Characterization of bacterial pectinolytic strains involved in the water retting process

Pectinolytic microorganisms involved in the water retting process were characterized. Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material. A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified. Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively. We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA). Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups. 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes. Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype. All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus. Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C. acetobutylicum-C. felsineum and C. saccharobutylicum species. Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B. subtilisT and B. pumilusT.     

Dissociation between rate of hepatic lipoprotein secretion and hepatocyte microtubule content

The fact that colchicines inhibits hepatic secretion of very low density lipoprotein (VLDL) particles has been interpreted to mean that microtubules are involved in hepatic VLDL secretion. To further define this relationship, we have attempted to see if changes in hepatic VLDL secretion are associated with changes in hepatocyte microtubule or tubulin content. Accordingly, hepatic secretion of VLDL was increased in rats, and the hepatocyte content of both microtubules (using quantitative morphometric methods) and tubulin (using a time-decay colchicine binding assay) was determined. In acute experiments, VLDL secretion was increased by perfusion of isolated rat livers for 2 h with varying concentrations of free fatty acids (FFA). Results indicate that hepatic VLDL triglyceride (TG) secretion at perfusate FFA levels of 0.7 μEq/ml is threefold greater (P < 0.01) than when livers are perfused without added FFA. However, no differences are observed in the content of microtubules in these livers: specifically, microtubules occupy 0.029 percent of hepatocyte cytoplasm in livers perfused without FFA and 0.030 percent of cytoplasm in livers perfused with FFA. In chronic experiments, rats were fed for 1 wk with either standard rat chow or a hyperlipidemic (sucrose/lard) diet. With the experimental diet, plasma triglyceride levels increase threefold over controls, and liver VLDL-TG production, as determined by [(3)H]glycerol turnover studies, is 55 percent greater (P < 0.01) than controls. However, microtubules occupy 0.027 percent of the cytoplasm of hepatocyte cytoplasm whether rats are on standard or hyperlipidemic diets. Furthermore, the tubulin content of isolated hepatocytes does change, and represents 1 percent of hepatocyte soluble protein, irrespective of diet. These results suggest that increases in hepatic VLDL secretion can occur without any demonstrable change in hepatocyte assembled microtubule or tubulin content, and raise questions as to the role played by microtubules in hepatic VLDL secretion.     

Isolation and Partial Characterization of Antifungal Metabolites Produced by <Emphasis Type="Italic">Bacillus</Emphasis> sp. IBA 33

Antifungal proteins produced by Bacillus sp. IBA 33 were purified by ammonium sulfate precipitation and DEAE-Sephacel column chromatography. The two purified proteins inhibited the growth of Geotrichum candidum, the sour rot disease agent in lemon. The proteins were stable at 20 (3 months), 40, 60 and 100°C (30 min) and remained active after sterilization at 121°C for 15 min. Their hydrophobic nature was proved and when were developed with ninhydrin they did not show any free amino groups. The infrared spectrum showed vibrational modes corresponding to peptide, ester or ketone links and saturated CH links corresponding to long chain fatty acids. UV scan spectroscopy showed tyrosine and or tryptophan amino acids in their composition. The remarkable thermo-resistance of proteins may be a good feature to be used in the development of a new biocontrol method of Geotrichum candidum.     

Daily physical activity in ankylosing spondylitis: validity and reliability of the IPAQ and SQUASH and the relation with clinical assessments

Introduction

The aim of this study was to investigate the construct validity and test-retest reliability of the International Physical Activity Questionnaire (IPAQ; long form) and the Short QUestionnaire to Assess Health-enhancing physical activity (SQUASH) and to investigate the relation between daily physical activity and clinical assessments in patients with ankylosing spondylitis (AS).

Methods

For validity, the self-report questionnaires IPAQ and SQUASH were compared with daily physical activity assessed with the ActiGraph accelerometer during 7 consecutive days in 63 AS outpatients. For reliability, the IPAQ and SQUASH were administered twice approximately 1 week apart in 52 AS outpatients. In all 115 patients, clinical assessments were performed at the outpatient clinic.

Results

IPAQ and SQUASH total scores correlated significantly with accelerometer outcome: ρ = 0.38 and r = 0.35, respectively. Intraclass correlation coefficients between first and second assessments of the IPAQ and SQUASH were 0.83 and 0.89, respectively. Bland-Altman analyses showed no systemic bias, but in particular for the IPAQ the 95% limits of agreement were wide. Daily physical activity assessed by accelerometer, IPAQ, and SQUASH correlated significantly with disease activity, physical activity, and quality of life. A relation with spinal mobility was found only for the accelerometer and SQUASH. The direction of these correlations indicates that higher daily physical activity is related to lower disease activity and better physical function, spinal mobility and quality of life.

Conclusions

Both physical activity questionnaires showed modest construct validity. The SQUASH showed good test-retest reliability, superior to the IPAQ. These results indicate that the SQUASH is more suitable than the IPAQ to assess daily physical activity in AS population studies. However, it is desirable to add questions on AS-specific physical activity. Further studies are needed to investigate the causality of the relation between daily physical activity and clinical assessments.     

Evaluation of APOE polymorphisms and the risk for age-related macular degeneration in a Southeastern Brazilian population

This study aimed to evaluate the role of APOE polymorphisms (rs429358 and rs7412) in the risk of age-related macular degeneration in a sample of the Southeastern Brazilian population. Seven hundred and five unrelated individuals were analyzed, 334 with age-related macular degeneration (case group), and 371 without the disease (control group). In the case group, patients were further stratified according to disease phenotypes, divided into dry and wet age-related macular degeneration, and non-advanced and advanced age-related macular degeneration. APOE polymorphisms (rs429358 and rs7412) were evaluated through polymerase chain reaction and direct sequencing. In the comparison of cases vs. controls, none of the associations reached statistical significance, considering the Bonferroni-adjusted P-value, although there was a suggestive protection for the E3/E4 genotype (OR = 0.626; P-value = 0.037) and E4 carriers (OR = 0.6515; P-value = 0.047). Statistically significant protection for both the E3/E4 genotype and E4 carriers was observed in the comparisons: advanced age-related macular degeneration vs. controls (OR = 0.3665, P-value = 0.491 × 10⁻³ and OR = 0.4031, P-value = 0.814 × 10⁻³, respectively), advanced age-related macular degeneration vs. non-advanced age-related macular degeneration (OR = 0.2529, P-value = 0.659 × 10⁻⁴ and OR = 0.2692, P-value = 0.631 × 10⁻⁴, respectively). In the comparison of wet age-related macular degeneration vs. control, protection was statistically significant only for E3/E4 (OR = 0.4052, P-value = 0.001). None of the comparisons demonstrated any significant association for E2 genotypes or E2 carriers in age-related macular degeneration risk in this study. Findings suggest a protective role of the E4 haplotype in the APOE gene in the risk for advanced and wet forms of age-related macular degeneration, in a sample of the Brazilian population. To our knowledge, this is the first Brazilian study to show the association between APOE polymorphisms and age-related macular degeneration.     

Living with myotonic dystrophy; what can be learned from couples? a qualitative study

Background  

Myotonic dystrophy type 1 (MD1) is one of the most prevalent neuromuscular diseases, yet very little is known about how MD1 affects the lives of couples and how they themselves manage individually and together. To better match health care to their problems, concerns and needs, it is important to understand their perspective of living with this hereditary, systemic disease.     

Regenerative potential of human muscle stem cells in chronic inflammation

Introduction

Chronic inflammation is a profound systemic modification of the cellular microenvironment which could affect survival, repair and maintenance of muscle stem cells. The aim of this study was to define the role of chronic inflammation on the regenerative potential of satellite cells in human muscle.

Methods

As a model for chronic inflammation, 11 patients suffering from rheumatoid arthritis (RA) were included together with 16 patients with osteoarthritis (OA) as controls. The mean age of both groups was 64 years, with more females in the RA group compared to the OA group. During elective knee replacement surgery, a muscle biopsy was taken from the distal musculus vastus medialis. Cell populations from four RA and eight OA patients were used for extensive phenotyping because these cell populations showed no spontaneous differentiation and myogenic purity greater than 75% after explantation.

Results

After mononuclear cell explantation, myogenic purity, viability, proliferation index, number of colonies, myogenic colonies, growth speed, maximum number of population doublings and fusion index were not different between RA and OA patients. Furthermore, the expression of proteins involved in replicative and stress-induced premature senescence and apoptosis, including p16, p21, p53, hTERT and cleaved caspase-3, was not different between RA and OA patients. Mean telomere length was shorter in the RA group compared to the OA group.

Conclusions

In the present study we found evidence that chronic inflammation in RA does not affect the in vitro regenerative potential of human satellite cells. Identification of mechanisms influencing muscle regeneration by modulation of its microenvironment may, therefore, be more appropriate.     

A Monte Carlo Study of the Relative Stability of Protein Helical Forms

We present simulation results on a simple model to describe the hydrogen bonding in proteins with helical structures. The approximation distinguishes between ! helices, where each amino acid interacts with another one located four residues apart, 3 10 structures, where the number of amino acids in between is three, and the ? arrangement, in which that number is five. We found that the main features of the system are determined by the most stable structure (the ! helix) and that the other type of hydrogen bonds appears just below the denaturation temperature of the peptide. The probability of finding a 3 10 -type bond is greater at the beginning or at the end of the peptide chain, irrespectively of its length, while in short peptides the existence of those bonds increases appreciably the denaturation temperature, promoting stability. On the other hand, the temperature of denaturation decreases with the length of the peptide to reach a value independent of the number of amino acid residues.     

First Evidence of Sternal Wound Biofilm following Cardiac Surgery

Management of deep sternal wound infection (SWI), a serious complication after cardiac surgery with high morbidity and mortality incidence, requires invasive procedures such as, debridement with primary closure or myocutaneous flap reconstruction along with use of broad spectrum antibiotics. The purpose of this clinical series is to investigate the presence of biofilm in patients with deep SWI. A biofilm is a complex microbial community in which bacteria attach to a biological or non-biological surface and are embedded in a self-produced extracellular polymeric substance. Biofilm related infections represent a major clinical challenge due to their resistance to both host immune defenses and standard antimicrobial therapies. Candidates for this clinical series were patients scheduled for a debridement procedure of an infected sternal wound after a cardiac surgery. Six patients with SWI were recruited in the study. All cases had marked dehiscence of all layers of the wound down to the sternum with no signs of healing after receiving broad spectrum antibiotics post-surgery. After consenting patients, tissue and/or extracted stainless steel wires were collected during the debridement procedure. Debrided tissues examined by Gram stain showed large aggregations of Gram positive cocci. Immuno-fluorescent staining of the debrided tissues using a specific antibody against staphylococci demonstrated the presence of thick clumps of staphylococci colonizing the wound bed. Evaluation of tissue samples with scanning electron microscope (SEM) imaging showed three-dimensional aggregates of these cocci attached to the wound surface. More interestingly, SEM imaging of the extracted wires showed attachment of cocci aggregations to the wire metal surface. These observations along with the clinical presentation of the patients provide the first evidence that supports the presence of biofilm in such cases. Clinical introduction of the biofilm infection concept in deep SWI may advance the current management strategies from standard antimicrobial therapy to anti-biofilm strategy.     

Vibrational Circular Dichroism Discrimination of Diastereomeric Cedranol Acetates

The reliability of vibrational circular dichroism (VCD) spectroscopy to discriminate four diastereomeric cedranol acetates 1 , 2 , 3 , 4 by means of their absolute configuration is examined. The usage of CompareVOA software to quantify comparisons of the measured infrared (IR) and VCD spectra with the corresponding simulated spectra at the B3LYP/DGDZVP and B3PW91/DGDZVP levels of theory for each diastereomer enabled the B3PW91 functional to be qualified as superior to the B3LYP functional for vibrational calculations of 1 , 2 , 3 , 4 . Analogously, a set of quantitative VCD spectra cross‐comparisons of 1 , 2 , 3 , 4 unambiguously distinguished the diastereomers using B3PW91 and failed using B3LYP. Remarkably, quantitative IR spectra cross‐comparisons of 1 , 2 , 3 , 4 using B3PW91 or B3LYP functionals demonstrated that the achiral spectroscopic IR technique is not able to distinguish cedranol acetate diastereomers. VCD comparisons using anisotropy g‐factor values of bands in the 1550–950 cm^‐1 region of the spectra were of aid to facilitate visual spectra matching for each diastereomer. Chirality 25:939‐951, 2013. © 2013 Wiley Periodicals, Inc.