DVPred: a disease-specific prediction tool for variant pathogenicity classification for hearing loss.

Numerous computational prediction tools have been introduced to estimate the functional impact of variants in the human genome based on evolutionary constraints and biochemical metrics. However, their implementation in diagnostic settings to classify variants faced challenges with accuracy and validity. Most existing tools are pan-genome and pan-diseases, which neglected gene- and disease-specific properties and limited the accessibility of curated data. As a proof-of-concept, we developed a disease-specific prediction tool named Deafness Variant deleteriousness Prediction tool (DVPred) that focused on the 157 genes reportedly causing genetic hearing loss (HL). DVPred applied the gradient boosting decision tree (GBDT) algorithm to the dataset consisting of expert-curated pathogenic and benign variants from a large in-house HL patient cohort and public databases. With the incorporation of variant-level and gene-level features, DVPred outperformed the existing universal tools. It boasts an area under the curve (AUC) of 0.98, and showed consistent performance (AUC = 0.985) in an independent assessment dataset. We further demonstrated that multiple gene-level metrics, including low complexity genomic regions and substitution intolerance scores, were the top features of the model. A comprehensive analysis of missense variants showed a gene-specific ratio of predicted deleterious and neutral variants, implying varied tolerance or intolerance to variation in different genes. DVPred explored the utility of disease-specific strategy in improving the deafness variant prediction tool. It can improve the prioritization of pathogenic variants among massive variants identified by high-throughput sequencing on HL genes. It also shed light on the development of variant prediction tools for other genetic disorders.

LOVD-DASH: A comprehensive LOVD database coupled with diagnosis and an at-risk assessment system for hemoglobinopathies.

Hemoglobinopathies are the most common monogenic disorders worldwide. Substantial effort has been made to establish databases to record complete mutation spectra causing or modifying this group of diseases. We present a variant database which couples an online auxiliary diagnosis and at-risk assessment system for hemoglobinopathies (DASH). The database was integrated into the Leiden Open Variation Database (LOVD), in which we included all reported variants focusing on a Chinese population by literature peer review-curation and existing databases, such as HbVar and IthaGenes. In addition, comprehensive mutation data generated by high-throughput sequencing of 2,087 hemoglobinopathy patients and 20,222 general individuals from southern China were also incorporated into the database. These sequencing data enabled us to observe disease-causing and modifier variants responsible for hemoglobinopathies in bulk. Currently, 371 unique variants have been recorded; 265 of 371 were described as disease-causing variants, whereas 106 were defined as modifier variants, including 34 functional variants identified by a quantitative trait association study of this high-throughput sequencing data. Due to the availability of a comprehensive phenotype-genotype data set, DASH has been established to automatically provide accurate suggestions on diagnosis and genetic counseling of hemoglobinopathies. LOVD-DASH will inspire us to deal with clinical genotyping and molecular screening for other Mendelian disorders.

Synergistic antibacterial effects of closantel and its enantiomers in combination with colistin against multidrug resistant gram-negative bacteria.

Drug combinations and repurposing have recently provided promising alternatives to cope with the increasingly severe issue of antibiotic resistance and depletion of natural drug molecular repertoires that undermine traditional antibacterial strategies. Closantel, an effective adjuvant, reverses antibiotic resistance in gram-negative bacteria. Herein, the combined antibacterial enantioselectivity of closantel is presented through separate enantiomer studies. Despite yielding unexpected differences, two closantel enantiomers (R, S) increased colistin activity against gram-negative bacteria both in vitro and in vivo. The fractional inhibitory concentration indices of R-closantel and S-closantel combined with colistin against Pseudomonas aeruginosa, Klebsiella pneumoniae, and Escherichia coli ranged from 0.0087 to 0.5004 and from 0.0117 to 0.5312, respectively. This difference was further demonstrated using growth inhibition assays and time-killing curves. Mechanistically, a higher intracellular concentration of R-CLO is more effective in enhancing the antimicrobial activity of combination. A mouse cutaneous infection model confirmed the synergistic stereoselectivity of closantel. This discovery provides novel insights for developing precision medication and containment of increasing antibiotic resistance.

Stretch of human mesothelial cells increases cytokine expression.

Peritoneal dialysis requires large solution volumes that increase abdominal girth and stretch the mesothelial cells of the abdominal wall. To address the hypothesis that stretch stimulates those cells to increase synthesis and production of inflammatory cytokines, we grew MeT-5A human mesothelial cells to confluence and placed the cells in growth arrest on BioFlex Collagen membranes (Flexcell International Corporation, Hillsborough, NC, U.S.A.). After 48 hours, cells were either left stationary (STA) or cycled using a 3000T system (Flexcell International Corporation) with a sinusoidal stretch (STR) frequency of 10 cycles per minute and an amplitude of 30%. The supernatant and cells of individual wells were removed at 0, 4, 12, 24, 48, 72, and 96 hours. Supernatant was assayed by ELISA for transforming growth factor beta (TGF-beta), interleukin 6 (IL-6), and vascular endothelial growth factor (VEGF). After trypsinization, the total number of viable cells in each well was estimated from the lack of trypan blue staining. Total RNA from the cells was extracted, and real-time reverse-transcriptase polymerase chain reaction was used to determine messenger RNA (mRNA) for IL-6, TGF-beta, VEGF, TGF-beta receptors 2 and 3 (TGF-beta-R2, -R3), kinase insert domain receptor (KDR), and FMS-related tyrosine kinase 1 (Flt1). Because of decline of cell numbers and viability, results for STR were compared with those for STA at each time interval up to 72 hours. The mRNA for TGF-beta, VEGF, TGF-beta-R3, and KDR were significantly higher in the STR group throughout the 72 hours, and STR IL-6 mRNA declined nonsignificantly. Normalized to the number of viable cells, supernatant IL-6, TGF-beta, and VEGF were not significantly different between the groups. We conclude that mechanical stress from mesothelial stretch does not enhance mesothelial cell secretion oflL-6, TGF-f, or VEGF, but does increase expression ofTGF-P, VEGF, and their corresponding cell receptors TGF-f-R3 and KDR.

Mouse model of foreign body reaction that alters the submesothelium and transperitoneal transport.

To address the hypothesis that sterile intraperitoneal (ip) catheters alone promote a progressive foreign body reaction (FBR), silicone catheters were surgically implanted in C57BL mice. Controls (CON) underwent sham operations. After 1-5 wk (E1-E5 for catheter-bearing mice), catheters were recovered, and the adherent cell layer (ACL) was separated and cultured to demonstrate sterility. Transperitoneal transport experiments were performed to determine the mass transfer coefficients of mannitol (MTCM) and albumin (MTCA) and the osmotic filtration flux (Josm). After euthanasia, tissue samples were analyzed for submesothelial thickness, angiogenesis, and cytokine immunohistochemistry (IHC). Progressive increases with time were observed in submesothelial thickness (µm: CON, 18.8±12.3; E1, 46.1±20.0; E2, 72.0±17.9; E4, 97.3±20.0; E5, 131.7±10.3; P<0.003), angiogenesis (no. of vessels/mm of peritoneum: CON, 10.7±9.4; E1, 15.4±15.6; E2, 27.0±14.0; E4, 39.8±15.7; E5, 90.1±8.1; P<0.0003), MTCA (6.5±1.5×10(-5) cm/min, mean CON; 18.0±1.1×10(-5) cm/min, mean E1-E5, P<0.0001), Josm (0.0013±0.0001 cm/min, mean CON; 0.0017±0.0001 cm/min, mean E1-E5, P<0.01). No significant differences were found for MTCM. IHC demonstrated strong staining for all treated animals and correlated with the ACL. This mouse model demonstrates that ip silicone catheters result in progressive FBR, altering the submesothelial anatomy and transperitoneal transport, and will form the basis for mechanistic studies in genetically-altered animals.

Similitude of transperitoneal permeability in different rodent species.

Transgenic mice facilitate mechanistic studies of altered peritoneal transport, but the majority of transport studies have been carried out in rats. We hypothesized that mouse transport parameters, normalized to the peritoneal contact area, would be similar to those of the rat. To address this, we affixed small ( approximately 10-mm diameter) plastic chambers to the serosa of the abdominal wall of anesthetized CD1 and C57BL mice. The chamber constrained transfer across the area of the chamber base and facilitated mixing, volumetric, and concentration measurements vs. time for mannitol, serum albumin, and osmotic and hydrostatic pressure-driven convection. The mass transfer coefficient of mannitol (MTC(M)) and of serum albumin (MTC(BSA)), hydrostatic pressure-driven flux (J(P)), and osmotic filtration (J(osm)) were calculated from the time-dependent volume and concentration data. The units of all parameters (microl x min(-1) x cm(-2)) were compared with previously derived parameters from SD rats with a one-way ANOVA. Results indicated small but significant differences in MTC(BSA) (x10(2)): CD1, 9.72 +/- 1.97, n = 6; C57BL, 7.13 +/- 1.52, n = 10; rat, 12.5 +/- 1.6, n = 17 (P = 0.03). ANOVAs of all other parameters were not significant and confirmed our hypothesis: MTC(M) (CD1, 3.20 +/- 0.38, n = 7; C57BL, 2.34 +/- 0.41, n = 6; rat, 2.72 +/- 0.23 n = 19), J(P) (CD1, 0.77 +/- 0.15, n = 10; C57BL, 0.33 +/- 0.13, n = 15; rat, 0.51 +/- 0.16, n = 9), or J(osm) (CD1, 0.92 +/- 0.35, n = 6; C57BL, 0.49 +/- 0.35, n = 6; rat 1.72 +/- 0.35, n = 6). We conclude that elimination of the variable peritoneal transfer area normalizes calculated transport characteristics and facilitates comparison between species.

In vivo determination of diffusive transport parameters in a superfused tissue.

To address the hypothesis that functional changes in tissue transport can be related to structural alterations, we combined mathematical modeling with in vivo experimentation. The model concept includes interstitial diffusion and removal by a distributed microvasculature. Transport of solute and water across the peritoneum is measured via a plastic chamber affixed to the abdominal wall of anesthetized Sprague-Dawley rats. Solutions containing [(14)C]mannitol, with or without vasoactive compounds [control (C; n = 10), C + nitroprusside (NP; n = 10), C + norepinephrine (NE; n = 10)], were infused into the chamber, and the volume and tracer concentrations were determined over 60 min to calculate the mass transfer coefficient (MTC) and the water flux. At 60 min, FITC-dextran (500 kDa) was given to mark the perfused vasculature. After euthanasia, the tissue under the chamber was frozen, dried, sliced with a cryomicrotome, and examined with fluorescent microscopy and quantitative autoradiography. The microvessel density (x10(3)/cm(2): NE, 50 +/- 10; C, 180 +/- 7.0; NP, 225 +/- 15) resulted in marked differences (P < 0.05) in water flux (mul.min(-1).cm(-2): NE, 0.1 +/- 0.1; C, 1.6 +/- 0.4; NP, 1.0 +/- 0.2) and in mannitol MTC (x10(3) cm/min: NE, 0.9 +/- 0.3; C, 3.8 +/- 0.3; NP, 3.6 +/- 0.6). Concentration profiles and calculated capillary permeability and tissue diffusivity were significantly different among the groups. These results demonstrate a direct correlation of mass transfer, diffusion, capillary permeability, and water flux with peritoneal vascular density and validate a method by which mechanistic changes in transport may be measured.