Alternative mRNA splicing events and regulators in epidermal differentiation.

Alternative splicing (AS) of messenger RNAs occurs in ∼95% of multi-exon human genes and generates diverse RNA and protein isoforms. We investigated AS events associated with human epidermal differentiation, a process crucial for skin function. We identified 6,413 AS events, primarily involving cassette exons. We also predicted 34 RNA-binding proteins (RBPs) regulating epidermal AS, including 19 previously undescribed candidate regulators. From these results, we identified FUS as an RBP that regulates the balance between keratinocyte proliferation and differentiation. Additionally, we characterized the function of a cassette exon AS event in MAP3K7, which encodes a kinase involved in cell signaling. We found that a switch from the short to long isoform of MAP3K7, triggered during differentiation, enforces the demarcation between proliferating basal progenitors and overlying differentiated strata. Our findings indicate that AS occurs extensively in the human epidermis and has critical roles in skin homeostasis.

Approximately half of pediatric or adolescent patients undergoing revision anterior cruciate ligament reconstruction return to the same level of sport or higher: A systematic review.

PURPOSE: To summarise the surgical techniques and clinical outcomes in paediatric and adolescent patients undergoing revision anterior cruciate ligament reconstruction (r-ACLR). METHODS: Three databases (MEDLINE, PubMed and EMBASE) were searched from inception to 29 July 2023. The authors adhered to the PRISMA and R-AMSTAR guidelines as well as the Cochrane Handbook for Systematic Reviews of Interventions. Data on demographics, surgical details, patient-reported outcome measures (PROMs), rates of instability, rupture and return to sport (RTS) were extracted. RESULTS: Eight studies comprising 706 (711 knees) patients were included (48.7% female). The mean age at r-ACLR was 17.1 years (range: 16.5-18.0). Autografts (67.5%) were more common than allografts (32.2%) in revision, with bone-patellar tendon-bone (BPTB) being the most prevalent autograft source (59.6%). Bone grafts were used in seven patients (4.8% of 146 patients). The most common femoral and tibial fixation techniques were interference screws (37.6% and 38.1%, of 244 patients, respectively). The most common tunnelling strategy was anatomic (69.1% of 236 patients), and meniscus repairs were performed in 39.7% of 256 patients. The re-rupture rate was 13.0% in 293 patients. RTS at the same level or higher was 51.6% in 219 patients. The mean (SD) Lysholm score was 88.1 (12.9) in 78 patients, the mean (SD) Tegner score was 6.0 (1.6) in 78 patients, and the mean (SD) IKDC score was 82.6 (16.0) in 126 patients. CONCLUSION: R-ACLR in paediatric and adolescent patients predominantly uses BPTB autografts and interference screw femoral and tibial fixation with concomitant meniscal procedures. Rates of re-rupture and RTS at the same level or higher were 13.0% and 51.6%, respectively. Information from this review can provide orthopaedic surgeons with a comprehensive understanding of the most commonly used operative techniques and their outcomes for revision ACLR in this population. LEVEL OF EVIDENCE: Level IV.

The role of enhancers in psoriasis and atopic dermatitis.

Regulatory elements, particularly enhancers, play a crucial role in disease susceptibility and progression. Enhancers are DNA sequences that activate gene expression and can be affected by epigenetic modifications, interactions with transcription factors (TFs) or changes to the enhancer DNA sequence itself. Altered enhancer activity impacts gene expression and contributes to disease. In this review, we define enhancers and the experimental techniques used to identify and characterize them. We also discuss recent studies that examine how enhancers contribute to atopic dermatitis (AD) and psoriasis. Articles in the PubMed database were identified (from 1 January 2010 to 28 February 2023) that were relevant to enhancer variants, enhancer-associated TFs and enhancer histone modifications in psoriasis or AD. Most enhancers associated with these conditions regulate genes affecting epidermal homeostasis or immune function. These discoveries present potential therapeutic targets to complement existing treatment options for AD and psoriasis.

Non-coding RNAs as skin disease biomarkers, molecular signatures, and therapeutic targets.

Non-coding RNAs (ncRNAs) are emerging as biomarkers, molecular signatures, and therapeutic tools and targets for diseases. In this review, we focus specifically on skin diseases to highlight how two classes of ncRNAs-microRNAs and long noncoding RNAs-are being used to diagnose medical conditions of unclear etiology, improve our ability to guide treatment response, and predict disease prognosis. Furthermore, we explore how ncRNAs are being used as both as drug targets and associated therapies have unique benefits, risks, and challenges to development, but offer a distinctive promise for improving patient care and outcomes.

Association of physician burnout with perceived EHR work stress and potentially actionable factors.

OBJECTIVE: Physicians of all specialties experienced unprecedented stressors during the COVID-19 pandemic, exacerbating preexisting burnout. We examine burnout's association with perceived and actionable electronic health record (EHR) workload factors and personal, professional, and organizational characteristics with the goal of identifying levers that can be targeted to address burnout. MATERIALS AND METHODS: Survey of physicians of all specialties in an academic health center, using a standard measure of burnout, self-reported EHR work stress, and EHR-based work assessed by the number of messages regarding prescription reauthorization and use of a staff pool to triage messages. Descriptive and multivariable regression analyses examined the relationship among burnout, perceived EHR work stress, and actionable EHR work factors. RESULTS: Of 1038 eligible physicians, 627 responded (60% response rate), 49.8% reported burnout symptoms. Logistic regression analysis suggests that higher odds of burnout are associated with physicians feeling higher level of EHR stress (odds ratio [OR], 1.15; 95% confidence interval [CI], 1.07-1.25), having more prescription reauthorization messages (OR, 1.23; 95% CI, 1.04-1.47), not feeling valued (OR, 3.38; 95% CI, 1.69-7.22) or aligned in values with clinic leaders (OR, 2.81; 95% CI, 1.87-4.27), in medical practice for ≤15 years (OR, 2.57; 95% CI, 1.63-4.12), and sleeping for <6 h/night (OR, 1.73; 95% CI, 1.12-2.67). DISCUSSION: Perceived EHR stress and prescription reauthorization messages are significantly associated with burnout, as are non-EHR factors such as not feeling valued or aligned in values with clinic leaders. Younger physicians need more support. CONCLUSION: A multipronged approach targeting actionable levers and supporting young physicians is needed to implement sustainable improvements in physician well-being.

Temporal Association of Reduced Serum Vitamin D with COVID-19 Infection: Two Single-Institution Case-Control Studies.

(1) Background: Vitamin D supplementation has been proposed for the prevention and treatment of COVID-19, but it is not clear if reduced serum vitamin D predisposes individuals to COVID-19 and/or is a secondary consequence of infection. This study assessed the temporal association between serum vitamin D and COVID-19 with two single-institution case-control studies through the University of California San Diego (UCSD) Health System. (2) Methods: This study included patients who tested positive for COVID-19 from 1 January to 30 September 2020 with serum 25-hydroxy-vitamin D (25(OH)D) measured within 180 days of diagnosis. Patients were separated based on whether 25(OH)D was measured before (n = 107 cases, 214 controls) or after (n = 203 cases, 406 controls) COVID-19 diagnosis. COVID-19 infection status was the outcome variable in the pre-diagnosis study, whereas serum 25(OH)D level was the outcome variable in the post-diagnosis study. (3) Results: Serum 25(OH)D levels were not associated with the odds of subsequent COVID-19 infection (OR 1.0, 95% CI: 1.0 to 1.0, p = 0.98). However, COVID-19-positive individuals had serum 25(OH)D measurements that were 2.7 ng/mL lower than the controls (95% CI: -5.2 to -0.2, p = 0.03). (4) Conclusions: In our study population, serum 25(OH)D levels were not associated with the risk of acquiring COVID-19 infection but were reduced in subjects after COVID-19 infection. These results support the possibility that reduced serum 25(OH)D is a consequence and not a cause of COVID-19 infection.

AhR Regulates Peptidoglycan-Induced Inflammatory Gene Expression in Human Keratinocytes.

Bacterial peptidoglycan (PGN) stimulates toll-like receptor 2 (TLR2) on the surface of keratinocytes (KCs), triggering signaling pathways that promote an innate immune response. However, excessive TLR2 activation can lead to inappropriate inflammation, which contributes to skin conditions such as rosacea. To better treat these conditions, there is a need to understand the molecular mechanisms that regulate the cellular response to TLR2 activation in the skin. Aryl hydrocarbon receptor (AhR) is a transcription factor that modulates the immune response in KCs and is a promising therapeutic target for inflammatory skin diseases. Here, we investigated the role of the AhR in regulating the transcriptional response of human KCs to PGN. We performed whole-transcriptome sequencing in wild-type and AhR-depleted KCs after PGN stimulation. AhR depletion altered the expression of 72 genes in response to PGN, leading to increased expression of 48 genes and repression of 24 genes, including interleukin (IL)-1ß. Chromatin immunoprecipitation showed that PGN stimulation resulted in AhR binding the promoters of IL-1ß and IL-6 to activate them. More broadly, AhR promoted inflammatory gene expression by increasing JNK/mitogen-activated protein kinase signaling and FosB expression. Finally, we observed that AhR depletion increased TLR2 expression itself, raising the hypothesis that AhR may serve to restrain TLR2-mediated inflammation in KCs through negative feedback. Viewed together, our findings demonstrate a significant and complex role for AhR in modulating the expression of inflammatory genes in KCs in response to PGN.

Research Techniques Made Simple: CRISPR Genetic Screens.

CRISPR and Cas proteins, often referred to as CRISPR/Cas, are the components of a bacterial genome editing system that can be used to perturb genes in cells and tissues. A classic application is to use CRISPR/Cas to generate genetic loss-of-function. When performed at large scale and combined with deep sequencing techniques, CRISPR-based perturbations can be performed in a high throughput setting to screen many candidate genomic elements for their roles in a phenotype of interest. Here, we discuss major considerations in the design, execution, and analysis of CRISPR screens. We focus on CRISPR knockout screens but also review adaptations to the CRISPR/Cas system that highlight the versatility of the system to make other types of experimental genetic changes as well. We also discuss examples of CRISPR genetic screens in investigative dermatology and how they may be used to answer key scientific questions in the field.

A genome-wide long noncoding RNA CRISPRi screen identifies PRANCR as a novel regulator of epidermal homeostasis.

Genome-wide association studies indicate that many disease susceptibility regions reside in non-protein-coding regions of the genome. Long noncoding RNAs (lncRNAs) are a major component of the noncoding genome, but their biological impacts are not fully understood. Here, we performed a CRISPR interference (CRISPRi) screen on 2263 epidermis-expressed lncRNAs and identified nine novel candidate lncRNAs regulating keratinocyte proliferation. We further characterized a top hit from the screen, progenitor renewal associated non-coding RNA (PRANCR), using RNA interference-mediated knockdown and phenotypic analysis in organotypic human tissue. PRANCR regulates keratinocyte proliferation, cell cycle progression, and clonogenicity. PRANCR-deficient epidermis displayed impaired stratification with reduced expression of differentiation genes that are altered in human skin diseases, including keratins 1 and 10, filaggrin, and loricrin. Transcriptome analysis showed that PRANCR controls the expression of 1136 genes, with strong enrichment for late cell cycle genes containing a CHR promoter element. In addition, PRANCR depletion led to increased levels of both total and nuclear CDKN1A (also known as p21), which is known to govern both keratinocyte proliferation and differentiation. Collectively, these data show that PRANCR is a novel lncRNA regulating epidermal homeostasis and identify other lncRNA candidates that may have roles in this process as well.

Agminated blue nevus with a GNAQ mutation: A case report and review of the literature.

Agminated blue nevi are dermal melanocytic proliferations that classically present as dark blue macules or papules in a grouped, linear, or blaschkoid distribution. In their more common sporadic form, blue nevi manifest in young adulthood as solitary blue papules or macules on the scalp, face, hands, or feet. By contrast, agminated blue nevi tend to manifest earlier in life, and are distributed more evenly across anatomic sites. Recent studies have identified mutations in sporadic blue nevi in the genes encoding G Protein subunit alpha Q and G protein subunit alpha 11 (GNAQ and GNA11). It is unknown whether agminated blue nevi share the same genetic changes. In the present paper, we present a case of agminated blue nevus on the wrist, and identify an activating mutation (c.626A > T, p.Glu209Leu) in GNAQ. We hypothesize that GNAQ/GNA11 activating mutations arising earlier during development may trigger agminated blue nevi, explaining the broader field of involvement in these cutaneous lesions.

CALML5 is a ZNF750- and TINCR-induced protein that binds stratifin to regulate epidermal differentiation.

Outward migration of epidermal progenitors occurs with induction of hundreds of differentiation genes, but the identities of all regulators required for this process are unknown. We used laser capture microdissection followed by RNA sequencing to identify calmodulin-like 5 (CALML5) as the most enriched gene in differentiating outer epidermis. CALML5 mRNA was up-regulated by the ZNF750 transcription factor and then stabilized by the long noncoding RNA TINCR. CALML5 knockout impaired differentiation, abolished keratohyalin granules, and disrupted epidermal barrier function. Mass spectrometry identified SFN (stratifin/14-3-3σ) as a CALML5-binding protein. CALML5 interacts with SFN in suprabasal epidermis, cocontrols 13% of late differentiation genes, and modulates interaction of SFN to some of its binding partners. A ZNF750-TINCR-CALML5-SFN network is thus essential for epidermal differentiation.

Advances in skin grafting and treatment of cutaneous wounds.

The ability of the skin to repair itself after injury is vital to human survival and is disrupted in a spectrum of disorders. The process of cutaneous wound healing is complex, requiring a coordinated response by immune cells, hematopoietic cells, and resident cells of the skin. We review the classic paradigms of wound healing and evaluate how recent discoveries have enriched our understanding of this process. We evaluate current and experimental approaches to treating cutaneous wounds, with an emphasis on cell-based therapies and skin transplantation.