Parasympathetic Stimuli on Bronchial and Cardiovascular Systems in Humans

Background

It is not known whether parasympathetic outflow simultaneously acts on bronchial tone and cardiovascular system waxing and waning both systems in parallel, or, alternatively, whether the regulation is more dependent on local factors and therefore independent on each system. The aim of this study was to evaluate the simultaneous effect of different kinds of stimulations, all associated with parasympathetic activation, on bronchomotor tone and cardiovascular autonomic regulation.

Methods

Respiratory system resistance (Rrs, forced oscillation technique) and cardio-vascular activity (heart rate, oxygen saturation, tissue oxygenation index, blood pressure) were assessed in 13 volunteers at baseline and during a series of parasympathetic stimuli: O₂ inhalation, stimulation of the carotid sinus baroreceptors by neck suction, slow breathing, and inhalation of methacholine.

Results

Pure cholinergic stimuli, like O₂ inhalation and baroreceptors stimulation, caused an increase in Rrs and a reduction in heart rate and blood pressure. Slow breathing led to bradycardia and hypotension, without significant changes in Rrs. However slow breathing was associated with deep inhalations, and Rrs evaluated at the baseline lung volumes was significantly increased, suggesting that the large tidal volumes reversed the airways narrowing effect of parasympathetic activation. Finally inhaled methacholine caused marked airway narrowing, while the cardiovascular variables were unaffected, presumably because of the sympathetic activity triggered in response to hypoxemia.

Conclusions

All parasympathetic stimuli affected bronchial tone and moderately affected also the cardiovascular system. However the response differed depending on the nature of the stimulus. Slow breathing was associated with large tidal volumes that reversed the airways narrowing effect of parasympathetic activation.     

Can Observation Skills of Citizen Scientists Be Estimated Using Species Accumulation Curves?

Volunteers are increasingly being recruited into citizen science projects to collect observations for scientific studies. An additional goal of these projects is to engage and educate these volunteers. Thus, there are few barriers to participation resulting in volunteer observers with varying ability to complete the project’s tasks. To improve the quality of a citizen science project’s outcomes it would be useful to account for inter-observer variation, and to assess the rarely tested presumption that participating in a citizen science projects results in volunteers becoming better observers. Here we present a method for indexing observer variability based on the data routinely submitted by observers participating in the citizen science project eBird, a broad-scale monitoring project in which observers collect and submit lists of the bird species observed while birding. Our method for indexing observer variability uses species accumulation curves, lines that describe how the total number of species reported increase with increasing time spent in collecting observations. We find that differences in species accumulation curves among observers equates to higher rates of species accumulation, particularly for harder-to-identify species, and reveals increased species accumulation rates with continued participation. We suggest that these properties of our analysis provide a measure of observer skill, and that the potential to derive post-hoc data-derived measurements of participant ability should be more widely explored by analysts of data from citizen science projects. We see the potential for inferential results from analyses of citizen science data to be improved by accounting for observer skill.     

Using an Adenosine Triphosphate Bioluminescent Assay to Determine Effective Antibiotic Combinations against Carbapenem-Resistant Gram Negative Bacteria within 24 Hours

Background

Current in vitro combination testing methods involve enumeration by bacterial plating, which is labor-intensive and time-consuming. Measurement of bioluminescence, released when bacterial adenosine triphosphate binds to firefly luciferin-luciferase, has been proposed as a surrogate for bacterial counts. We developed an ATP bioluminescent combination testing assay with a rapid turnaround time of 24h to determine effective antibiotic combinations.

Methods

100 strains of carbapenem-resistant (CR) GNB [30 Acinetobacter baumannii (AB), 30 Pseudomonas aeruginosa (PA) and 40 Klebsiella pneumoniae (KP)] were used. Bacterial suspensions (10⁵ CFU/ml) were added to 96-well plates containing clinically achievable concentrations of multiple single and two-antibiotic combinations. At 24h, the luminescence intensity of each well was measured. Receiver operator characteristic curves were plotted to determine optimal luminescence threshold (T_RLU) to discriminate between inhibitory/non-inhibitory combinations when compared to viable plating. The unweighted accuracy (UA) [(sensitivity + specificity)/2] of T_RLU values was determined. External validation was further done using 50 additional CR-GNB.

Results

Predictive accuracies of T_RLU were high for when all antibiotic combinations and species were collectively analyzed (T_RLU = 0.81, UA = 89%). When individual thresholds for each species were determined, UA remained high. Predictive accuracy was highest for KP (T_RLU = 0.81, UA = 91%), and lowest for AB (T_RLU = 0.83, UA = 87%). Upon external validation, high overall accuracy (91%) was observed. The assay distinguished inhibitory/non-inhibitory combinations with UA of 80%, 94% and 93% for AB, PA and KP respectively.

Conclusion

We developed an assay that is robust at identifying useful combinations with a rapid turn-around time of 24h, and may be employed to guide the timely selection of effective antibiotic combinations.     

A Serine-Threonine Kinase (StkP) Regulates Expression of the Pneumococcal Pilus and Modulates Bacterial Adherence to Human Epithelial and Endothelial Cells In Vitro

The pneumococcal serine threonine protein kinase (StkP) acts as a global regulator in the pneumococcus. Bacterial mutants deficient in StkP are less virulent in animal models of infection. The gene for this regulator is located adjacent to the gene for its cognate phosphatase in the pneumococcal genome. The phosphatase dephosphorylates proteins phosphorylated by StkP and has been shown to regulate a number of key pneumococcal virulence factors and to modulate adherence to eukaryotic cells. The role of StkP in adherence of pneumococci to human cells has not previously been reported. In this study we show StkP represses the pneumococcal pilus, a virulence factor known to be important for bacterial adhesion. In a serotype 4 strain regulation of the pilus by StkP modulates adherence to human brain microvascular endothelial cells (HBMEC) and human lung epithelial cells. This suggests that the pneumococcal pilus may play a role in adherence during infections such as meningitis and pneumonia. We show that regulation of the pilus occurs at the population level as StkP alters the number of pili-positive cells within a single culture. As far as we are aware this is the first gene identified outside of the pilus islet that regulates the biphasic expression of the pilus. These findings suggest StkPs role in cell division may be linked to regulation of expression of a cell surface adhesin.     

Correlation between Body Composition and Walking Capacity in Severe Obesity

Background

Obesity is associated with mobility reduction due to mechanical factors and excessive body fat. The six-minute walk test (6MWT) has been used to assess functional capacity in severe obesity.

Objective

To determine the association of BMI, total and segmental body composition with distance walked (6MWD) during the six-minute walk test (6MWT) according to gender and obesity grade.

Setting

University of São Paulo Medical School, Brazil; Public Practice.

Methods

Functional capacity was assessed by 6MWD and body composition (%) by bioelectrical impedance analysis in 90 patients.

Results

The mean 6MWD was 514.9 ± 50.3 m for both genders. The male group (M: 545.2 ± 46.9 m) showed a 6MWD higher (p = 0.002) than the female group (F: 505.6 ± 47.9 m). The morbid obese group (MO: 524.7 ± 44.0 m) also showed a 6MWD higher (p = 0.014) than the super obese group (SO: 494.2 ± 57.0 m). There was a positive relationship between 6MWD and fat free mass (FFM), FFM of upper limps (FFM_UL), trunk (FFM_TR) and lower limbs (FFM_LL). Female group presented a positive relationship between 6MWD and FFM, FFM_UL and FFM_LL and male group presented a positive relationship between 6MWD and FFM_TR. In morbid obese group there was a positive relationship between 6MWD with FFM, FFM_UL, FFM_TR and FFM_LL. The super obese group presented a positive relationship between 6MWD with FFM, FFM_TR and FFM_LL.

Conclusions

Total and segmental FFM is associated with a better walking capacity than BMI.     

Llama Nanoantibodies with Therapeutic Potential against Human Norovirus Diarrhea

Noroviruses are a major cause of acute gastroenteritis, but no vaccines or therapeutic drugs are available. Llama-derived single chain antibody fragments (also called VHH) are small, recombinant monoclonal antibodies of 15 kDa with several advantages over conventional antibodies. The aim of this study was to generate recombinant monoclonal VHH specific for the two major norovirus (NoV) genogroups (GI and GII) in order to investigate their potential as immunotherapy for the treatment of NoV diarrhea. To accomplish this objective, two llamas were immunized with either GI.1 (Norwalk-1968) or GII.4 (MD2004) VLPs. After immunization, peripheral blood lymphocytes were collected and used to generate two VHH libraries. Using phage display technology, 10 VHH clones specific for GI.1, and 8 specific for GII.4 were selected for further characterization. All VHH recognized conformational epitopes in the P domain of the immunizing VP1 capsid protein, with the exception of one GII.4 VHH that recognized a linear P domain epitope. The GI.1 VHHs were highly specific for the immunizing GI.1 genotype, with only one VHH cross-reacting with GI.3 genotype. The GII.4 VHHs reacted with the immunizing GII.4 strain and showed a varying reactivity profile among different GII genotypes. One VHH specific for GI.1 and three specific for GII.4 could block the binding of homologous VLPs to synthetic HBGA carbohydrates, saliva, and pig gastric mucin, and in addition, could inhibit the hemagglutination of red blood cells by homologous VLPs. The ability of Nov-specific VHHs to perform well in these surrogate neutralization assays supports their further development as immunotherapy for NoV treatment and immunoprophylaxis.     

Exploration of the Canyon-Incised Continental Margin of the Northeastern United States Reveals Dynamic Habitats and Diverse Communities

The continental margin off the northeastern United States (NEUS) contains numerous, topographically complex features that increase habitat heterogeneity across the region. However, the majority of these rugged features have never been surveyed, particularly using direct observations. During summer 2013, 31 Remotely-Operated Vehicle (ROV) dives were conducted from 494 to 3271 m depth across a variety of seafloor features to document communities and to infer geological processes that produced such features. The ROV surveyed six broad-scale habitat features, consisting of shelf-breaching canyons, slope-sourced canyons, inter-canyon areas, open-slope/landslide-scar areas, hydrocarbon seeps, and Mytilus Seamount. Four previously unknown chemosynthetic communities dominated by Bathymodiolus mussels were documented. Seafloor methane hydrate was observed at two seep sites. Multivariate analyses indicated that depth and broad-scale habitat significantly influenced megafaunal coral (58 taxa), demersal fish (69 taxa), and decapod crustacean (34 taxa) assemblages. Species richness of fishes and crustaceans significantly declined with depth, while there was no relationship between coral richness and depth. Turnover in assemblage structure occurred on the middle to lower slope at the approximate boundaries of water masses found previously in the region. Coral species richness was also an important variable explaining variation in fish and crustacean assemblages. Coral diversity may serve as an indicator of habitat suitability and variation in available niche diversity for these taxonomic groups. Our surveys added 24 putative coral species and three fishes to the known regional fauna, including the black coral Telopathes magna, the octocoral Metallogorgia melanotrichos and the fishes Gaidropsarus argentatus, Guttigadus latifrons, and Lepidion guentheri. Marine litter was observed on 81% of the dives, with at least 12 coral colonies entangled in debris. While initial exploration revealed the NEUS region to be both geologically dynamic and biologically diverse, further research into the abiotic conditions and the biotic interactions that influence species abundance and distribution is needed.     

Reduced Functional Connectivity of Default Mode and Set-Maintenance Networks in Ornithine Transcarbamylase Deficiency

Background and Purpose

Ornithine transcarbamylase deficiency (OTCD) is an X-chromosome linked urea cycle disorder (UCD) that causes hyperammonemic episodes leading to white matter injury and impairments in executive functioning, working memory, and motor planning. This study aims to investigate differences in functional connectivity of two resting-state networks—default mode and set-maintenance—between OTCD patients and healthy controls.

Methods

Sixteen patients with partial OTCD and twenty-two control participants underwent a resting-state scan using 3T fMRI. Combining independent component analysis (ICA) and region-of-interest (ROI) analyses, we identified the nodes that comprised each network in each group, and assessed internodal connectivity.

Results

Group comparisons revealed reduced functional connectivity in the default mode network (DMN) of OTCD patients, particularly between the anterior cingulate cortex/medial prefrontal cortex (ACC/mPFC) node and bilateral inferior parietal lobule (IPL), as well as between the ACC/mPFC node and the posterior cingulate cortex (PCC) node. Patients also showed reduced connectivity in the set-maintenance network, especially between right anterior insula/frontal operculum (aI/fO) node and bilateral superior frontal gyrus (SFG), as well as between the right aI/fO and ACC and between the ACC and right SFG.

Conclusion

Internodal functional connectivity in the DMN and set-maintenance network is reduced in patients with partial OTCD compared to controls, most likely due to hyperammonemia-related white matter damage. Because several of the affected areas are involved in executive functioning, it is postulated that this reduced connectivity is an underlying cause of the deficits OTCD patients display in this cognitive domain.     

Validation of Next-Generation Sequencing of Entire Mitochondrial Genomes and the Diversity of Mitochondrial DNA Mutations in Oral Squamous Cell Carcinoma

Background

Oral squamous cell carcinoma (OSCC) is mainly caused by smoking and alcohol abuse and shows a five-year survival rate of ~50%. We aimed to explore the variation of somatic mitochondrial DNA (mtDNA) mutations in primary oral tumors, recurrences and metastases.

Methods

We performed an in-depth validation of mtDNA next-generation sequencing (NGS) on an Illumina HiSeq 2500 platform for its application to cancer tissues, with the goal to detect low-level heteroplasmies and to avoid artifacts. Therefore we genotyped the mitochondrial genome (16.6 kb) from 85 tissue samples (tumors, recurrences, resection edges, metastases and blood) collected from 28 prospectively recruited OSCC patients applying both Sanger sequencing and high-coverage NGS (~35,000 reads per base).

Results

We observed a strong correlation between Sanger sequencing and NGS in estimating the mixture ratio of heteroplasmies (r = 0.99; p<0.001). Non-synonymous heteroplasmic variants were enriched among cancerous tissues. The proportions of somatic and inherited variants in a given gene region were strongly correlated (r = 0.85; p<0.001). Half of the patients shared mutations between benign and cancerous tissue samples. Low level heteroplasmies (<10%) were more frequent in benign samples compared to tumor samples, where heteroplasmies >10% were predominant. Four out of six patients who developed a local tumor recurrence showed mutations in the recurrence that had also been observed in the primary tumor. Three out of five patients, who had tumor metastases in the lymph nodes of their necks, shared mtDNA mutations between primary tumors and lymph node metastases. The percentage of mutation heteroplasmy increased from the primary tumor to lymph node metastases.

Conclusions

We conclude that Sanger sequencing is valid for heteroplasmy quantification for heteroplasmies ≥10% and that NGS is capable of reliably detecting and quantifying heteroplasmies down to the 1%-level. The finding of shared mutations between primary tumors, recurrences and metastasis indicates a clonal origin of malignant cells in oral cancer.     

Escherichia coli Heat-Stable Enterotoxin Mediates Na+/H+ Exchanger 4 Inhibition Involving cAMP in T84 Human Intestinal Epithelial Cells

The enterotoxigenic Escherichia coli strains lead to diarrhoea in humans due to heat-labile and heat-stable (STa) enterotoxins. STa increases Cl^-release in intestinal cells, including the human colonic carcinoma T₈₄ cell line, involving increased cGMP and membrane alkalization due to reduced Na⁺/H⁺ exchangers (NHEs) activity. Since NHEs modulate intracellular pH (pH_i), and NHE1, NHE2, and NHE4 are expressed in T₈₄ cells, we characterized the STa role as modulator of these exchangers. pH_i was assayed by the NH₄Cl pulse technique and measured by fluorescence microscopy in BCECF–preloaded cells. pH_i recovery rate (dpHi/dt) was determined in the absence or presence of 0.25 μmol/L STa (30 minutes), 25 μmol/L HOE-694 (concentration inhibiting NHE1 and NHE2), 500 μmol/L sodium nitroprusside (SNP, spontaneous nitric oxide donor), 100 μmol/L dibutyryl cyclic GMP (db-cGMP), 100 nmol/L H89 (protein kinase A inhibitor), or 10 μmol/L forskolin (adenylyl cyclase activator). cGMP and cAMP were measured in cell extracts by radioimmunoassay, and buffering capacity (ßi) and H⁺ efflux (J _H ⁺) was determined. NHE4 protein abundance was determined by western blotting. STa and HOE-694 caused comparable reduction in dpHi/dt and J _H ⁺ (~63%), without altering basal pH_i (range 7.144–7.172). STa did not alter ßi value in a range of 1.6 pH_i units. The dpHi/dt and J _H ⁺ was almost abolished (~94% inhibition) by STa + HOE-694. STa effect was unaltered by db-cGMP or SNP. However, STa and forskolin increased cAMP level. STa–decreased dpHi/dt and J _H ⁺ was mimicked by forskolin, and STa + HOE-694 effect was abolished by H89. Thus, incubation of T₈₄ cells with STa results in reduced NHE4 activity leading to a lower capacity of pH_i recovery requiring cAMP, but not cGMP. STa effect results in a causal phenomenon (STa/increased cAMP/increased PKA activity/reduced NHE4 activity) ending with intracellular acidification that could have consequences in the gastrointestinal cells function promoting human diarrhoea.