The organization and composition of the extracellular matrix (ECM) have been shown to impact the propagation of electrical signals in multiple tissue types. To date, many studies with electroactive biomaterial substrates have relied upon passive electrical stimulation of the ionic media to affect cell behavior. However, development of cell culture systems in which stimulation can be directly applied to the material – thereby isolating the signal to the cell-material interface and cell-cell contracts – would provide a more physiologically-relevant paradigm for investigating how electrical cues modulate lineage-specific stem cell differentiation. In the present study, we have employed unmodified, directly-stimulated, (un)patterned graphene as a cell culture substrate to investigate how extrinsic electrical cycling influences the differentiation of naïve human mesenchymal stem cells (hMSCs) without the bias of exogenous biochemicals. We first demonstrated that cyclic stimulation does not deteriorate the cell culture media or result in cytotoxic pH, which are critical experiments for correct interpretation of changes in cell behavior. We then measured how the expression of osteogenic and neurogenic lineage-specific markers were altered simply by exposure to electrical stimulation and/or physical patterns. Expression of the early osteogenic transcription factor RUNX2 was increased by electrical stimulation on all graphene substrates, but the mature marker osteopontin was only modulated when stimulation was combined with physical patterns. In contrast, the expression of the neurogenic markers MAP2 and β3-tubulin were enhanced in all electrical stimulation conditions, and were less responsive to the presence of patterns. These data indicate that specific combinations of non-biological inputs – material type, electrical stimulation, physical patterns – can regulate hMSC lineage specification. This study represents a substantial step in understanding how the interplay of electrophysical stimuli regulate stem cell behavior and helps to clarify the potential for graphene substrates in tissue engineering applications.

The endothelial cell adhesion molecule, CD146, is expressed on ≈ 2% of normal circulating T cells, correlating with T cell activation, endothelial interactions and T helper type 17 (Th17) effector functions. In this study, we have characterized CD146 expression in circulating T cells from healthy controls and patients with stable, well-controlled autoimmune connective tissue diseases (CTDs). In vitro, anti-CD3/anti-CD28 stimulation induced CD146 expression in both CD4 and CD8 T cells. In healthy controls and CTD patients, CD146 was associated with expression of recent and chronic activation markers (CD25(+), OX-40(+), CD69(+), CD27(-)) and was confined to CD45RO(+)/RA(-)/CD28(+) populations within the CD4 subset. Except for CD69, these markers were not associated with CD146 in the CD8 subset. Surprisingly, most CTD patients exhibited no T cell hyperactivation ex vivo. In five of five patients with secondary Sjögren's syndrome circulating T cells appeared activated despite therapy, and CD146 up-regulation, associated with activation markers, was observed both on CD4 and CD8 T cells. There was no association between CD146 and putative pro-atherogenic T cell subsets. In conclusion, the relationship of CD146 expression to T cell activation differs between T cell subsets in healthy subjects and correlates with systemic hyperactivity, where present, in patients with CTDs, as exemplified by the patients with secondary Sjögren's syndrome in this study.

ImportanceMost studies of autosomal dominant polycystic kidney disease (ADPKD) genetics have used kidney specialty cohorts, focusing on PKD1 and PKD2. These can lead to biased estimates of population prevalence of ADPKD-associated gene variants and their phenotypic expression.ObjectiveTo determine the prevalence of ADPKD and contributions of PKD1, PKD2, and other genes related to cystic kidney disease in a large, unselected cohort.Design, Setting, and ParticipantsThis retrospective observational study used an unselected health system–based cohort in central and northeast Pennsylvania with exome sequencing (enrolled from 2004 to 2020) and electronic health record data (up to October 2021). The genotype-first approach included the entire cohort and the phenotype-first approach focused on patients with ADPKD diagnosis codes, confirmed by chart and imaging review.ExposuresLoss-of-function (LOF) variants in PKD1, PKD2, and other genes associated with cystic kidney disease (ie, ALG8, ALG9, DNAJB11, GANAB, HNF1B, IFT140, SEC61B, PKHD1, PRKCSH, SEC63); likely pathogenic missense variants in PKD1 and PKD2.Main Outcomes and MeasuresGenotype-first analysis: ADPKD diagnosis code (Q61.2, Q61.3, 753.13, 753.12); phenotype-first analysis: presence of a rare variant in PKD1, PKD2, or other genes associated with cystic kidney disease.ResultsOf 174 172 patients (median age, 60 years; 60.6% female; 93% of European ancestry), 303 patients had ADPKD diagnosis codes, including 235 with sufficient chart review data for confirmation. In addition to PKD1 and PKD2, LOF variants in IFT140, GANAB, and HNF1B were associated with ADPKD diagnosis after correction for multiple comparisons. Among patients with LOF variants in PKD1, 66 of 68 (97%) had ADPKD; 43 of 43 patients (100%) with LOF variants in PKD2 had ADPKD. In contrast, only 24 of 77 patients (31.2%) with a PKD1 missense variant previously classified as “likely pathogenic” had ADPKD, suggesting misclassification or variable penetrance. Among patients with ADPKD diagnosis confirmed by chart review, 180 of 235 (76.6%) had a potential genetic cause, with the majority being rare variants in PKD1 (127 patients) or PKD2 (34 patients); 19 of 235 (8.1%) had variants in other genes associated with cystic kidney disease. Of these 235 patients with confirmed ADPKD, 150 (63.8%) had a family history of ADPKD. The yield for a genetic determinant of ADPKD was higher for those with a family history of ADPKD compared with those without family history (91.3% [137/150] vs 50.6% [43/85]; difference, 40.7% [95% CI, 29.2%-52.3%]; P < .001). Previously unreported PKD1, PKD2, and GANAB variants were identified with pedigree data suggesting pathogenicity, and several PKD1 missense variants previously reported as likely pathogenic appeared to be benign.Conclusions and RelevanceThis study demonstrates substantial genetic and phenotypic variability in ADPKD among patients within a regional health system in the US.

Background and study aims: Capsule endoscopy is an attractive alternative to colorectal cancer screening by conventional colonoscopy, but is currently limited by compromised mucosal visibility because of the lack of safe, controlled colonic insufflation. We have therefore developed a novel system of untethered, wireless-controlled carbon dioxide (CO2) insufflation for use in colonic capsule endoscopy, which this study aims to assess in vivo. Material and methods: This observational, nonsurvival, in vivo study used five Yorkshire-Landrace cross swine. A novel insufflation capsule was placed in the porcine colons, and we recorded volume of insufflation, time, force, visualization, and a pathologic assessment of the colon. Results: The mean (standard deviation [SD]) diameter of insufflation was 32.1 (3.9)?mm. The volume of CO2 produced successfully allowed complete endoscopic visualization of the mucosa and safe proximal passage of the endoscope. Pathologic examination demonstrated no evidence of trauma caused by the capsule. Conclusions: These results demonstrate the feasibility of a novel method of controlled colonic insufflation via an untethered capsule in vivo. This technological innovation addresses a critical need in colon capsule endoscopy.

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BackgroundLarge clinical cohorts with electronic health records (EHR) and genetic data allow estimating population prevalence of rare genetic disorders like polycystic kidney disease (PKD) and deciphering their genetics, allowing more precise diagnosis and management.MethodsWe performed genotype- and phenotype-based analyses to gain insight into the prevalence and genetic basis of PKD in an unselected health system-based cohort of 173,954 patients with EHR and exome sequencing data. We determined the diagnostic rate of PKD amongst patients with PKD1/2 variants, reviewed EHR data including imaging and family history when available to phenotype PKD cases of various severity, determined disease prevalence, and identified the genetic basis for most of these cases.ResultsIn genotype-first analyses, individuals with a protein-truncating (PTV) or copy number variant deletion (CNV) in PKD1 and PKD2 had ADPKD rates of 94.3% (66/70) and 97.7% (43/44) respectively. For individuals with missense or in-frame deletion variants previously classified as likely pathogenic (LP) or hypomorphic, ADPKD diagnosis was infrequent (34/425 for PKD1 and 0 /12 for PKD2). Phenotype-first analyses identified 235 ADPKD cases (1.35/1,000), which were further subclassified by imaging findings (196 moderate/severe typical ADPKD, 16 mild typical ADPKD, 23 atypical ADPKD). The genetic diagnostic rate for moderate/severe typical ADPKD was 69% (135/196) and significantly lower for mild typical and atypical ADPKD (25.0% and 21.7% respectively). Specifically, for typical ADPKD, 125/196 (63.7%) had a diagnostic (CNV/PTV/LP) variant in PKD1 or PKD2, two had PKD1 missense variants previously classified as VUS, and 24 had novel PKD1 or PKD2 missense variants, 8 of which segregated with ADPKD in pedigree analysis. Genetic diagnostic yield was much higher in those with a family history of ADPKD than those without a family history.ConclusionsData from a large carefully phenotyped clinical cohort provides the population-based prevalence of PKD and establishes the utility of exome sequencing in ADPKD diagnosis which should lead to earlier diagnosis and improved care for PKD.

Monogenic causes in over 300 kidney-associated genes account for roughly 12% of end stage kidney disease (ESKD) cases. Advances in next generation sequencing, and large customized panels enable the diagnosis of monogenic kidney disease noninvasively at relatively low cost, allowing for more precise management for patients and their families. A major challenge is interpreting rare variants, many of which are classified as variants of unknown significance (VUS). We present a framework in which we thoroughly evaluated and provided evidence of pathogenicity for HNF1B-p.Arg303His, a VUS returned from clinical genetic testing for a kidney transplant candidate. This blueprint, designed by a multi-disciplinary team of clinicians, molecular biologists, and diagnostic geneticists, includes using a health system-based cohort with genetic and clinical information to perform deep phenotyping of VUS carriers, examination of existing genetic databases, as well as functional testing. With our approach, we demonstrate evidence for pathogenicity for HNF1B-p.Arg303His by showing similar burden of kidney manifestations in this variant to known HNF1B pathogenic variants, and greater burden compared to non-carriers. Determination of a molecular diagnosis for the example family allows for proper surveillance and management of HNF1B-related manifestations such as kidney disease, diabetes, and hypomagnesemia with important implications for safe living-related kidney donation. The candidate gene-variant pair also allows for clinical biomarker testing for aberrations of linked pathways. This working model may be applicable other diseases of genetic etiology.