Low-Cost Polyester-Tipped Three-Dimensionally Printed Nasopharyngeal Swab for the Detection of Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2).

Case identification, isolation, and contact tracing are fundamental strategies used to control the spread of coronavirus disease 2019 (COVID-19). This has led to widespread testing that interrupted the supply chain for testing materials around the world. A prospective study was conducted to compare inexpensive and easily sourced 3-dimensionally (3D)-printed polylactic acid and polyester nasopharyngeal swabs to commercially manufactured swabs for the detection of severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2). During the study period, 287 laboratory-confirmed hospitalized COVID-19 patients, at multiple stages of their illness, were enrolled. The median age for the study population was 47.6 years (interquartile range [IQR], 34.4 to 56.6 years), and two-thirds (67.6%) of the subjects were male. The median duration of hospitalization, at the time of sampling, was 13 days (IQR, 10 to 16 days). Overall concordance between the prototype and control swabs was 80.8% (Cohen's kappa coefficient, 0.61). Most discrepant results were due to prototype-positive control-negative results. When considering all positive results to be true positives, the prototype swab had a higher sensitivity (90.6% versus 80.8%; 95% confidence interval [CI], 85.7% to 94.0% and 74.7% to 85.7%, respectively; P < 0.015). The cost to produce the prototype swab was estimated to be $0.05 per swab. Polylactic acid 3D-printed polyester-tipped swabs were shown to be effective for nasopharyngeal sample collection. We believe that this design can easily be adopted in countries where commercial swabs are not readily available and can play a vital role in public health efforts for disease control in low-income countries.

A heterozygous mutation in GJB2 (Cx26F142L) associated with deafness and recurrent skin rashes results in connexin assembly deficiencies.

Mutations in GJB2 encoding Connexin 26 (CX26) are associated with hearing loss and hyperproliferative skin disorders of differing severity including keratitis-ichthyosis-deafness (KID) and Vohwinkel syndrome. A 6-year-old Caucasian girl who presented with recurrent skin rashes and sensorineural hearing loss harboured a heterozygous point mutation in GJB2 (c.424T > C; p.F142L). To characterize the impact of CX26F142L on cellular events. Plasmids CX26WT, CX26F142L, CX26G12R (KID) or CX26D66H (Vohwinkel) were transfected into HeLa cells expressing Cx26 or Cx43 or into HaCaT cells, a model keratinocyte cell line. Confocal microscopy determined protein localization. MTT assays assessed cell viability in the presence or absence of carbenoxolone, a connexin-channel blocker. Co-immunoprecipitation/Western blot analysis determined Cx43:Cx26 interactions. Quantitative real-time polymerase chain reaction assessed changes in gene expression of ER stress markers. Dye uptake assays determined Connexin-channel functionality. F142L and G12R were restricted to perinuclear areas. Collapse of the microtubule network, rescued by co-treatment with paclitaxel, occurred. ER stress was not involved. Cell viability was reduced in cells expressing F142L and G12R but not D66H. Unlike G12R that forms "leaky" hemichannels, F142L had restricted permeability. Cell viability of F142L and G12R transfected cells was greater in HeLa cells expressing Cx43 than in native Cx-free HeLa cells. Co-immunoprecipitation suggested a possible interaction between Cx43 and the three mutations. Expression of CX26F142L and G12R results in microtubule collapse, rescued by interaction with Cx43. The GJB2 mutations interacted with Cx43 suggesting that unique Cx43:Cx26 channels are central to the diverse phenotype of CX26 skin-related channelopathies.

A rare missense mutation in GJB3 (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death.

Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (Cx30.3). We identified a rare GJB3 missense mutation, c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics. Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype.

Connexins and the Epithelial Tissue Barrier: A Focus on Connexin 26.

Epithelial tissue responds rapidly to environmental triggers and is constantly renewed. This tissue is also highly accessible for therapeutic targeting. This review highlights the role of connexin mediated communication in avascular epithelial tissue. These proteins form communication conduits with the extracellular space (hemichannels) and between neighboring cells (gap junctions). Regulated exchange of small metabolites less than 1kDa aide the co-ordination of cellular activities and in spatial communication compartments segregating tissue networks. Dysregulation of connexin expression and function has profound impact on physiological processes in epithelial tissue including wound healing. Connexin 26, one of the smallest connexins, is expressed in diverse epithelial tissue and mutations in this protein are associated with hearing loss, skin and eye conditions of differing severity. The functional consequences of dysregulated connexin activity is discussed and the development of connexin targeted therapeutic strategies highlighted.