (BD Diagnostic Systems) as the `gold standard'. Of the 945 specimens processed; 731 had interpretable results from both tests and therefore were included in the analysis. The overall sensitivities of the MTBDRplus in detecting individual resistance to rifampicin (RMP) and isoniazid (INH); as well as MDR were 95.0; 93.4and 100; respectively. The specificities were 99.7for RMP; and 100for INH and MDR. The Genotyper MTBDRplus assay showed excellent concordance with the conventional `gold standard' MGIT DST; and it detected all the MDR-TB cases analysed

Traumatic abdominal wall hernia--four cases and a review of the literature.

OBJECTIVE: To review blunt traumatic abdominal wall hernias (TAWHs) in our institution. METHOD: Retrospective review of blunt abdominal trauma cases over a 6-month period. RESULTS: Four patients with TAWH were identified. The mean age was 36 years. Three had been involved in vehicular collisions, and 1 had been assaulted with a large stone. All were diagnosed on presentation, 3 by computed tomography scan and 1 clinically. Two were repaired as emergencies, and 1 was repaired after 4 months. The 4th patient refused surgery. CONCLUSION: This uncommon injury requires a high index of suspicion and a low threshold for intervention. CT scan offers the best imaging potential.

The role of protein kinase A pathway and cAMP responsive element-binding protein in androgen receptor-mediated transcription at the prostate-specific antigen locus.

Androgen-independent prostate cancer is a lethal form of the disease that is marked by metastasis and rapid proliferation in its final stages. As no effective therapy for this aggressive tumor currently exists, it is imperative to elucidate and target the mechanisms involved in the progression to androgen independence. Accumulating evidence indicates that aberrant activation of androgen receptor (AR) via signal transduction pathways, AR gene mutation and/or amplification, and/or coregulator alterations may contribute to the progression of prostate cancer. In the present study, the effects of protein kinase A (PKA) signaling and its downstream factors on AR activity at the prostate-specific antigen (PSA) gene were tested. Activation of PKA by forskolin resulted in enhanced androgen-induced expression of the PSA gene, an effect that was blocked by the AR antagonist, bicalutamide. Interestingly, when either p300 or CBP was overexpressed, PKA activation was sufficient to stimulate PSA promoter-driven transcription in the absence of androgen, which was not inhibited by bicalutamide. PKA activation did not significantly alter AR protein levels but significantly increased the phosphorylated form of its downstream effector, cAMP responsive element-binding protein (CREB) in the presence of androgen. Furthermore, chromatin immunoprecipitation showed that the combination of androgen and forskolin increased phosphorylated CREB occupancy, which was accompanied by histone acetylation, at the putative cAMP responsive element located in the 5' upstream regulatory region of the PSA gene. Remarkably, mammalian two-hybrid assay indicated that p300/CBP may bridge the interaction between AR and CREB, suggesting a novel enhanceosomal cooperation. These results demonstrate an intriguing interplay between a signal transduction pathway, coactivator overexpression and AR signaling as a possible combined mechanism of progression to androgen-independent prostate cancer.

Traumatic abdominal wall hernia - four cases and a review of the literature : trauma

Objective. To review blunt traumatic abdominal wall hernias (TAWHs) in our institution. Method. Retrospective review of blunt abdominal trauma cases over a 6-month period. Results. Four patients with TAWH were identified. The mean age was 36 years. Three had been involved in vehicular collisions; and 1 had been assaulted with a large stone. All were diagnosed on presentation; 3 by computed tomography scan and 1 clinically. Two were repaired as emergencies; and 1 was repaired after 4 months. The 4th patient refused surgery. Conclusion. This uncommon injury requires a high index of suspicion and a low threshold for intervention. CT scan offers the best imaging potential

Eliminating protein from reusable laryngeal mask airways. A study comparing routinely cleaned masks with three alternative cleaning methods.

Laryngeal mask airways (LMAs) have the potential to act as a vector for the transmission of prion diseases. This study was undertaken to define the problem of protein contamination and to investigate three alternative cleaning methods. Forty-eight LMAs were allocated to one of four groups, stained with erythrosin and given a total stain score and a grid stain score in order to determine the degree of protein contamination. Eighteen randomly selected LMAs that had been routinely cleaned and sterilised (group 1) were compared with 12 LMAs that had been washed and scrubbed with the benefit of prior staining (group 2), 13 that had been washed and scrubbed without any visual guide (group 3) and 13 that had been cleaned using a Biosonic ultrasonic cleaning system (group 4). The results show that none of the cleaning methods achieved optimal results, as all methods left proteinaceous material on some masks. The study showed that: (i) staining as a guide to cleaning does not lead to more effective removal of proteinaceous material; (ii) systematic cleaning and scrubbing does lead to more effective removal of proteinaceous material from surfaces other than the grid area; and (iii) ultrasonic cleaning was more effective than other methods of cleaning for the removal of proteinaceous material from those areas of the mask most inaccessible, such as the grid.

Vitamin D receptor polymorphisms and risk of colorectal adenomas (United States).

OBJECTIVE: The purpose of this study was to determine whether vitamin D receptor (VDR) gene polymorphisms influence risk of colorectal adenoma. METHODS: Polymorphisms in the 5' and 3' ends of the VDR gene were genotyped for 373 colorectal adenoma cases and 394 controls. RESULTS: Overall, there was no significant association between the 5' (FokI) or the 3' (BsmI) polymorphisms and adenoma risk. However, risk of large (>1 cm) adenomas decreased with increasing copies of the FokI f allele (p = 0.04). Compared to the FF genotype, odds ratios for the Ff and ff genotypes were 0.79 (95% CI 0.44-1.41) and 0.32 (95% CI 0.11-0.91), respectively. FokI genotype was more strongly related to large adenoma risk among subjects with low dietary calcium intake (ORFf=0.48; 95% CI 0.17-1.3; ORff=0.21: 95% CI 0.04-1.3), low dietary vitamin D intake (ORFf=0.25; 95% CI 0.09-0.69; ORff= 0.22; 95% CI 0.04-1.2), or dark skin color (ORFf=0.66; 95% CI 0.27-1.6; ORff=0.10; 95% CI 0.01-1.0). CONCLUSION: These results suggest that VDR FokI genotype influences development of colorectal adenomas. and that the effect may be modified by calcium and vitamin D status.

Genetic determinants of serum prostate-specific antigen levels in healthy men from a multiethnic cohort.

We recently reported an association between prostate cancer risk and polymorphisms in the prostate-specific antigen (PSA) and androgen receptor (AR) genes. The purpose of this study is to test whether these two polymorphisms, AR CAG and PSA ARE1, influence serum PSA levels in healthy men. Serum PSA and the two genotypes were assayed for 420 healthy men from a multiethnic cohort, and regression models were fit to estimate the effects of AR CAG genotype and PSA ARE1 genotype on serum PSA levels. Predicted serum PSA decreased 3.5% with each additional AR CAG repeat decile (P = 0.01). Serum PSA was also associated with PSA ARE1 genotype, with PSA levels higher among men with the PSA AA genotype compared with men with the AG or GG genotypes (P = 0.02). The relationship between serum PSA level and AR CAG length differed according to PSA genotype (P = 0.049): for genotype GG, the slope was not significantly different from zero (P = 0.74); for genotype AG, serum PSA increased 4.5% with each decrease of one CAG repeat decile (P = 0.03); for genotype AA, serum PSA increased 7% with each decrease of one CAG repeat decile (P = 0.02). These results indicate that in healthy men, genetic variants in the PSA and AR genes contribute to variation in serum PSA levels. Men with the PSA AA genotype and short AR CAG alleles have, on average, higher serum PSA levels.

Genetic, dietary, and other lifestyle determinants of plasma homocysteine concentrations in middle-aged and older Chinese men and women in Singapore.

BACKGROUND: Epidemiologic studies have identified the plasma homocysteine concentration as a risk factor for atherothrombotic vascular disease. There is little information on the distributions and determinants of homocysteine concentrations in Asian populations. OBJECTIVE: The present study was designed to examine the relations between genetic and lifestyle factors and plasma homocysteine concentrations among Chinese in Singapore. DESIGN: Plasma total homocysteine, folate, vitamin B-12, and vitamin B-6 concentrations and genetic variation at the methylenetetrahydrofolate reductase (MTHFR) locus were measured in 486 Chinese men and women aged 45-74 y in Singapore. Data on dietary and other lifestyle factors were collected in face-to-face interviews. RESULTS: Men had higher plasma concentrations of total homocysteine than women (P = 0.0001). Age was positively associated with plasma homocysteine in both sexes (P for trend = 0.0001). Plasma concentrations of folate, vitamin B-12, and vitamin B-6 were inversely associated with homocysteine concentrations. Among individuals with low plasma folate, those possessing 2 copies of MTHFR mutant alleles had significantly higher homocysteine concentrations than did those with > or = 1 copy of the wild-type allele. Cigarette smoking, daily coffee consumption, and physical inactivity were positively related to plasma homocysteine concentrations in both sexes (P < 0.05). However, these associations disappeared after adjustment for plasma folate concentrations. CONCLUSIONS: Age, sex, plasma folate, vitamin B-12 and B-6 concentrations, and MTHFR genotype are independent determinants of plasma homocysteine in middle-aged and older Chinese in Singapore. These factors combined could account for up to 40% of the total variation in homocysteine concentrations in this Asian population.

Building a multigenic model of breast cancer susceptibility: CYP17 and HSD17B1 are two important candidates.

We conducted a nested case-control study to evaluate whether polymorphisms in two genes involved in estrogen metabolism, CYP17 and HSD17B1, were useful in developing a breast cancer risk model that could help discriminate women who are at higher risk of breast cancer. If polymorphisms in these genes affect the level of circulating estrogens, they may directly influence breast cancer risk. The base population for this study is a multiethnic cohort study that includes African-American, Non-Latina White, Japanese, Latina, and Native Hawaiian women. For this analysis, 1508 randomly selected controls and 850 incident breast cancer cases of the first four ethnic groups who agreed to provide a blood specimen were included (76 and 80% response rates, respectively). The CYP17 A2 allele and the HSD17B1 A allele were considered "high-risk" alleles. Subjects were then classified according to number of high-risk alleles. After adjusting for age, weight, and ethnicity, we found that carrying one or more high-risk alleles increases the risk of advanced breast cancer in a dose-response fashion. The risk among women carrying four high-risk alleles was 2.21 [95% confidence interval (CI), 0.98-5.00; P for trend = 0.03] compared with those who carried none. This risk was largely limited to women who were not taking hormone replacement therapy (relative risk, 2.60; 95% CI, 0.95-7.14) and was most pronounced among those weighing 170 pounds or less (RR, 3.05; 95% CI, 1.29-7.25). These findings suggest that breast cancer risk has a strong genetic component and supports the theory that the underlying mechanism of "complex traits" can be understood using a multigenic model of candidate genes.

Risk of endometrial cancer and estrogen replacement therapy history by CYP17 genotype.

Common variants among genes coding for enzymes in sex steroid biosynthetic pathways may influence the risk of endometrial cancer. We examined the association between endometrial cancer risk and estrogen replacement therapy (ERT) by CYP17 genotype using 51 incident cases and 391 randomly selected controls from a multiethnic cohort in Hawaii and Los Angeles, California. The relative risk of endometrial cancer was calculated for ever use versus never use of ERT by CYP17 genotype (TT, TC, and CC). We found that women who reported ever taking ERT were more than twice as likely to develop endometrial cancer as women who never took ERT [odds ratio (OR), 2.24; 95% confidence interval (CI), 1.19-4.23]. Among these women, the risk of endometrial cancer was higher for women homozygous for the CYP17 T allele (OR, 4.10; 95% CI, 1.64-10.3), but not for women with the C allele (OR, 1.31; 95% CI, 0.53-3.21). These preliminary findings suggest that CYP17 or other variants in estrogen biosynthesis or metabolism pathways may be potential markers of endometrial cancer susceptibility due to ERT.