A personalized, multi-platform nutrition, exercise, and lifestyle coaching program: A pilot in women.

The aim of this pilot study was to examine if a personalized web-based multi-platform nutrition, exercise, and lifestyle coaching program, supported weight loss and the reduction of chronic disease risk factors in overweight or obese women. Twenty-eight women completed the program, which represented 50% of those who provided baseline data. The program consisted of a one-year curriculum with daily exercise, nutritional habits, and health behaviour lessons along with access to a one-on-one coach. The workouts, habits, and lessons were available via computer, tablet, and mobile device which, along with coaching, facilitated self-monitoring and accountability. At baseline and 12-months, weight, waist circumference, fat mass, muscle mass, blood pressure, total cholesterol, low density lipoproteins, high density lipoproteins, triglycerides, C reactive protein, and fasting glucose were collected. Over the 12 months, women who completed the program, (average age 49.64 (SD 10.99) years), lost 16.52 (SD 13.63) lbs (P < 0.001), and reduced waist circumference by 3.56 (SD 2.31) in (P < 0.0001). Diastolic blood pressure decreased by 3.77 (SD 7.25) mm Hg (P = 0.02) and high density lipoproteins increased by 0.16 (SD 0.28) mmol/L (P = 0.01). No other risk factors changed significantly. Compliance was a significant predictor of weight loss (P < 0.01). In conclusion, women who completed the web-based program experienced significant weight loss (8.62% of initial body weight) coming predominantly from body fat. Chronic disease risk factors also improved.

Adenovirus membrane penetration activates the NLRP3 inflammasome.

Adenovirus type 5 (Ad5) infection of macrophages results in rapid secretion of interleukin-1ß (IL-1ß) and is dependent on the inflammasome components NLRP3 and ASC and the catalytic activity of caspase-1. Using lentivirus-expressed short hairpin RNA (shRNA) and competitive inhibitors, we show that Ad-induced IL-1ß release is dependent upon Toll-like receptor 9 (TLR9) sensing of the Ad5 double-stranded DNA (dsDNA) genome in human cell lines and primary monocyte-derived macrophages but not in mouse macrophages. Additionally, a temperature-sensitive mutant of Ad5 unable to penetrate endosomal membranes, ts1, is unable to induce IL-1ß release in TLR2-primed THP-1 cells, suggesting that penetration of endosomal membranes is required for IL-1ß release. Disruption of lysosomal membranes and the release of cathepsin B into the cytoplasm are required for Ad-induced NLRP3 activation. Ad5 cell entry also induces reactive oxygen species (ROS) production, and inhibitors of ROS prevent Ad-induced IL-1ß release. Ad5 activation of NLRP3 also induces necrotic cell death, resulting in the release of the proinflammatory molecule HMGB1. This work further defines the mechanisms of virally induced inflammasome activation.

The challenge of low physical activity during the school day: at recess, lunch and in physical education.

PURPOSE: To describe physical activity (PA) intensity across a school day and assess the percentage of girls and boys achieving recommended guidelines. METHODS: The authors measured PA via accelerometry in 380 children (8-11 years) and examined data representing (1) the whole school day, (2) regular class time, (3) recess, (4) lunch and (5) scheduled physical education (PE). Activity was categorised as sedentary (SED), light physical activity (LPA) or moderate to vigorous physical activity (MVPA) using age-specific thresholds. They examined sex differences across PA intensities during each time period and compliance with recommended guidelines. RESULTS: Girls accumulated less MVPA and more SED than boys throughout the school day (MVPA -10.6 min; SED +13.9 min) recess (MVPA -1.6 min; SED +1.7 min) and lunch (MVPA -3.1 min; SED +2.9 min). Girls accumulated less MVPA (-6.2 min), less LPA (-2.5 min) and more SED (+9.4 min) than boys during regular class time. Fewer girls than boys achieved PA guidelines during school (90.9% vs 96.2%), recess (15.7% vs 34.1%) and lunch (16.7% vs 37.4%). During PE, only 1.8% of girls and 2.9% of boys achieved the PA guidelines. Girls and boys accumulated similar amounts of MVPA, LPA and SED. CONCLUSION: The MVPA deficit in girls was due to their sedentary behaviour as opposed to LPA. Physical activity strategies that target girls are essential to overcome this deficit. Only a very small percentage of children met physical activity guidelines during PE. There is a great need for additional training and emphasis on PA during PE. In addition schools should complement PE with PA models that increase PA opportunities across the school day.

beta-Ketoacyl-[acyl carrier protein] synthase I of Escherichia coli: aspects of the condensation mechanism revealed by analyses of mutations in the active site pocket.

beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309, Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers.

Stability and function of interdomain linker variants of glucoamylase 1 from Aspergillus niger.

Several variants of glucoamylase 1 (GA1) from Aspergillus niger were created in which the highly O-glycosylated peptide (aa 468--508) connecting the (alpha/alpha)(6)-barrel catalytic domain and the starch binding domain was substituted at the gene level by equivalent segments of glucoamylases from Hormoconis resinae, Humicola grisea, and Rhizopus oryzae encoding 5, 19, and 36 amino acid residues. Variants were constructed in which the H. resinae linker was elongated by proline-rich sequences as this linker itself apparently was too short to allow formation of the corresponding protein variant. Size and isoelectric point of GA1 variants reflected differences in linker length, posttranslational modification, and net charge. While calculated polypeptide chain molecular masses for wild-type GA1, a nonnatural proline-rich linker variant, H. grisea, and R. oryzae linker variants were 65,784, 63,777, 63,912, and 65,614 Da, respectively, MALDI-TOF-MS gave values of 82,042, 73,800, 73,413, and 90,793 Da, respectively, where the latter value could partly be explained by an N-glycosylation site introduced near the linker C-terminus. The k(cat) and K(m) for hydrolysis of maltooligodextrins and soluble starch, and the rate of hydrolysis of barley starch granules were essentially the same for the variants as for wild-type GA1. beta-Cyclodextrin, acarbose, and two heterobidentate inhibitors were found by isothermal titration calorimetry to bind to the catalytic and starch binding domains of the linker variants, indicating that the function of the active site and the starch binding site was maintained. The stability of GA1 linker variants toward GdnHCl and heat, however, was reduced compared to wild-type.

Osteomyelitis of the temporomandibular joint.

Osteomyelitis of the temporomandibular joint is very uncommon, and osteomyelitis as a result of Aspergillus niger infection has not previously been reported. A case report of skull base and condylar osteomyelitis is presented. Previously reported cases of temporomandibular joint osteomyelitis are reviewed, and management is discussed. Because of the significant morbidity possible with infections in this region, otolaryngologists should be familiar with the anatomy, diagnostic modalities, and therapeutic options. [Editorial comment: This unusual case presents unique aspects of the pathophysiology of osteomyelitis of the skull base.]

Restoration of catalytic activity beyond wild-type level in glucoamylase from Aspergillus awamori by oxidation of the Glu400-->Cys catalytic-base mutant to cysteinesulfinic acid.

Glucoamylase catalyzes the hydrolysis of glucosidic bonds with inversion of the anomeric configuration. Site-directed mutagenesis and three-dimensional structure determination of the glucoamylase from Aspergillus awamori previously identified Glu179 and Glu400 as the general acid and base catalyst, respectively. The average distance between the two carboxyl groups was measured to be 9.2 A, which is typical for inverting glycosyl hydrolases. In the present study, this distance was increased by replacing the catalytic base Glu400 with cysteine which was then oxidized to cysteinesulfinic acid. Initially, this oxidation occurred during attempts to carboxyalkylate the Cys400 residue with iodoacetic acid, 3-iodopropionic acid, or 4-bromobutyric acid. However, endoproteinase Lys-C digestion of modified glucoamylase followed by high-pressure liquid chromatography in combination with matrix-assisted laser desorption ionization/time-of-flight mass spectrometry on purified peptide fragments demonstrated that all enzyme derivatives contained the cysteinesulfinic acid oxidation product of Cys400. Subsequently, it was demonstrated that treatment of Glu400-->Cys glucoamylase with potassium iodide in the presence of bromine resulted in complete conversion to the cysteinesulfinic acid product. As expected, the catalytic base mutant Glu400-->Cys glucoamylase had very low activity, i.e., 0.2% compared to wild-type. The oxidation of Cys400 to cysteinesulfinic acid, however, restored activity (kcat) on alpha-1,4-linked substrates to levels up to 160% of the wild-type glucoamylase which corresponded to approximately a 700-fold increase in the kcat of the Glu400-->Cys mutant glucoamylase. Whereas Glu400-->Cys glucoamylase was much less thermostable and more sensitive to guanidinium chloride than the wild-type enzyme, the oxidation to cysteinesulfinic acid was accompanied by partial recovery of the stability.

High yield overexpression and characterization of human recombinant proapolipoprotein A-I.

Human apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) where it defines the particle structure and stability and functions as the main activator of the enzyme lecithin:cholesterol acyltransferase (LCAT). ApoA-I is expressed in the liver as a preproprotein that is targeted to the endoplasmic reticulum for secretion; in plasma, an unknown protease removes the six amino acid long propeptide. In this study, the cDNA coding the human proapoA-I was cloned into an Escherichia coli vector; the overexpressed protein was purified to 99% homogeneity and was extensively characterized together with mature apoA-I purified from plasma. SDS-PAGE, mass spectrometry, and Edman sequence analysis showed that the initial Met residue needed for translation in E. coli is posttranslationally removed from the N-terminal sequence of the proapoA-I. The structural and functional analyses were carried out on the lipid-free and the lipid-bound proteins. ProapoA-I self associated, interacted with dimyristoyl phosphatidylcholine vesicles, and formed secondary structures very similar to the lipid-free apoA-I. Reconstituted HDL particles made with two initial molar ratios of palmitoyloleoyl phosphatidylcholine/cholesterol/apolipoprotein/Na-cholate had identical particle sizes and distributions when apoA-I or proapoA-I were used. Particles having diameters of 79 A and 98 A, containing two apoA-I or proapoA-I molecules per particle, were isolated and characterized. The particles contained the same amounts of alpha-helical structure, had very similar fluorescence properties, and activated LCAT equally well. We conclude that proapoA-I expressed and purified from E. coli is functionally and structurally indistinguishable from mature apoA-I purified from plasma when analyzed in vitro. Therefore, this recombinant proapoA-I and mutants derived from it will be important sources of protein for analyzing apoA-I structure and function, as well as for studies of proapoA-I processing.

Proptosis as the initial presentation of fungal sinusitis in immunocompetent patients.

BACKGROUND: Fungal sinusitis typically occurs in immunocompromised patients. The authors report four cases of fungal sinusitis in immunocompetent young adults, all of whom had proptosis. METHODS: The diagnosis in all four patients was determined after orbital imaging and sinus biopsies. RESULTS: All four patients required surgical removal of the fungal source and anti-fungal chemotherapy postoperatively. CONCLUSION: Patients with proptosis, ocular pain, or other symptoms suggestive of orbital cellulitis unresponsive to antibiotic treatment should undergo radiographic imaging. If sinus disease is present, biopsy and culture may lead to the diagnosis of fungal disease. Surgical debridement and the appropriate systemic antifungal therapy usually lead to cure.

Dimensionality and clustering in the semantic network of patients with Alzheimer's disease.

The organization of semantic memory in patients with Alzheimer's disease (AD) was investigated with a triadic comparison task. A multidimensional scaling statistic was used to analyze proximity data and to generate 3-dimensional cognitive maps that were then compared by a discriminant function analysis. The results suggest that the structure of semantic memory in AD patients differs from that of elderly normal controls (NC) in 2 ways. First, AD patients are less consistent in using the attributes (predation, domesticity, and size) of concepts. Second, AD patients focus primarily on concrete perceptual information (size), whereas NC Ss stress abstract conceptual knowledge (domesticity). These results are consistent with the notion that AD is characterized by a breakdown in the structure of semantic knowledge.