Dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior

Mupanomunda, Maria, Jeffrey F. Williams, Charles D. Mackenzie, and Lana Kaiser. Dirofilaria immitis:heartworm infection alters pulmonary artery endothelial cell behavior.J. Appl. Physiol. 82(2): 389-398, 1997.Thepathogenesis of filariasis has generally been attributed to eitherphysical presence of the adult parasites or the host's immune responseto the parasites. However, the spectrum of filariasis cannot beentirely explained by these causes, and other mechanisms must beoperative. It is now evident that factors released by filarialparasites likely contribute to the pathogenesis of filarial diseases.Adult heartworms (Dirofilaria immitis) reside in the rightheart and pulmonary artery, so the pulmonary artery should be exposedto the highest concentration of filarial factors. We tested thehypothesis that endothelium-dependent relaxation is altered in the invitro pulmonary artery from heartworm-infected dogs. Relaxationresponses to endothelium-dependent vasodilators (methacholine,bradykinin, substance P, and A-23187) and the non-endothelium-dependent vasodilator nitroglycerin and contractile responses were measured inrings of pulmonary artery from control and heartworm-infected dogs.Endothelium-dependent relaxation was assessed in the presence andabsence of inhibitors of nitric oxide synthase, cyclooxygenase, andguanylate cyclase. Responses to methacholine, substance P, and A-23187,but not to bradykinin, nitroglycerin, norepinephrine, or KCl, weredepressed in pulmonary artery from heartworm-infected dogs whencompared with control, suggesting that changes in endothelial cell andnot vascular smooth muscle behavior are involved in altered relaxation.The mechanism of endothelium-dependent relaxation in control pulmonaryartery appears to involve nitric oxide in the case of methacholine andboth nitric oxide and a cyclooxygenase product in the case ofbradykinin and A-23187. The mechanism of endothelium-dependentrelaxation in pulmonary artery from heartworm-infected dogs was notclearly elucidated. These data provide no evidence that heartworminfection globally influences either endothelial cell receptor functionor the vascular smooth muscle guanylate cyclase guanosine 3,5-cyclicmonophosphate system, making it likely that changes in intracellularsignaling are primarily responsible for the observed alteration ofendothelium-mediated relaxation. Alteration of endothelial cellfunction by filarial parasites may be an important component inthe pathology associated with filariasis.

     

Recombinant human prorenin from CHO cells: Expression and purification

The gene for human preprorenin was obtained from total RNA prepared from primary human chorion cells. An expression vector was constructed containing an SV40 early promoter, a human preprorenin cDNA, bovine growth hormone poly-A addition signal, and a dihydrofolate reductase (dhfr) expression cassette. This vector was inserted into the DXB-11 Chinese hamster ovary (CHO) cell line. The recombinant protein was exported by CHO cells into the tissue culture media. At harvest the prorenin levels ranged from 1–5 mg/L. For prorenin isolation the cell culture supernatants were processed by filtration, concentration, dialysis, and batch extraction. Preparative-scale isolation of prorenin was accomplished using blue-dye chromatography and size-exclusion chromatography. The isolated prorenin yielded a single SDS-gel band with Mr 40,000. The proprotein was characterized with respect to N-terminal sequence and N-linked sugar composition. Trypsin-activated renin prepared from the proprotein was characterized with respect to N-terminal sequence andpH-activity profile. Enzyme activity was measured with a newly developed fluorogenic peptide substrate containing the P₆-P₃ sequence of human angiotensinogen.     

Gap junctions assemble in the presence of cytoskeletal inhibitors, but enhanced assembly requires microtubules

The role of cytoskeletal elements in gap junction (GJ) assembly has been studied using Novikoff hepatoma cells treated with cytochalasin B (CB) to disrupt actin filaments or with colchicine or nocodazole to disrupt microtubules. After 60 min of cell reaggregation, freeze-fracture was used to evaluate quantitatively the "initiation," "maturation," and "growth" phases of GJ assembly. The development of junctional permeability to fluorescent dyes was also analyzed. The only effects of CB on the structure or permeability of the developing junctions involved an elongation of GJ aggregates and a small decrease in formation plaque areas. Colchicine (but not the inactive form, lumicolchicine) prevented the enhancement of GJ growth by cholesterol, but its effect on basal growth was equivocal. Nocodazole inhibited the growth of GJ, even under basal conditions, without an effect on initiation. Nocodazole also blocked the forskolin-enhanced increase in the growth of GJs and, in living MDCK cells, reduced the movement of transport intermediates containing green fluorescent protein-tagged connexin43. Thus, neither actin filaments nor microtubules appear to restrict GJ assembly by anchoring intramembrane GJ proteins, nor are they absolutely required for functional GJs to form. However, microtubules are necessary for enhanced GJ growth and likely for facilitating connexin trafficking under basal conditions.     

Intertidal zonation of benthic macrofauna in a subtropical salt marsh and nearby unvegetated flat (SE, Brazil)

Intertidal zonation and seasonal variability of benthic macrofaunawere analysed along a Spartina alterniflora (Loisel) marshand nearby unvegetated flat in a subtropical bay. Fivereplicate samples were taken along six tidal levels from the uppermarsh, limited by mangroves, to the lower unvegetated flat.Sediment composition, live and dead above- and below-ground biomassof S. alterniflora and detritus biomass from the vegetatedand unvegetated areas were determined to evaluate whethervariations on plant structure and detritus along the 25 m transectaffect the dynamics of benthic invertebrates. Composition andabundance of invertebrates varied with the elevation and plantcover clearly plays a key role on the distribution patterns of themacrofauna. Below-ground and dead above-ground biomass presentedthe highest correlation with the densities of the invertebrates.Vertical distribution of benthic fauna, however, appears not to beaffected by bellow-ground fraction. Higher detritus biomass in theupper unvegetated flat coincided with higher densities ofdetritivorous or omnivorous species in this level. An eruptivealgal growth during summer affected positively most of the dominantspecies.     

Comparative biological characterization of Berenice and Berenice-78 strains of Trypanosoma cruzi isolated from the same patient at different times

The Berenice-78 strain of T. cruzi is very different from the Berenice strain isolated 16 years earlier from the same patient. The authors verified its high infectivity and low virulence for C3H inbred mice that survived the acute phase of infection. In these animals, it was verified that the tropism of parasites was more accentuated for cardiac and skeletal musculature and the parasitaemic level progressively increased with successive blood passages with posterior stability. In relation to Berenice strain the same characteristics were observed as described by Brener, Chiari & Alvarenga (1974). The increase in its virulence for albino mice was again demonstrated. The authors discussed the possibility of reinfection of the patient called Berenice and the importance of knowledge about T. cruzi strains of low virulence for laboratory animals.     

Evaluation of Cell Type Annotation R Packages on Single-cell RNA-seq Data

Annotating cell types is a critical step in single-cell RNA sequencing(scRNA-seq) data analysis. Some supervised or semi-supervised classification methods have recently emerged to enable automated cell type identification. However, comprehensive evaluations of these methods are lacking. Moreover, it is not clear whether some classification methods originally designed for analyzing other bulk omics data are adaptable to scRNA-seq analysis. In this study, we evaluated ten cell type annotation methods publicly available as R packages. Eight of them are popular methods developed specifically for single-cell research, including Seurat, scmap, SingleR, CHETAH, SingleCellNet, scID, Garnett, and SCINA. The other two methods were repurposed from deconvoluting DNA methylation data, i.e., linear constrained projection(CP) and robust partial correlations(RPC). We conducted systematic comparisons on a wide variety of public scRNA-seq datasets as well as simulation data. We assessed the accuracy through intra-dataset and inter-dataset predictions; the robustness over practical challenges such as gene filtering, high similarity among cell types, and increased cell type classes; as well as the detection of rare and unknown cell types. Overall, methods such as Seurat, SingleR, CP, RPC, and SingleCellNet performed well, with Seurat being the best at annotating major cell types. Additionally, Seurat, SingleR, CP, and RPC were more robust against downsampling. However, Seurat did have a major drawback at predicting rare cell populations, and it was suboptimal at differentiating cell types highly similar to each other,compared to SingleR and RPC. All the code and data are available from https://github.com/qianhuiSenn/scRNA_cell_deconv_benchmark.     

Radiotherapy during the COVID-19: a review about management and treatment strategies

BackgroundThe administration of radiotherapy should be encouraged despite the emergency of COVID-19; therefore, our aim is to analyze management and therapeutic interventions to be implemented in a Radiotherapy department to allow patients to continue their treatment and health professionals to continue their work safely.Materials and methodsA Pubmed search was performed, in which all articles specific to Radiotherapy and COVID-19 were included. Those articles that were too specific about the COVID-19, surgery and chemiotherapy, were excluded.Results315 articles were selected, of which 35 were about therapeutic strategies and 25 about management strategies. In the first category, 5 articles were about how radiotherapy could be a weapon to be used for COVID-19 positive patients with important lung problems. While 30 articles described priorities and new treatment plans for oncology patients who have to undergo radiotherapy during the pandemic. In the second category, almost all the articles explained how triage can be a preventive and monitoring way against COVID-19 in an operating unit with many patients and professionals, and other articles developed a telemedicine system, too, which allows patients to make scheduled visits without coming to the hospital and also for the staff, who can work remotely. In addition, 5 articles concerning psychological aspects of both patients and health care providers were included.ConclusionThis document can be used as a summary in the coming months/years, during the recovery phase from COVID-19 pandemic outbreak and as a starting point to be used in case of further pandemic break-out.     

Genome-Wide Association Study of Serum Creatinine Levels during Vancomycin Therapy

Vancomycin, a commonly used antibiotic, can be nephrotoxic. Known risk factors such as age, creatinine clearance, vancomycin dose / dosing interval, and concurrent nephrotoxic medications fail to accurately predict nephrotoxicity. To identify potential genomic risk factors, we performed a genome-wide association study (GWAS) of serum creatinine levels while on vancomycin in 489 European American individuals and validated findings in three independent cohorts totaling 439 European American individuals. In primary analyses, the chromosome 6q22.31 locus was associated with increased serum creatinine levels while on vancomycin therapy (most significant variant rs2789047, risk allele A, β = -0.06, p = 1.1 x 10^-7). SNPs in this region had consistent directions of effect in the validation cohorts, with a meta-p of 1.1 x 10^-7. Variation in this region on chromosome 6, which includes the genes TBC1D32/C6orf170 and GJA1 (encoding connexin43), may modulate risk of vancomycin-induced kidney injury.