Changes in fecal microbiota with CFTR modulator therapy: A pilot study.

Studies have demonstrated that people with CF with pancreatic insufficiency (PI) have fecal dysbioses. Evidence suggests the causes of these dysbioses are multifactorial, and that important drivers include antibiotic exposure, dietary intake, and CF gastrointestinal tract dysfunction, including nutrient malabsorption. In this pilot study, we tested whether initiation of the CFTR modulator treatments ivacaftor (in a cohort of pancreatic sufficient (PS) people with CF and an R117H CFTR variant) or lumacaftor/ivacaftor (in a cohort of PI people with CF and an F508del variant) changed fecal measures of malabsorption or fecal microbiomes. While we identified no statistically significant fecal changes with either treatment, we detected trends in the PI cohort when initiating lumacaftor/ivacaftor towards decreased fecal fat content and towards fecal microbiomes that more closely resembled the fecal microbiota of people without PI. While these findings support a model in which nutrient malabsorption resulting from CF-induced PI drives fecal dysbiosis, they must be validated in future, larger studies of fecal microbiome and malabsorption outcomes with highly effective CFTR modulator therapies.

The effect of dry needling on gastrocnemius muscle stiffness and strength in participants with latent trigger points.

Abnormal muscle stiffness is a potential complication after injury and identifying interventions that modify muscle stiffness may be useful to promote recovery. The purpose of this study was to identify the short-term effects of dry needling (DN) on resting and contracted gastrocnemius muscle stiffness and strength of the triceps surae in individuals with latent myofascial trigger points (MTrPs). In this randomized controlled trial, 52 individuals received two DN treatment sessions to latent MTrPs and 50 individuals received two sham needling sessions. Resting and contracted muscle stiffness were assessed both at the treatment site and a standardized central site in the medial gastrocnemius head immediately post-treatment and one week after the last session. There were significant group by time interactions for resting muscle stiffness at the site of the MTrP (p = .03), but not at the central site (p = .29). Post-needling between group comparison indicated that the DN group had significantly lower resting muscle stiffness at the site of the MTrP than the sham group after adjusting for baseline differences. There were no significant between group differences in contracted muscle stiffness or muscle strength. Identifying strategies that can reduce aberrant muscle stiffness may help to guide management of individuals with neuromuscular pain-related conditions. Level of evidence: Therapy, level 2.

The microbiota in bronchoalveolar lavage from young children with chronic lung disease includes taxa present in both the oropharynx and nasopharynx.

BACKGROUND: Invasive methods requiring general anaesthesia are needed to sample the lung microbiota in young children who do not expectorate. This poses substantial challenges to longitudinal study of paediatric airway microbiota. Non-invasive upper airway sampling is an alternative method for monitoring airway microbiota; however, there are limited data describing the relationship of such results with lung microbiota in young children. In this study, we compared the upper and lower airway microbiota in young children to determine whether non-invasive upper airway sampling procedures provide a reliable measure of either lung microbiota or clinically defined differences. RESULTS: The microbiota in oropharyngeal (OP) swabs, nasopharyngeal (NP) swabs and bronchoalveolar lavage (BAL) from 78 children (median age 2.2 years) with and without lung disease were characterised using 16S rRNA gene sequencing. Permutational multivariate analysis of variance (PERMANOVA) detected significant differences between the microbiota in BAL and those in both OP swabs (p = 0.0001, Pseudo-F = 12.2, df = 1) and NP swabs (p = 0.0001; Pseudo-F = 21.9, df = 1) with the NP and BAL microbiota more different than the OP and BAL, as indicated by a higher Pseudo-F value. The microbiota in combined OP and NP data (upper airways) provided a more comprehensive representation of BAL microbiota, but significant differences between the upper airway and BAL microbiota remained, albeit with a considerably smaller Pseudo-F (PERMANOVA p = 0.0001; Pseudo-F = 4.9, df = 1). Despite this overall difference, paired BAL and upper airway (OP and NP) microbiota were >50 % similar among 69 % of children. Furthermore, canonical analysis of principal coordinates (CAP analysis) detected significant differences between the microbiota from clinically defined groups when analysing either BAL (eigenvalues >0.8; misclassification rate 26.5 %) or the combined OP and NP data (eigenvalues >0.8; misclassification rate 12.2 %). CONCLUSIONS: Upper airway sampling provided an imperfect, but reliable, representation of the BAL microbiota for most children in this study. We recommend inclusion of both OP and NP specimens when non-invasive upper airway sampling is needed to assess airway microbiota in young children who do not expectorate. The results of the CAP analysis suggest lower and upper airway microbiota profiles may differentiate children with chronic suppurative lung disease from those with persistent bacterial bronchitis; however, further research is needed to confirm this observation.

OBSERVATIONS ON THE FINE STRUCTURE OF OEDOGONIUM. III. MICROTUBULAR ELEMENTS IN THE CHLOROPLASTS OF OE. CARDIACUM(1) (2).

The expansive, reticulate cliloroplasts of the filamentous green alga Oedogonium tardiacum were found to contain microtubular elements with an outside diameter of about 270 A. These microtubules have been examined in the chloroplast stroma of vegetative cells, zoospores, zoospore germlings, and eggs. They may occur singly or, more commonly, in groups or bundles of 2 to as many as 30. In longitudinal section these microtubules show a regular pattern of slightly oblique cross striations and the margins appear rough. They bear little structural resemblance to the more familiar and slightly smaller microtubules found in the cytoplasm external to the cliloroplasts. Their overall appearance suggests that each is constructed of 2 helically wound, ribbon-like units that are approximately 155-175 A broad and 45-55 A thick. The 2 units of this proposed double helix appear to lie in juxtaposition at a pitch of 17-27 deg. The dimensions of each unit, or helix, are such that it could conceivably be constructed of 3 filamentous subunits similar to those already described in the literature for other microtubular elements.

OBSERVATIONS ON THE FINE STRUCTURE OF OEDOGONIUM. V. EVIDENCE FOR THE DE NOVO FORMATION OF PYRENOIDS IN ZOOSPORES OF OE. CARDIACUM(1) (2) (3).

Ultrastructural studies of the chloroplasts of zoospores and developing zoospores of Oedogonium carcliacum have disclosed the occurrence of numerous incipient pyrenoids. A single developing zoospore may possess several score of these structures which appear to arise de novo in the chloroplast stroma and seem to lack any direct association with mature pyrenoids which are also present in the cells. The incipient pyrenoids lack the associated starch grains and the membrane-limited channels characteristic of mature pyrenoids, but they are readily recognized in the chloroplasts since they demonstrate a greater granularity and electron density than the surrounding chloroplast stroma. The granularity and electron density of the incipient pyrenoids match the ultra-structural appearance of the matrix of mature pyrenoids. The smallest of the incipient pyrenoids examined from serial sections had a maximum diameter of less than 0.3 µ. This may be compared with the size of mature pyrenoids, many with a maximum diameter of over 5.0 µ. In all the zoospores and developing zoospores examined, only one mature pyrenoid was observed in an apparent stage of division.

Altered regulation of regional sucrase-isomaltase expression in diabetic rat intestine.

Increased sucrase-isomaltase (SI) expression is a prominent feature of adaptive changes observed in the small intestine of streptozocin-treated chronically diabetic (CD) rats. In this study, we examine the cellular and molecular basis of increased SI expression in CD rats by determining SI specific activities and mRNA abundance in sequentially isolated enterocytes along the villus-to-crypt axis of proximal jejunum and distal ileum. In all regions, two- to fourfold increases in sucrase activity in diabetic rat enterocytes were paralleled by increases in SI mRNA. However, analogous to nondiabetic rat intestine, no differences in SI mRNA abundance were observed between corresponding enterocyte fractions from ileum and jejunum of diabetic rat intestine. By nuclear run-on assays, differences in rates of SI gene transcription were not observed in diabetic and nondiabetic intestinal tissues. We conclude that diabetes induces increased total and specific activities and mRNA abundance of intestinal SI, largely through the stabilization of SI mRNA. Furthermore, analogous to nondiabetic small intestine, differences in proximal-to-distal SI expression appear to be determined at the translational or posttranslational level.

Determinants of regional sucrase-isomaltase expression in adult rat small intestine.

Sucrase-isomaltase (SI) expression along the longitudinal and vertical axis of the small intestine was studied by sequentially isolating enterocytes from villus to crypt of rat proximal jejunum and distal ileum. Gradients of sucrase activity were observed with greatest activity occurring in jejunal and villus regions. Along the villus-to-crypt axis, gradients of SI mRNA abundance corresponded with activity. However, along the longitudinal axis no differences in SI mRNA levels were observed, thus not accounting for the observed 3-5-fold difference in SI activities between jejunum and ileum. Comparison of SI immunoprecipitates from jejunal and ileal mucosal scrapings showed significant differences in gel mobilities of the more mature forms, which did not appear to affect SI functional activities. When relative rates of de novo SI protein synthesis were compared, [35S]methionine incorporation into all SI forms was observed to be 3-5-fold greater in jejunum than in ileum at all time points. Because these results suggested differences in regional translational regulation, subcellular distribution of SI mRNA in jejunal and ileal epithelial cells was compared. A greater proportion of jejunal SI mRNA was found to be associated with membrane-bound polyribosomes. We conclude 1) sucrase expression along the villus-to-crypt axis correlates with SI mRNA abundance, 2) post-translational processing of SI differ in ileum and jejunum, but appear not to determine SI expression, and 3) differences in translational processing in distal ileum and proximal jejunum may determine sucrase activity along the longitudinal axis of rat small intestine.

TWO NEW SPECIES OF OEDOCLADIUM(1).

Two new gynandrosporous species of Oedocladium from the United States were isolated into unialgal culture and comparative studies were made. The new taxa described, O. carolinianum sp. nov. and O. cir-ratum sp. nov., are morphologically very similar. Both species have an opercufate oogonium with inferior division, oospore walls with the middle layer ungulate, and hyaline suffultory cells containing a small amount of vacuolated cytoplasm. Conditions for sexuality in culture zuere determined for both species. Zoospores and akinetes serve as means for asexual reproduction.

Structure of N,N-[bis(2-pyridylmethyl)-gamma-aminobutyrato]copper(II) bromide methanol solvate.

[Cu(C16H18N3O2)]Br.CH4O, Mr = 459.83, orthorhombic, P2(1)2(1)2(1), a = 10.452 (4), b = 12.197 (7), c = 14.984 (6) A, V = 1910 (3) A3, Z = 4, Dm = 1.61 (1), Dx = 1.599 g cm-3, lambda(Mo K alpha) = 0.71073 A, mu = 32.37 cm-1, F(000) = 932, T = 174 K, R = 0.0396, wR = 0.0419, 1146 observed reflections [I greater than 3 sigma(I)]. The title compound is a polymeric species in the solid state, with a unit cell consisting of two segments of one-dimensional chains. The ligand, a derivative of gamma-aminobutyric acid in which the amino group is substituted with two 2-pyridylmethyl moieties, coordinates to one copper atom through the three nitrogen atoms and to another copper atom through the two carboxylate oxygen atoms. The copper(II) atom has a pseudo square-pyramidal geometry, distorted by a distant sixth interaction to a carboxylate oxygen atom [Cu-O(2), 2.770 (7) A].

Regional alterations in intestinal sucrase expression in streptozocin-treated chronically diabetic rats.

In ad libitum-fed diabetic rats, sucrase specific activities in jejunum and ileum were significantly increased two- to threefold compared to controls, a response unaltered by pair-feeding. Gradients of sucrase activities along the ileal villus-to-crypt axis were readily measured in crypt regions in diabetic, but not in nondiabetic, rats. Changes in sucrase activities were commensurate with increases in sucrase immunoreactivity and not a result of altered functional activity. Insulin treatment reversed these effects, although insulin-deficiency, studied in food-deprived, nondiabetic rats, did not affect sucrase expression. We conclude that chronic diabetes significantly stimulates sucrase expression along the proximal-to-distal and villus-to-crypt axes of rat small intestine. In ileum, these changes suggest marked alterations in phenotypic development of enterocytes along the villus-to-crypt axis. Alterations in sucrase expression do not appear to correlate with insulin states and are not a consequence of altered functional activity.

Structure-based identification of an inducer of the low-pH conformational change in the influenza virus hemagglutinin: irreversible inhibition of infectivity.

Past efforts to employ a structure-based approach to design an inhibitor of the fusion-inducing conformational change in the influenza virus hemagglutinin (HA) yielded a family of small benzoquinones and hydroquinones. The most potent of these, tert-butyl hydroquinone (TBHQ), inhibits both the conformational change in HA from strain X:31 influenza virus and viral infectivity in tissue culture cells with 50% inhibitory concentrations in the micromolar range (D. L. Bodian, R. B. Yamasaki, R. L. Buswell, J. F. Stearns, J. M. White, and I. D. Kuntz, Biochemistry 32:2967-2978, 1993). A new structure-based inhibitor design search was begun which involved (i) the recently refined crystal structure (2.1-A resolution) of the HA ectodomain, (ii) new insights into the conformational change, and (iii) improvements in the molecular docking program, DOCK. As a result, we identified new inhibitors of HA-mediated membrane fusion. Like TBHQ, most of these molecules inhibit the conformational change. One of the new compounds, however, facilitates rather than inhibits the HA conformational change. Nonetheless, the facilitator, diiodofluorescein, inhibits HA-mediated membrane fusion and, irreversibly, infectivity. We further characterized the effects of inhibitors from both searches on the conformational change and membrane fusion activity of HA as well as on viral infectivity. We also isolated and characterized several mutants resistant to each class of inhibitor. The implications of our results for HA-mediated membrane fusion, anti-influenza virus therapy, and structure-based inhibitor design are discussed.

Isolation and partial chemical characterization of a virus-like particle from a eukaryotic alga.

A virus-like particle (VLP) associated with the green alga Cylindrocapsa geminella has been purified and partially characterized. The Cylindrocapsa VLP (C-VLP) has a polygonal profile in sectional view, ranges in diameter from 200 to 230 nm, and has a complex, multilayered capsid. Purified VLPs, when analyzed by SDS-polyacrylamide gel electrophoresis, contained at least 10 proteins ranging in molecular weight from 10,000 to 160,000. The C-VLP contains DNA, most probably double-stranded, having an estimated molecular weight of 175-190 x 10(6). Intracellular VLPs were only observed within single-celled germlings and all cultures examined produced the VLP. Generally, only 4-6% of the single-celled germlings contained VLPs, which resulted in cell death. The physicochemical and biological characteristics of the C-VLP support the conclusion that the C-VLP is, in fact, a virus.

Virus-cell and cell-cell fusion.

Significant progress has been made in elucidating the mechanisms of viral membrane fusion proteins; both those that function at low, as well as those that function at neutral, pH. For many viral fusion proteins evidence now suggests that a triggered conformational change that exposes a previously cryptic fusion peptide, along with a rearrangement of the fusion protein oligomer, allows the fusion peptide to gain access to the target bilayer and thus initiate the fusion reaction. Although the topologically equivalent process of cell-cell fusion is less well understood, several cell surface proteins, including members of the newly described ADAM gene family, have emerged as candidate adhesion/fusion proteins.