Methotrexate Use and Risk of Interstitial Lung Disease in Dermatomyositis.

This cohort study examines the association between methotrexate use and interstitial lung disease in patients with dermatomyositis.

Hospitalization and Continuity of Care in Anti-MDA5 Dermatomyositis.

This cohort study describes the clinical features, patient characteristics, and treatment of anti-melanoma differentiation­associated gene 5 (MDA5) dermatomyositis.

Gene Therapy and Spinal Fusion: Systematic Review and Meta-Analysis of the Available Data.

OBJECTIVE: To analyze the extant literature describing the application of gene therapy to spinal fusion. METHODS: A systematic review of the English-language literature was performed. The search query was designed to include all published studies examining gene therapy approaches to promote spinal fusion. Approaches were classified as ex vivo (delivery of genetically modified cells) or in vivo (delivery of growth factors via vectors). The primary endpoint was fusion rate. Random effects meta-analyses were performed to calculate the overall odds ratio (OR) of fusion using a gene therapy approach and overall fusion rate. Subgroup analyses of fusion rate were also performed for each gene therapy approach. RESULTS: Of 1179 results, 35 articles met criteria for inclusion (all preclinical), of which 26 utilized ex vivo approaches and 9 utilized in vivo approaches. Twenty-seven articles (431 animals) were included in the meta-analysis. Gene therapy use was associated with significantly higher fusion rates (OR 77; 95% confidence interval {CI}: [31, 192]; P < 0.001); ex vivo strategies had a greater effect (OR 136) relative to in vivo strategies (OR 18) (P = 0.017). The overall fusion rate using a gene therapy approach was 80% (95% CI: [62%, 93%]; P < 0.001); overall fusion rates were significantly higher in subjects treated with ex vivo compared to in vivo strategies (90% vs. 42%; P = 0.011). For both ex vivo and in vivo approaches, the effect of gene therapy on fusion was independent of animal model. CONCLUSIONS: Gene therapy may augment spinal fusion; however, future investigation in clinical populations is necessary.

BTB domain mutations perturbing KCTD15 oligomerisation cause a distinctive frontonasal dysplasia syndrome.

INTRODUCTION: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome. METHODS: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. RESULTS: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. CONCLUSION: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex.

Clinical Characteristics of Erythema Nodosum and Associations With Chronicity and Recurrence.

This cohort study characterizes the presentation, causes, treatment, and disease course of erythema nodosum, as well as identifies associations with chronicity and recurrence.

Design, Synthesis, and Evaluation of New 1H-Benzo[d]imidazole Based PqsR Inhibitors as Adjuvant Therapy for Pseudomonas aeruginosa Infections.

Pseudomonas aeruginosa is one of the top priority pathogens that requires immediate attention according to the World Health Organisation (WHO). Due to the alarming shortage of novel antimicrobials, targeting quorum sensing (QS), a bacterial cell to cell signaling system controlling virulence, has emerged as a promising approach as an antibiotic adjuvant therapy. Interference with the pqs system, one of three QS systems in P. aeruginosa, results in reduction of bacterial virulence gene expression and biofilm maturation. Herein, we report a hit to lead process to fine-tune the potency of our previously reported inhibitor 1 (IC50 3.2 µM in P. aeruginosa PAO1-L), which led to the discovery of 2-(4-(3-((6-chloro-1-isopropyl-1H-benzo[d]imidazol-2-yl)amino)-2-hydroxypropoxy)phenyl)acetonitrile (6f) as a potent PqsR antagonist. Compound 6f inhibited the PqsR-controlled PpqsA-lux transcriptional reporter fusion in P. aeruginosa at low submicromolar concentrations. Moreover, 6f showed improved efficacy against P. aeruginosa CF isolates with significant inhibition of pyocyanin, 2-alkyl-4(1H)-quinolones production.

Osteoimmunology: Interactions With the Immune System in Spinal Fusion.

Spinal fusion is important for the clinical success of patients undergoing surgery, and the immune system plays an increasingly recognized role. Osteoimmunology is the study of the interactions between the immune system and bone. Inflammation impacts the osteogenic, osteoconductive, and osteoinductive properties of bone grafts and substitutes and ultimately influences the success of spinal fusion. Macrophages have emerged as important cells for coordinating the immune response following spinal fusion surgery, and macrophage-derived cytokines impact each phase of bone graft healing. This review explores the cellular and molecular immune processes that regulate bone homeostasis and healing during spinal fusion.

SAGES/AHPBA guidelines for the use of microwave and radiofrequency liver ablation for the surgical treatment of hepatocellular carcinoma or colorectal liver metastases less than 5 cm.

BACKGROUND: Primary hepatocellular carcinoma (HCC) and colorectal liver metastases (CRLM) represent the liver's two most common malignant neoplasms. Liver-directed therapies such as ablation have become part of multidisciplinary therapies despite a paucity of data. Therefore, an expert panel was convened to develop evidence-based recommendations regarding the use of microwave ablation (MWA) and radiofrequency ablation (RFA) for HCC or CRLM less than 5 cm in diameter in patients ineligible for other therapies. METHODS: A systematic review was conducted for six key questions (KQ) regarding MWA or RFA for solitary liver tumors in patients deemed poor candidates for first-line therapy. Subject experts used the GRADE methodology to formulate evidence-based recommendations and future research recommendations. RESULTS: The panel addressed six KQs pertaining to MWA vs. RFA outcomes and laparoscopic vs. percutaneous MWA. The available evidence was poor quality and individual studies included both HCC and CRLM. Therefore, the six KQs were condensed into two, recognizing that these were two disparate tumor groups and this grouping was somewhat arbitrary. With this significant limitation, the panel suggested that in appropriately selected patients, either MWA or RFA can be safe and feasible. However, this recommendation must be implemented cautiously when simultaneously considering patients with two disparate tumor biologies. The limited data suggested that laparoscopic MWA of anatomically more difficult tumors has a compensatory higher morbidity profile compared to percutaneous MWA, while achieving similar overall 1-year survival. Thus, either approach can be appropriate depending on patient-specific factors (very low certainty of evidence). CONCLUSION: Given the weak evidence, these guidelines provide modest guidance regarding liver ablative therapies for HCC and CRLM. Liver ablation is just one component of a multimodal approach and its use is currently limited to a highly selected population. The quality of the existing data is very low and therefore limits the strength of the guidelines.

Vascular smooth muscle cell mechanotransduction through serum and glucocorticoid inducible kinase-1 promotes interleukin-6 production and macrophage accumulation in murine hypertension.

Objective: The objective of this investigation was to demonstrate that in vivo induction of hypertension (HTN) and in vitro cyclic stretch of aortic vascular smooth muscle cells (VSMCs) can cause serum and glucocorticoid-inducible kinase (SGK-1)-dependent production of cytokines to promote macrophage accumulation that may promote vascular pathology. Methods: HTN was induced in C57Bl/6 mice with angiotensin II infusion (1.46 mg/kg/day × 21 days) with or without systemic infusion of EMD638683 (2.5 mg/kg/day × 21 days), a selective SGK-1 inhibitor. Systolic blood pressure was recorded. Abdominal aortas were harvested to quantify SGK-1 activity (pSGK-1/SGK-1) by immunoblot. Flow cytometry quantified the abundance of CD11b+/F480+ cells (macrophages). Plasma interleukin (IL)-6 and monocyte chemoattractant protein-1 (MCP-1) was assessed by enzyme-linked immunosorbent assay. Aortic VSMCs from wild-type mice were subjected to 12% biaxial cyclic stretch (Stretch) for 3 or 12 hours with or without EMD638683 (10 µM) and with or without SGK-1 small interfering RNA with subsequent quantitative polymerase chain reaction for IL-6 and MCP-1 expression. IL-6 and MCP-1 in culture media were analyzed by enzyme-linked immunosorbent assay. Aortic VSMCs from SGK-1flox+/+ mice were transfected with Cre-Adenovirus to knockdown SGK-1 (SGK-1KD VSMCs) and underwent parallel tension experimentation. Computational modeling was used to simulate VSMC signaling. Statistical analysis included analysis of variance with significance at a P value of <.05. Results: SGK-1 activity, abundance of CD11b+/F4-80+ cells, and plasma IL-6 were increased in the abdominal aorta of mice with HTN and significantly reduced by treatment with EMD638683. This outcome mirrored the increased abundance of IL-6 in media from Stretch C57Bl/6 VSMCs and attenuation of the effect with EMD638683 or SGK-1 small interfering RNA. C57Bl/6 VSMCs also responded to Stretch with increased MCP-1 expression and secretion into the culture media. Further supporting the integral role of mechanical signaling through SGK-1, target gene expression and cytokine secretion was unchanged in SGK-1KD VSMCs with Stretch, and computer modeling confirmed SGK-1 as an intersecting node of signaling owing to mechanical strain and angiotensin II. Conclusions: Mechanical activation of SGK-1 in aortic VSMCs can promote inflammatory signaling and increased macrophage abundance, therefore this kinase warrants further exploration as a pharmacotherapeutic target to abrogate hypertensive vascular pathology.

Oligodendrocyte dynamics dictate cognitive performance outcomes of working memory training in mice.

Previous work has shown that motor skill learning stimulates and requires generation of myelinating oligodendrocytes (OLs) from their precursor cells (OLPs) in the brains of adult mice. In the present study we ask whether OL production is also required for non-motor learning and cognition, using T-maze and radial-arm-maze tasks that tax spatial working memory. We find that maze training stimulates OLP proliferation and OL production in the medial prefrontal cortex (mPFC), anterior corpus callosum (genu), dorsal thalamus and hippocampal formation of adult male mice; myelin sheath formation is also stimulated in the genu. Genetic blockade of OL differentiation and neo-myelination in Myrf conditional-knockout mice strongly impairs training-induced improvements in maze performance. We find a strong positive correlation between the performance of individual wild type mice and the scale of OLP proliferation and OL generation during training, but not with the number or intensity of c-Fos+ neurons in their mPFC, underscoring the important role played by OL lineage cells in cognitive processing.

Management of autofluorescence in formaldehyde-fixed myocardium: choosing the right treatment.

Autofluorescence (AF) poses challenges for detecting proteins of interest in situ when employing immunofluorescence (IF) microscopy. This interference is particularly pronounced in strongly autofluorescent tissues such as myocardium, where tissue AF can be comparable to IF. Although various histochemical methods have been developed to achieve effective AF suppression in different types of tissue, their applications on myocardial  samples have not been well validated. Due to inconsistency across different autofluorescent structures in sometypes of tissue, it is unclear if these methods can effectively suppress AF across all autofluorescent structures within the myocardium. Here, we quantitatively evaluated the performance of several commonly used quenching treatments on formaldehyde-fixed myocardial samples, including 0.3 M glycine, 0.3% Sudan Black B (SBB), 0.1% and 1% sodium borohydride (NaBH4), TrueVIEW® and TrueBlack®. We further assessed their quenching performance by employing the pre-treatment and post-treatment protocols, designed to cover two common IF staining scenarios where buffers contained detergents or not. The results suggest that SBB and TrueBlack® outperform other reagents in AF suppression on formaldehyde-fixed myocardial samples in both protocols. Furthermore, we inspected the quenching performance of SBB and TrueBlack® on major autofluorescent myocardial structures and evaluated their influence on IF imaging. The results suggest that SBB outperforms TrueBlack® in quenching major autofluorescent structures, while TrueBlack® excels in preserving IF labeling signal. Surprisingly, we found the treatment of NaBH4 increased AF signal and enhanced the AF contrast of major autofluorescent structures. This finding suggests that NaBH4 has the potential to act as an AF enhancer and may facilitate the interpretation of myocardial structures without the need for counterstaining.

Computational screen for sex-specific drug effects in a cardiac fibroblast signaling network model.

Heart disease is the leading cause of death in both men and women. Cardiac fibrosis is the uncontrolled accumulation of extracellular matrix proteins, which can exacerbate the progression of heart failure, and there are currently no drugs approved specifically to target matrix accumulation in the heart. Computational signaling network models (SNMs) can be used to facilitate discovery of novel drug targets. However, the vast majority of SNMs are not sex-specific and/or are developed and validated using data skewed towards male in vitro and in vivo samples. Biological sex is an important consideration in cardiovascular health and drug development. In this study, we integrate a cardiac fibroblast SNM with estrogen signaling pathways to create sex-specific SNMs. The sex-specific SNMs demonstrated high validation accuracy compared to in vitro experimental studies in the literature while also elucidating how estrogen signaling can modulate the effect of fibrotic cytokines via multi-pathway interactions. Further, perturbation analysis and drug screening uncovered several drug compounds predicted to generate divergent fibrotic responses in male vs. female conditions, which warrant further study in the pursuit of sex-specific treatment recommendations for cardiac fibrosis. Future model development and validation will require more generation of sex-specific data to further enhance modeling capabilities for clinically relevant sex-specific predictions of cardiac fibrosis and treatment.

Shifts in sage-grouse arthropod food sources across grazing and environmental gradients in upland meadow communities.

Groundwater dependent systems are extremely important habitats for a wide variety of taxa in the Great Basin of North America. The impacts of grazing on these habitats cause shifts in resources and subsequent change in species composition. The Greater sage-grouse, a keystone species of Great Basin ecosystems, rear offspring in these areas during spring and summer months using forbs and arthropods. To examine the impact of grazing on arthropod abundance in these ecosystems, seven meadows, each made up of three unique vegetative communities, were grazed at three intensities across two years (2019-2020) and monitored for environmental variables and abundance of arthropods during peak sage-grouse utilization periods. Additionally, the relationship of field measurements and near-surface digital cameras (phenocams) was examined to better understand how remote sensing technologies can be used to monitor these insect abundance shifts on larger scales. Arthropod taxa abundance responded differently to grazing management and environmental variables. Coleoptera abundance during peak sage-grouse usage periods increased roughly 50% in some meadows with increased grazing intensity. For year-to-year environmental variability in precipitation, Lepidoptera abundance was 114% higher in the drier year, while Coleoptera was 39% lower. Near-surface cameras had varied success with predicting peak insect abundance levels. Lepidoptera and Coleoptera capture rates had strong correlations with phenological indices derived from phenocams, while Formicidae had much weaker relationships.

Surgical management of an ossified giant lumbar pseudomeningocele: A case report.

Background: Pseudomeningoceles (PMs) are infrequent complications of spine surgery resulting from incidental durotomy and subsequent extravasation of cerebrospinal fluid. Giant PMs (GPMs), defined as ≥8 cm in major diameter, are rarely reported in the literature and present a challenge due to a lack of clear guidelines for surgical management. Case Description: Here, the authors discuss the successful surgical management of a 25.3 cm lumbar GPM that became calcified 3 years following an initial T10-S2 laminectomy with instrumented fusion performed at an outside-hospital. Conclusion: This report focuses on the successful 3-year delayed surgical intervention for the management of an ossified GPM.

A Single-Institution 5-Year Retrospective Analysis of Laparoscopic Sleeve Gastrectomy Staple-Line Reinforcement: Bioabsorbable Mesh Versus Oversewing.

Background: Staple-line reinforcement has been used to decrease complications such as staple-line bleeding (SLB) and staple-line leaks (SLLs) in patients undergoing laparoscopic sleeve gastrectomy (SG). There is little data comparing bioabsorbable mesh reinforcement (BMR) with oversewing the staple line (OSL). The aim of our study was to compare BMR with OSL in SG. Materials and Methods: This is a single-institution retrospective analysis comparing risks and benefits of BMR (group a) with those of OSL (group b) for SG staple-line reinforcement between 2015 and 2020. Results: In total, 857 patients were identified. There were 452 (52.74%) in group a and 405 (47.26%) in group b. SLB requiring transfusion occurred in 6 (1.32%) patients in group a and 6 (1.48%) patients in group b, NS (P = .848). Zero SLL was identified in either group. One-year mean direct cost of SG in group a was $7881 compared with $6677 in group b. Conclusion: This retrospective study showed that there was low risk of bleeding or leak with either technique of staple-line reinforcement and there was no significant difference in SLB or leak rate with bioabsorbable mesh versus oversewing. The use of bioabsorbable mesh was more expensive than oversewing.

Discovery and characterization of a specific inhibitor of serine-threonine kinase cyclin-dependent kinase-like 5 (CDKL5) demonstrates role in hippocampal CA1 physiology.

Pathological loss-of-function mutations in cyclin-dependent kinase-like 5 (CDKL5) cause CDKL5 deficiency disorder (CDD), a rare and severe neurodevelopmental disorder associated with severe and medically refractory early-life epilepsy, motor, cognitive, visual, and autonomic disturbances in the absence of any structural brain pathology. Analysis of genetic variants in CDD has indicated that CDKL5 kinase function is central to disease pathology. CDKL5 encodes a serine-threonine kinase with significant homology to GSK3ß, which has also been linked to synaptic function. Further, Cdkl5 knock-out rodents have increased GSK3ß activity and often increased long-term potentiation (LTP). Thus, development of a specific CDKL5 inhibitor must be careful to exclude cross-talk with GSK3ß activity. We synthesized and characterized specific, high-affinity inhibitors of CDKL5 that do not have detectable activity for GSK3ß. These compounds are very soluble in water but blood-brain barrier penetration is low. In rat hippocampal brain slices, acute inhibition of CDKL5 selectively reduces postsynaptic function of AMPA-type glutamate receptors in a dose-dependent manner. Acute inhibition of CDKL5 reduces hippocampal LTP. These studies provide new tools and insights into the role of CDKL5 as a newly appreciated key kinase necessary for synaptic plasticity. Comparisons to rodent knock-out studies suggest that compensatory changes have limited the understanding of the roles of CDKL5 in synaptic physiology, plasticity, and human neuropathology.

Ropivacaine-Epinephrine-Clonidine-Ketorolac Cocktail as a Local Anesthetic for Lumbar Decompression Surgery: A Single Institutional Experience.

OBJECTIVE: The goal of this study is to discuss our initial experience with a multimodal opioid-sparing cocktail containing ropivacaine, epinephrine, clonidine, and ketorolac (RECK) in the postoperative management of lumbar decompression surgeries. METHODS: Patients were either administered no local anesthetic at the incision site or were administered a weight-based amount of RECK into the paraspinal musculature and subdermal space surrounding the operative site once the fascia was closed. We performed a retrospective chart review of all patients 18 years of age or older undergoing lumbar laminectomy and lumbar diskectomy surgeries between December 2019 and April 2021. Outcomes including total opioid use, measured as morphine milligram equivalent, length of stay, and postoperative visual analog scores for pain, were collected. Relationships between variables were analyzed with Student's t-test, chi-square tests, and Fisher exact tests. RESULTS: A total of 121 patients undergoing 52 lumbar laminectomy and 69 lumbar diskectomy surgeries were identified. For lumbar laminectomy, patients who were administered RECK had decreased opioid use in the postoperative period (11.47 ± 12.32 vs. 78.51 ± 106.10 morphine milligram equivalents, P = 0.019). For patients undergoing lumbar diskectomies, RECK administration led to a shorter length of stay (0.17 ± 0.51 vs. 0.79 ± 1.45 days, P = 0.019) and a lower 2-hour postoperative pain score (3.69 ± 2.56 vs. 5.41 ± 2.28, P = 0.006). CONCLUSIONS: The RECK cocktail has potential to be an effective therapeutic option for the postoperative management of lumbar decompression surgeries.