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Epidermal keratinocytes are enriched with at least nine connexins that are key regulators of epidermal homeostasis. The role of Cx30.3 in keratinocytes and epidermal health became evident when fourteen autosomal dominant mutations in the Cx30.3-encoding GJB4 gene were linked to a rare and incurable skin disorder called erythrokeratodermia variabilis et progressiva (EKVP). While these variants are linked to EKVP, they remain largely uncharacterized hindering therapeutic options. In this study, we characterize the expression and functional status of three EKVP-linked Cx30.3 mutants (G12D, T85P, and F189Y) in tissue-relevant and differentiation-competent rat epidermal keratinocytes. We found that GFP-tagged Cx30.3 mutants were non-functional likely due to their impaired trafficking and primary entrapment within the endoplasmic reticulum (ER). However, all mutants failed to increase BiP/GRP78 levels suggesting they were not inducing an unfolded protein response. FLAG-tagged Cx30.3 mutants were also trafficking impaired yet occasionally exhibited some capacity to assemble into gap junctions. The pathological impact of these mutants may extend beyond their trafficking deficiencies as keratinocytes expressing FLAG-tagged Cx30.3 mutants exhibited increased propidium iodide uptake in the absence of divalent cations. Attempts to rescue the delivery of trafficking impaired GFP-tagged Cx30.3 mutants into gap junctions by chemical chaperone treatment were ineffective. However, co-expression of wild type Cx30.3 greatly enhanced the assembly of Cx30.3 mutants into gap junctions, although endogenous levels of Cx30.3 do not appear to prevent the skin pathology found in patients harboring these autosomal dominant mutations. In addition, a spectrum of connexin isoforms (Cx26, Cx30, and Cx43) exhibited the differential ability to trans-dominantly rescue the assembly of GFP-tagged Cx30.3 mutants into gap junctions suggesting a broad range of connexins found in keratinocytes may favourably interact with Cx30.3 mutants. We conclude that selective upregulation of compatible wild type connexins in keratinocytes may have potential therapeutic value in rescuing epidermal defects invoked by Cx30.3 EKVP-linked mutants.

Pannexin 1 (PANX1) is upregulated in many cancers, where its channel activity and signalling promote tumorigenic properties. Here, we report that potential internal translation start sites exist in mouse and human PANX1 which have implications in trafficking and protein interaction. Using mouse PANX1 constructs for each internal methionine (M) we saw that the shorter PANX1 isoforms were glycosylated, able to traffic to the cell surface and PANX1-M37 formed channels which could be activated by C-terminus cleavage or α1-adrenoceptor stimulation. Furthermore, we report a novel ∼25 kDa isoform of human PANX1 (hPANX1-25K) which lacks the N-terminus and was detected in several human cancer cell lines including melanoma, osteosarcoma, breast cancer, and glioblastoma multiforme. This isoform was increased upon CRISPR/Cas9 deletion targeting the first exon near M1, and using Expasy PeptideCutter we did not find any evidence of hPANX1 cleavage sites which would produce a 25 kDa fragment, suggesting a potential alternative translation initiation site as the source of hPANX1-25K. hPANX1-25K was confirmed to be a hPANX1 isoform via mass spectrometry, can be N-linked glycosylated at multiple sites including the canonical N255 and novel N338 and N394 residues, and can interact with both β-catenin and full length hPANX1. Using cell surface biotinylation and immunocytochemistry, we also determined hPANX1-25K exhibits a predominantly intracellular localization. hPANX1-25K is prevalent throughout melanoma progression, and its levels are increased in squamous cell carcinoma cells and patient-derived tumours, compared to keratinocytes and patient-matched normal skin, indicating that it may be differentially regulated in normal and cancer cells.

In this study, we explored the intricate relationship between Pannexin 1 (PANX1) and the Hippo signaling pathway effector, Yes-associated protein (YAP). Analysis of The Cancer Genome Atlas (TCGA) data revealed a significant positive correlation between PANX1 mRNA and core Hippo components, YAP, TAZ, and Hippo scaffold, IQGAP1, in invasive cutaneous melanoma and breast carcinoma. Furthermore, we demonstrated that PANX1 expression is upregulated in invasive melanoma cell lines and is associated with increased YAP protein levels. Notably, our investigations uncovered a previously unrecognized interaction between endogenous PANX1 and the Hippo scaffold protein IQGAP1 in melanoma cells. Moreover, our findings revealed that IQGAP1 exhibits differential expression in melanoma cells and plays a regulatory role in cellular morphology. Functional studies involving PANX1 knockdown provided compelling evidence that PANX1 modulates YAP protein levels and its co-transcriptional activity in both melanoma and breast carcinoma cells. Importantly, our study showcases the potential therapeutic relevance of targeting PANX1, as pharmacological inhibition of PANX1 using selective FDA-approved inhibitors or PANX1 knockdown reduced YAP abundance in melanoma cells. Furthermore, our Clariom S analysis unveiled key genes implicated in cell proliferation, such as neuroglin1 (NRG1), β-galactoside binding protein, galectin-3 (LGALS3), that are affected in PANX1-deficient cells. In summary, our investigation delves into the intricate interplay between PANX1 and YAP in the context of invasive melanoma, offering valuable insights into potential therapeutic strategies for effective treatment.

Clinical outcome of patients with acute myeloid leukemia (AML) is associated with demographic and genetic features. Although the associations of acquired genetic alterations with patients’ sex have been recently analyzed, their impact on outcome of female and male patients has not yet been comprehensively assessed. We performed mutational profiling, cytogenetic and outcome analyses in 1726 adults with AML (749 female and 977 male) treated on frontline Alliance for Clinical Trials in Oncology protocols. A validation cohort comprised 465 women and 489 men treated on frontline protocols of the German AML Cooperative Group. Compared with men, women more often had normal karyotype, FLT3 -ITD, DNMT3A , NPM1 and WT1 mutations and less often complex karyotype, ASXL1 , SRSF2 , U2AF1 , RUNX1 , or KIT mutations. More women were in the 2022 European LeukemiaNet intermediate-risk group and more men in adverse-risk group. We found sex differences in co-occurring mutation patterns and prognostic impact of select genetic alterations. The mutation-associated splicing events and gene-expression profiles also differed between sexes. In patients aged <60 years, SF3B1 mutations were male-specific adverse outcome prognosticators. We conclude that sex differences in AML-associated genetic alterations and mutation-specific differential splicing events highlight the importance of patients’ sex in analyses of AML biology and prognostication.

Background and Study Aims. The presence of an implantable electromechanical cardiac device (IED) has long been considered a relative contraindication to the performance of video capsule endoscopy (CE). The primary aim of this study was to evaluate the safety of CE in patients with IEDs. A secondary purpose was to determine whether IEDs have any impact on images captured by CE. Patients and Methods. A retrospective chart review of all patients who had a capsule endoscopy at Mayo Clinic in Scottsdale, AZ, USA, or Rochester, MN, USA, (January 2002 to June 2010) was performed to identify CE studies done on patients with IEDs. One hundred and eighteen capsule studies performed in 108 patients with IEDs were identified and reviewed for demographic data, method of preparation, and study data. Results. The most common indications for CE were obscure gastrointestinal bleeding (77%), anemia (14%), abdominal pain (5%), celiac disease (2%), diarrhea (1%), and Crohn’s disease (1%). Postprocedure assessments did not reveal any detectable alteration on the function of the IED. One patient with an ICD had a 25-minute loss of capsule imaging due to recorder defect. Two patients with LVADs had interference with capsule image acquisition. Conclusions. CE did not interfere with IED function, including PM, ICD, and/or LVAD and thus appears safe. Additionally, PM and ICD do not appear to interfere with image acquisition but LVAD may interfere with capsule images and require that capsule leads be positioned as far away as possible from the IED to assure reliable image acquisition.

Daily administration of vitamin D is important for maintaining bone homeostasis. The orthopedic community has shown increased interest in vitamin D supplementation and patient outcomes after fracture. The current study used data from a large hip fracture trial to determine the proportion of patients who consistently used vitamin D after hip fracture surgery and to determine whether supplementation was associated with improved health-related quality of life and reduced reoperation rates. The FAITH study is a multicenter trial of elderly patients with femoral neck fracture treated with internal fixation. The current study asked a subset of patients included in the FAITH study about vitamin D supplementation and categorized them as consistent users, inconsistent users, or nonusers. This study also evaluated whether supplementation was associated with improved quality of life and reduced reoperation rates. The final analysis included 573 patients (mean age, 74.1 years; female, 66.3%; nondis-placed fractures, 72.4%). A total of 18.7% of participants reported no use of vitamin D, 35.6% reported inconsistent use, and 45.7% reported consistent use. Adjusted analysis found that consistent supplementation was associated with a 2.42 increase of the Short Form-12 physical component score 12 months postoperatively ( P =.033). However, supplementation was not associated with reduced reoperation rates ( P =.386). Despite guidelines recommending vitamin D supplementation, a low proportion of elderly patients with hip fracture use vitamin D consistently, suggesting a need for additional strategies to promote compliance. This study found that the use of vitamin D was associated with a statistically significant but not clinically significant improvement in health-related quality of life after hip fracture. Further research is needed to confirm these findings. [ Orthopedics. 2017; 40(5):e868–e875.]

Neuronal insulin resistance is linked to the pathogenesis of Parkinson’s disease through unclear, but potentially targetable, mechanisms. We delineated neuronal and glial mechanisms of insulin resistance and glucagon-like 1 peptide (GLP-1) receptor agonism in human iPSC models of synucleinopathy, and corroborated our findings in patient samples from a Phase 2 trial of a GLP-1R agonist in Parkinson’s (NCT01971242). Human iPSC models of synucleinopathy exhibit neuronal insulin resistance and dysfunctional insulin signalling, which is associated with inhibition of the neuroprotective Akt pathways, and increased expression of the MAPK-associated p38 and JNK stress pathways. Ultimately, this imbalance is associated with cellular stress, impaired proteostasis, accumulation of α-synuclein, and neuronal loss. The GLP-1R agonist exenatide led to restoration of insulin signalling, associated with restoration of Akt signalling and suppression of the MAPK pathways in neurons. GLP-1R agonism reverses the neuronal toxicity associated with the synucleinopathy, through reduction of oxidative stress, improved mitochondrial and lysosomal function, reduced aggregation of α-synuclein, and enhanced neuronal viability. GLP-1R agonism further suppresses synuclein induced inflammatory states in glia, leading to neuroprotection through non cell autonomous effects. In the exenatide-PD2 clinical trial, exenatide treatment was associated with clinical improvement in individuals with higher baseline MAPK expression (and thus insulin resistance). Exenatide treatment led to a reduction of α-synuclein aggregates, and a reduction in inflammatory cytokine IL-6. Taken together, our patient platform defines the mechanisms of GLP-1R action in neurons and astrocytes, identifies the population likely to benefit from GLP-1R agonism, and highlights the utility of GLP-1R agonism as a disease modifying strategy in synucleinopathies.