FOXM1 network in association with TREM1 suppression regulates NET formation in diabetic foot ulcers.

Diabetic foot ulcers (DFU) are a serious complication of diabetes mellitus and associated with reduced quality of life and high mortality rate. DFUs are characterized by a deregulated immune response with decreased neutrophils due to loss of the transcription factor, FOXM1. Diabetes primes neutrophils to form neutrophil extracellular traps (NETs), contributing to tissue damage and impaired healing. However, the role of FOXM1 in priming diabetic neutrophils to undergo NET formation remains unknown. Here, we found that FOXM1 regulates reactive oxygen species (ROS) levels in neutrophils and inhibition of FOXM1 results in increased ROS leading to NET formation. Next generation sequencing revealed that TREM1 promoted the recruitment of FOXM1+ neutrophils and reversed effects of diabetes and promoted wound healing in vivo. Moreover, we found that TREM1 expression correlated with clinical healing outcomes of DFUs, indicating TREM1 may serve as a useful biomarker or a potential therapeutic target. Our findings highlight the clinical relevance of TREM1, and indicates FOXM1 pathway as a novel regulator of NET formation during diabetic wound healing, revealing new therapeutic strategies to promote healing in DFUs.

IRE1α regulates ROS and immune responses after UVB irradiation.

Objective: UV irradiation of the skin induces photo damage and generates cytotoxic intracellular reactive oxygen species (ROS), activating the unfolded protein response (UPR) to adapt or reduce these UVB-mediated damages. This study was designed to understand the role of the UPR mediator IRE1α in the antioxidant response following UVB irradiation of mouse skin and keratinocytes. Methods: We used mice with an epidermal deletion of IRE1α and primary mouse keratinocytes to examine effects of UV on different parameters of the antioxidant response in the presence and absence of functional IRE1α. Results: In the absence of IRE1α, PERK activity and protein levels are significantly compromised following UVB irradiation. Additionally, the loss of IRE1α suppressed phosphorylation of the PERK target, nuclear factor erythroid-2-related factor 2 (NRF2), and NRF2-dependent antioxidant gene expression after UVB irradiation. Interestingly, IRE1α-deficient keratinocytes exhibit elevated basal ROS levels, while a robust ROS induction upon UVB exposure is abolished. Because UVB-induced ROS plays an essential role in regulating skin inflammation, we analyzed recruited immune cell populations and the expression of pro-inflammatory cytokines, Il-6 and Tnfα in mice with epidermally-targeted deletion of Ire1α. Following UVB irradiation, there was significantly less recruitment of neutrophils and leukocytes and reduced expression of pro-inflammatory cytokine genes in the skin of mice lacking IRE1α. Furthermore, keratinocyte proliferation was also significantly reduced after chronic UVB exposure in the skin of these mice. Conclusions: Collectively, our findings indicate that IRE1α is essential for basal and UVB-induced oxidative stress response, UV-induced skin immune responses, and keratinocyte proliferation. Significance: These findings shed new light on the protective function of IRE1α in the response to UV. IRE1α plays an important role in the regulation of ROS, PERK stability, and antioxidant gene expression in response to UVB in mouse keratinocytes and epidermis.

Adjuvant Stereotactic Radiosurgery for Clear Cell Meningiomas.

OBJECTIVE: Clear cell meningiomas (CCM) are an uncommon meningioma subtype marked by aggressive growth and high rates of recurrence despite initial resection. The present study evaluates the adjuvant benefit of stereotactic radiosurgery (SRS) for residual or recurrent tumors. METHODS: After review of our prospectively maintained database, we identified 6 patients (3 female) with histologically confirmed Grade 2 CCMs. The median age of the patients at the time of SRS was 45 years. Five patients had undergone prior gross total surgical resection and 1 patient had subtotal resection before SRS. The median SRS treatment volume was 4.7 cc and the median radiosurgical margin dose was 13 Gy (range: 10-15 Gy). RESULTS: The median follow-up time was 35.5 months (range 6-168 months). Three patients achieved tumor control after the first SRS procedure. Three patients experienced tumor progression at 4, 22, and 32 months after initial SRS. Tumor control was obtained in 2 of these patients after additional SRS. One patient with multiple SRS procedures had suspected adverse radiation effect that was successfully treated with corticosteroids followed by bevacizumab. CONCLUSIONS: Tumor control was maintained in 5 of 6 patients after one or more SRS procedures. SRS should be considered for early intervention after surgical resection of CCM. To maximize the tumor control rate, patients with diagnosed CCM should be treated more generously and higher margin dose should be prescribed. Patients with CCM should be counselled that more than one SRS may be necessary to provide sustained tumor control.

Single-Session Gamma Knife Radiosurgery for Patients With 20 or More Brain Metastases.

BACKGROUND: Stereotactic radiosurgery (SRS) is a widely accepted treatment modality for brain metastases. The role of SRS in patients with higher numbers of metastases remains controversial. OBJECTIVES: To define outcomes in patients with ≥20 brain metastases managed using single-session SRS. METHODS: This single-institution retrospective cohort study studied 75 patients (26 non-small-cell lung cancer, 21 small-cell lung cancer, 14 breast cancer, and 14 melanoma) undergoing single-session SRS. The median number of tumors per patient was 24, and the median cumulative tumor volume was 3.70 cc. The median margin dose prescribed to each individual tumor was 16 Gy. The median integral cranial dose was 5492 mJ. The median beam on time was 160 minutes. Univariate and multivariate analyses were performed with significance set at P < .05. RESULTS: The median overall survival after SRS was 8.8 months (patients with non-small-cell lung cancer), 4.6 months (patients with small-cell lung cancer), 11.3 months (patients with breast cancer), and 4.1 months (patients with melanoma). Primary cancer type, number of brain metastases, and concurrent immunotherapy were significant factors in predicting survival. Local tumor control rate per patient was 97.3% and 94.6% at 6 and 12 months after SRS, respectively. Thirty-six patients underwent additional SRS for new tumor development with a median time after SRS of 5 months. Three patients experienced adverse radiation events. CONCLUSION: Single-session SRS is a well-tolerated palliative treatment option even in patients with ≥20 brain metastases, achieving local control rate >90% with low risks of neurotoxicity while continuing concurrent systemic oncological care.

Assessment of anti-HIV-1 guide RNA efficacy in cells containing the viral target sequence, corresponding gRNA, and CRISPR/Cas9.

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene editing system has been shown to be effective at inhibiting human immunodeficiency virus type 1 (HIV-1). Studies have not consistently used a trackable dual reporter system to determine what cells received the Cas9/gRNA to determine the overall knockdown of HIV. Some studies have used stably transduced cells under drug selection to accomplish this goal. Here a two-color system was used that allows tracking of viral protein expression and which cells received the CRISPR/Cas9 system. These experiments ensured that each gRNA used was a perfect match to the intended target to remove this variable. The data showed that gRNAs targeting the transactivation response element (TAR) region or other highly conserved regions of the HIV-1 genome were effective at stopping viral gene expression, with multiple assays demonstrating greater than 95 percent reduction. Conversely, gRNAs targeting conserved sites of the 5' portion of the U3 region were largely ineffective, demonstrating that the location of edits in the long terminal repeat (LTR) matter with respect to function. In addition, it was observed that a gRNA targeting Tat was effective in a T-cell model of HIV-1 latency. Taken together, these studies demonstrated gRNAs designed to highly conserved functional regions have near 100% efficacy in vitro in cells known to have received the Cas9/gRNA pair.

Chromatin Landscape Governing Murine Epidermal Differentiation.

Chromatin landscape and regulatory networks are determinants in lineage specification and differentiation. To define the temporospatial differentiation axis in murine epidermal cells in vivo, we generated datasets profiling expression dynamics (RNA sequencing), chromatin accessibility (assay for transposase-accessible chromatin using sequencing), architecture (Hi-C), and histone modifications (chromatin immunoprecipitation followed by sequencing) in the epidermis. We show that many differentially regulated genes are suppressed during the differentiation process, with superenhancers controlling differentiation-specific epigenomic changes. Our data shows the relevance of the Dlx/Klf/Grhl combinatorial regulatory network in maintaining correct temporospatial gene expression during epidermal differentiation. We determined differential open compartments, topologically associating domain score, and looping in the basal cell and suprabasal cell epidermal fractions, with the evolutionarily conserved epidermal differentiation complex region showing distinct suprabasal cell-specific topologically associating domain and loop formation that coincided with superenhancer sites. Overall, our study provides a global genome-wide resource of chromatin dynamics that define unrecognized regulatory networks and the epigenetic control of Dlx3-bound superenhancer elements during epidermal differentiation.

The Endoplasmic Reticulum Stress Sensor IRE1α Regulates the UV DNA Repair Response through the Control of Intracellular Calcium Homeostasis.

The unfolded protein response is activated by UVB irradiation, but the role of a key mediator, IRE1α, is not clear. In this study, we show that mice with an epidermal IRE1α deletion are sensitized to UV with increased apoptosis, rapid loss of UV-induced cyclopyrimidine dimer‒positive keratinocytes, and sloughing of the epidermis. In vitro, Ire1α-deficient keratinocytes have increased UVB sensitivity, reduced cyclopyrimidine dimer repair, and reduced accumulation of γH2AX and phosphorylated ATR, suggesting defective activation of nucleotide excision repair. Knockdown of XBP1 or pharmacologic inhibition of the IRE1α ribonuclease did not phenocopy Ire1α deficiency. The altered UV response was linked to elevated intracellular calcium levels and ROS, and this was due to dysregulation of the endoplasmic reticulum calcium channel InsP3R. Pharmacologic, genetic, and biochemical studies linked the regulation of the Ins3PR, intracellular calcium, and normal UV DNA damage response to CIB1 and the IRE1α‒TRAF2‒ASK1 complex. These results suggest a model where IRE1α activation state drives CIB1 binding either to the InsP3R or ASK1 to regulate endoplasmic reticulum calcium efflux, ROS, and DNA repair responses after UV irradiation.

Therapeutic potential of ketamine for alcohol use disorder.

Excessive alcohol consumption is involved in 1/10 of deaths of U.S. working-age adults and costs the country around $250,000,000 yearly. While Alcohol Use Disorder (AUD) pathology is complex and involves multiple neurotransmitter systems, changes in synaptic plasticity, hippocampal neurogenesis, and neural connectivity have been implicated in the behavioral characteristics of AUD. Depressed mood and stress are major determinants of relapse in AUD, and there is significant comorbidity between AUD, depression, and stress disorders, suggesting potential for overlap in their treatments. Disulfiram, naltrexone, and acamprosate are current pharmacotherapies for AUD, but these treatments have limitations, highlighting the need for novel therapeutics. Ketamine is a N-methyl-D-Aspartate receptor antagonist, historically used in anesthesia, but also affects other neurotransmitters systems, synaptic plasticity, neurogenesis, and neural connectivity. Currently under investigation for treating AUDs and other Substance Use Disorders (SUDs), ketamine has strong support for efficacy in treating clinical depression, recently receiving FDA approval. Ketamine's effect in treating depression and stress disorders, such as PTSD, and preliminary evidence for treating SUDs further suggests a role for treating AUDs. This review explores the behavioral and neural evidence for treating AUDs with ketamine and clinical data on ketamine therapy for AUDs and SUDs.