Expression of IL-4 in Tumors: A Safety Surrogate to Predict Cancer Survival Associated With Biologic Therapies.

Interleukin (IL)-4-targeted therapies have revolutionized management of inflammatory dermatoses.

Optimized 13C-TrEnDi Enhances the Sensitivity of Plasmenyl Ether Glycerophospholipids and Demonstrates Compatibility with Other Derivatization Strategies.

The identification and quantitation of plasmalogen glycerophospholipids is challenging due to their isobaric overlap with plasmanyl ether-linked glycerophospholipids, susceptibility to acid degradation, and their typically low abundance in biological samples. Trimethylation enhancement using diazomethane (TrEnDi) can be used to significantly enhance the signal of glycerophospholipids through the creation of quaternary ammonium groups producing fixed positive charges using 13C-diazomethane in complex lipid extracts. Although TrEnDi requires a strong acid for complete methylation, we report an optimized protocol using 10 mM HBF4 with the subsequent addition of a buffer solution that prevents acidic hydrolysis of plasmalogen species and enables the benefits of TrEnDi to be realized for this class of lipids. These optimized conditions were applied to aliquots of bovine liver extract (BLE) to achieve permethylation of plasmalogen lipids within a complex mixture. Treating aliquots of unmodified and TrEnDi-derivatized BLE samples with 80% formic acid and comparing their liquid chromatography mass spectrometry (LCMS) results to analogous samples not treated with formic acid, enabled the identification of 29 plasmalogen species. On average, methylated plasmalogen species from BLE demonstrated 2.81-fold and 28.1-fold sensitivity gains over unmodified counterparts for phosphatidylcholine and phosphatidylethanolamine plasmalogen species, respectively. Furthermore, the compatibility of employing 13C-TrEnDi and a previously reported iodoacetalization strategy was demonstrated to effectively identify plasmenyl-ether lipids in complex biological extracts at greater levels of sensitivity. Overall, we detail an optimized 13C-TrEnDi derivatization strategy that enables the analysis of plasmalogen glycerophospholipids with no undesired cleavage of radyl groups, boosting their sensitivity in LCMS and LCMS/MS analyses.

High Community-Level Social Vulnerability is Associated with Worse Recurrence-Free Survival (RFS) After Resection of Extremity and Truncal Soft Tissue Sarcoma.

BACKGROUND: Although social vulnerability has been associated with worse postoperative and oncologic outcomes in other cancer types, these effects have not been characterized in patients with soft tissue sarcoma. This study evaluated the association of social vulnerability and oncologic outcomes. METHODS: The authors conducted a single-institution cohort study of adult patients with primary and locally recurrent extremity or truncal soft tissue sarcoma undergoing resection between January 2016 and December 2021. The social vulnerability index (SVI) was measured on a low (SVI 1-39%, least vulnerable) to high (60-100%, most vulnerable) SVI scale. The association of SVI with overall survival (OS) and recurrence-free survival (RFS) was evaluated by Kaplan-Meier analysis and Cox proportional hazard regression. RESULTS: The study identified 577 patients. The median SVI was 44 (interquartile range [IQR], 19-67), with 195 patients categorized as high SVI and 265 patients as low SVI. The median age, tumor size, histologic subtype, grade, comorbidities, stage, follow-up time, and perioperative chemotherapy and radiation utilization were similar between the high and low SVI cohorts. The patients with high SVI had worse OS (p = 0.07) and RFS (p = 0.016) than the patients with low SVI. High SVI was independently associated with shorter RFS in the multivariate analysis (hazard ratio, 1.64; 95% confidence interval, 1.06-2.54) but not with OS (HR, 1.47; 95% CI 0.84-2.56). CONCLUSION: High community-level social vulnerability appears to be independently associated with worse RFS for patients undergoing resection of extremity and truncal soft tissue sarcoma. The effect of patient and community-level social risk factors should be considered in the treatment of patients with extremity sarcoma.

Free labial mucocutaneous graft for eyelid reconstruction in four dogs.

PURPOSE: To describe a technique for eyelid margin reconstruction following large mass resection utilizing a free labial mucocutaneous graft. METHODS: Four dogs (4 eyes) underwent en bloc eyelid mass excision under general anesthesia. Measurements were made of the mass followed by free labial mucocutaneous graft retrieval, resection of the mass, and then transplantation of retrieved region of labial mucocutaneous tissue into the resulting defect. RESULTS: Three patients underwent eyelid margin reconstruction with a free labial mucocutaneous graft. One patient received a pedicle advancement graft combined with a free labial mucocutaneous graft. In all cases, a length of 120%-150% of the eyelid defect was retrieved from the oral labia. Postoperative follow-up ranged from 6 weeks to 4 months. All cases had superficial graft necrosis and depigmentation of the donor tissue with total healing time taking up to 8 weeks. All cases had an esthetic and functional reconstruction. CONCLUSIONS: This technique allows reconstruction of the majority of the eyelid margin, greater than that which can be closed primarily. Due to tissue sloughing, the healing time can be extended but cosmetic outcomes are good. Eyelid reconstruction utilizing a free labial graft restored a mucocutaneous margin and recreated a functional eyelid, thus avoiding trichiasis or secondary keratitis.

Gα12 signaling regulates transcriptional and phenotypic responses that promote glioblastoma tumor invasion.

In silico interrogation of glioblastoma (GBM) in The Cancer Genome Atlas (TCGA) revealed upregulation of GNA12 (Gα12), encoding the alpha subunit of the heterotrimeric G-protein G12, concomitant with overexpression of multiple G-protein coupled receptors (GPCRs) that signal through Gα12. Glioma stem cell lines from patient-derived xenografts also showed elevated levels of Gα12. Knockdown (KD) of Gα12 was carried out in two different human GBM stem cell (GSC) lines. Tumors generated in vivo by orthotopic injection of Gα12KD GSC cells showed reduced invasiveness, without apparent changes in tumor size or survival relative to control GSC tumor-bearing mice. Transcriptional profiling of GSC-23 cell tumors revealed significant differences between WT and Gα12KD tumors including reduced expression of genes associated with the extracellular matrix, as well as decreased expression of stem cell genes and increased expression of several proneural genes. Thrombospondin-1 (THBS1), one of the genes most repressed by Gα12 knockdown, was shown to be required for Gα12-mediated cell migration in vitro and for in vivo tumor invasion. Chemogenetic activation of GSC-23 cells harboring a Gα12-coupled DREADD also increased THBS1 expression and in vitro invasion. Collectively, our findings implicate Gα12 signaling in regulation of transcriptional reprogramming that promotes invasiveness, highlighting this as a potential signaling node for therapeutic intervention.

Using a Novel Connected Device for the Collection of Puffing Topography Data for the Vuse Solo Electronic Nicotine Delivery System in a Real-World Setting: Prospective Ambulatory Clinical Study.

BACKGROUND: Over the last decade, the use of electronic nicotine delivery systems (ENDSs) has risen, whereas studies that describe how consumers use these products have been limited. Most studies related to ENDS use have involved study designs focused on use in a central location environment or attempted to measure use outcomes through subjective self-reported end points. The development of accurate and reliable tools to collect data in a naturalistic real-world environment is necessary to capture the complexities of ENDS use. Using connected devices in a real-world setting provides a convenient and objective approach to collecting behavioral outcomes with ENDS. OBJECTIVE: The Product Use and Behavior instrument was developed and used to capture the use of the Vuse Solo ENDS in an ambulatory setting to best replicate real-world use behavior. This study aims to determine overall mean values for topography outcomes while also providing a definition for an ENDS use session. METHODS: A prospective ambulatory clinical study was performed with the Product Use and Behavior instrument. Participants (n=75) were aged between 21 and 60 years, considered in good health, and were required to be established regular users of ENDSs. To better understand use behavior within the population, the sample was sorted into percentiles with bins based on daily puff counts. To frame these data in the relevant context, they were binned into low-, moderate-, and high-use categories (10th to 40th, 40th to 70th, and 70th to 100th percentiles, respectively), with the low-use group representing the nonintense category, the high-use group representing the intense category, and the moderate-use group being reflective of the average consumer. RESULTS: Participants with higher daily use took substantially more puffs per use session (6.71 vs 4.40) and puffed more frequently (interpuff interval: 32.78 s vs 61.66 s) than participants in the low-use group. Puff duration remained consistent across the low-, moderate­, and high-use groups (2.10 s, 2.18 s, and 2.19 s, respectively). The moderate-use group had significantly shorter session lengths (P<.001) than the high- and low-use groups, which did not differ significantly from each other (P=.16). CONCLUSIONS: Using connected devices allows for a convenient and robust approach to the collection of behavioral outcomes related to product use in an ambulatory setting. By using the variables captured with these tools, it becomes possible to move away from predefined periods of use to better understand topography outcomes and define use sessions. The data presented here offer a possible method to define these sessions. These data also begin to frame international standards used for the analytical assessments of ENDSs in the correct context and begin to shed light on the differences between standardized testing regimens and actual use behavior. TRIAL REGISTRATION: Clinicaltrials.gov NCT04226404; https://clinicaltrials.gov/study/NCT04226404.

Mutation Burden Independently Predicts Survival in the Pan-Cancer Atlas.

PURPOSE: Long-standing clinical predictors of cancer survival have included histopathologic type, stage, and grade. We hypothesized that the principal categories of tumor somatic mutations might also portend survival. We investigated this hypothesis using the Pan-Cancer Atlas, encompassing clinical, genomic, and outcome data of 10,652 patients and 32 cancer types. METHODS: We evaluated the prognostic capability of cancer type, stage, grade and the burden of each major mutation category on overall and disease-specific survival. Mutation categories included short substitution and insertion-deletion mutations (SMs), copy number alterations (CNAs), and gene fusions. RESULTS: SM count and CNA fraction proved to be strong independent predictors of survival (joint P = 5.3e-95) that remained highly significant when adjusted for the traditional factors. Importantly, the relationship between mutation burden and survival proved to be nonlinear (P = 9.5e-56); survival improved at both low- and high-burden extremes. In clinically predictive modeling, SM count together with CNA fraction meaningfully distinguished survival even among patients sharing a given cancer type, stage, or grade. CONCLUSION: Burden of somatic mutation is a key index of survival of analogous clinical utility to these traditional factors.

Fob1-dependent condensin recruitment and loop extrusion on yeast chromosome III.

Despite recent advances in single-molecule and structural analysis of condensin activity in vitro, mechanisms of functional condensin loading and loop extrusion that lead to specific chromosomal organization remain unclear. In Saccharomyces cerevisiae, the most prominent condensin loading site is the rDNA locus on chromosome XII, but its repetitiveness deters rigorous analysis of individual genes. An equally prominent non-rDNA condensin site is located on chromosome III (chrIII). It lies in the promoter of a putative non-coding RNA gene called RDT1, which is in a segment of the recombination enhancer (RE) that dictates MATa-specific chrIII organization. Here, we unexpectedly find that condensin is recruited to the RDT1 promoter in MATa cells through hierarchical interactions with Fob1, Tof2, and cohibin (Lrs4/Csm1), a set of nucleolar factors that also recruit condensin to the rDNA. Fob1 directly binds to this locus in vitro, while its binding in vivo depends on an adjacent Mcm1/α2 binding site that provides MATa cell specificity. We also uncover evidence for condensin-driven loop extrusion anchored by Fob1 and cohibin at RDT1 that unidirectionally extends toward MATa on the right arm of chrIII, supporting donor preference during mating-type switching. S. cerevisiae chrIII therefore provides a new platform for the study of programmed condensin-mediated chromosome conformation.

Novel SMAD3 variant identified in a patient with familial aortopathy modeled using a zebrafish embryo assay.

In human, pathogenic variants in smad3 are one cause of familial aortopathy. We describe a novel SMAD3 variant of unknown significance (VUS), V244F, in a patient who presented with aortic root dilation, right coronary artery ectasia, abdominal aortic aneurysm, right vertebral artery atresia, and cavernoma. Determination of variant pathogenicity impacted multiple aspects of the patient's care, including the most appropriate surgical threshold for which to recommend a valve-sparing aortic root replacement. To determine whether the newly identified SMAD3 variant, and whether SMAD3 induced aortopathy in general, can be assayed in a zebrafish embryo model, we injected smad3a mRNA into Tg[kdrl:mCherry] zebrafish embryos. By measuring the size of the dorsal aorta at 48hpf we found a correlation between pathogenic SMAD3 variants and increased dorsal aortic diameter. The newly identified V244F variant increased dorsal aortic diameter (p < 0.0001) similar to that of the pathogenic control variant T261I (p < 0.0084). In addition, we examined several previously identified variants of uncertain significance and found P124T (p < 0.0467), L296P (p < 0.0025) and A349P (p < 0.0056) to behave like T261I. These results demonstrate that the zebrafish embryo assay was successful in validating known pathogenic variants, classifying our newly identified variant V244F as likely pathogenic, and classifying previously identified variants P124T, L296P, and A349P as likely pathogenic. Overall, our findings identify a novel SMAD3 variant that is likely pathogenic as well as offer a new mechanism to model SMAD3 VUSs in vivo.

An investigation into the development of qualitative tear film disorders in dogs following cryoepilation for distichiasis.

OBJECTIVE: The aim of this prospective study was to compare tear film quality between dogs who have previously undergone cryoepilation for distichiasis to a reference population. ANIMALS STUDIED: Nine dogs (17 eyes) were recruited after surgery and were compared to a reference population of 21 dogs (42 eyes). PROCEDURES: Canine patients who had previously undergone cryoepilation for distichiasis for a minimum of 1 month prior to examination were recruited. A complete ophthalmic examination was performed by an ABVO resident (BDR), with additional tear tests, including tear film interferometry, infra-red meibography, and a tear film break-up time (TFBUT) performed. The tear test results were compared to a reference population obtained from client-owned dogs with no history of ophthalmic complaints, a normal ophthalmic examination performed by an ABVO resident (BDR) and a Schirmer Tear Test-1 > 15 mm/min. Statistical analysis was performed of the results obtained. RESULTS: The treated group was significantly more affected with meibomian gland dropout (MG-dropout) in 11/17 (64.7%) cases, compared to the reference population of 2/21 (9.5%) (p < .01). The treated group had an odds ratio of 23.8 to develop MG-dropout compared to the reference population (p < .01). Tear film breakup time (TFBUT) was significantly shorter in the treatment group (5.8 ± 2.6 s) compared to the reference population (10.1 ± 1.1 s) (p < .001). In the treatment group, 12/17 (70.5%) of treated eyes had a TFBUT < 5 s compared to 2/21 (9.5%) of the reference population. CONCLUSION: Cryoepilation for distichaiasis appears to be a risk factor for developing MG-dropout and qualitative tear film disorders post-operatively in canines.

Targeted degradation of PCNA outperforms stoichiometric inhibition to result in programed cell death.

Biodegraders are targeted protein degradation constructs composed of mini-proteins/peptides linked to E3 ligase receptors. We gained deeper insights into their utility by studying Con1-SPOP, a biodegrader against proliferating cell nuclear antigen (PCNA), an oncology target. Con1-SPOP proved pharmacologically superior to its stoichiometric (non-degrading) inhibitor equivalent (Con1-SPOPmut) as it had more potent anti-proliferative effects and uniquely induced DNA damage, cell apoptosis, and necrosis. Proteomics showed that PCNA degradation gave impaired mitotic division and mitochondria dysfunction, effects not seen with the stoichiometric inhibitor. We further showed that doxycycline-induced Con1-SPOP achieved complete tumor growth inhibition in vivo. Intracellular delivery of mRNA encoding Con1-SPOP via lipid nanoparticles (LNPs) depleted endogenous PCNA within hours of application with nanomolar potency. Our results demonstrate the utility of biodegraders as biological tools and highlight target degradation as a more efficacious approach versus stoichiometric inhibition. Once in vivo delivery is optimized, biodegraders may be leveraged as an exciting therapeutic modality.

Single-Photon Emission Computed Tomographic Reverse Lymphatic Mapping for Groin Vascularized Lymph Node Transplant Planning.

BACKGROUND: Superficial inguinal (groin) vascularized lymph node transplantation is the most common option for the treatment of lymphedema, particularly in combination with free abdominal flap breast reconstruction. This study examines the utility of single-photon emission computed tomographic (SPECT/CT) lymphoscintigraphy for lower extremity reverse lymphatic mapping in presurgical planning for groin vascularized lymph node transplantation and appraises the physiologic lymphatic drainage to the superficial inguinal lymph nodes. METHODS: All patients who underwent bilateral lower extremity SPECT/CT reverse lymphatic mapping over a 5-year period were included. Retrospective case note analysis was performed to collect demographic, surgical, and outcomes data. RESULTS: The study included 84 patients; 56 of these subsequently underwent groin vascularized lymph node transplantation (58 flaps). Fifty-four of these flaps were combined with free abdominal flaps for breast reconstruction (55 flaps). Using SPECT/CT reverse lymphatic mapping investigation of 168 inguinal regions, drainage to at least one superficial inguinal region was visualized in 38.1 percent of patients; in 13.1 percent, drainage was visualized to both superficial inguinal regions. Using this information for presurgical planning, groin vascularized lymph node flap harvest was performed from the contralateral side in 57 of 58 cases (98.3 percent) using intraoperative gamma probe guidance, and no patient developed donor lower extremity lymphedema during follow-up (mean ± SD, 34.5 ± 15.4 months). CONCLUSIONS: The authors' use of presurgical SPECT/CT reverse lymphatic mapping together with limited flap dissection and intraoperative gamma probe guidance resulted in no clinical cases of iatrogenic donor lower extremity lymphedema. The high incidence of drainage from the lower extremity to the superficial inguinal region mandates the use of reverse lymphatic mapping when performing groin vascularized lymph node transplantation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, IV.

Treatment of Metastatic Melanoma with a Combination of Immunotherapies and Molecularly Targeted Therapies.

Melanoma possesses invasive metastatic growth patterns and is one of the most aggressive types of skin cancer. In 2021, it is estimated that 7180 deaths were attributed to melanoma in the United States alone. Once melanoma metastasizes, traditional therapies are no longer effective. Instead, immunotherapies, such as ipilimumab, pembrolizumab, and nivolumab, are the treatment options for malignant melanoma. Several biomarkers involved in tumorigenesis have been identified as potential targets for molecularly targeted melanoma therapy, such as tyrosine kinase inhibitors (TKIs). Unfortunately, melanoma quickly acquires resistance to these molecularly targeted therapies. To bypass resistance, combination treatment with immunotherapies and single or multiple TKIs have been employed and have been shown to improve the prognosis of melanoma patients compared to monotherapy. This review discusses several combination therapies that target melanoma biomarkers, such as BRAF, MEK, RAS, c-KIT, VEGFR, c-MET and PI3K. Several of these regimens are already FDA-approved for treating metastatic melanoma, while others are still in clinical trials. Continued research into the causes of resistance and factors influencing the efficacy of these combination treatments, such as specific mutations in oncogenic proteins, may further improve the effectiveness of combination therapies, providing a better prognosis for melanoma patients.

JAK in the [Black] Box: A Dermatology Perspective on Systemic JAK Inhibitor Safety.

Janus kinase (JAK) inhibitors are immunomodulatory agents with broad potential for use within dermatology. However, the US Food and Drug Administration has recently placed additional warning labels on JAK inhibitors given concern for an increased risk of major adverse cardiovascular events, malignancy, venous thromboembolism, and mortality. Here, we summarize recent efficacy and safety data of multiple JAK inhibitors including tofacitinib, upadacitinib, baricitinib, and abrocitinib. JAK inhibitors have high efficacy in treating psoriatic arthritis and atopic dermatitis, but carry an increased risk of venous thromboembolism and cardiovascular events relative to other approved treatments. Here, we provide current considerations on balancing the benefits of JAK inhibitors with potentially serious, but low-absolute risk, safety concerns.

The host inflammatory response contributes to disease severity in Crimean-Congo hemorrhagic fever virus infected mice.

Crimean-Congo hemorrhagic fever virus (CCHFV) is an important human pathogen. In cell culture, CCHFV is sensed by the cytoplasmic RNA sensor retinoic acid-inducible gene I (RIG-I) molecule and its adaptor molecule mitochondrial antiviral signaling (MAVS) protein. MAVS initiates both type I interferon (IFN-I) and proinflammatory responses. Here, we studied the role MAVS plays in CCHFV infection in mice in both the presence and absence of IFN-I activity. MAVS-deficient mice were not susceptible to CCHFV infection when IFN-I signaling was active and showed no signs of disease. When IFN-I signaling was blocked by antibody, MAVS-deficient mice lost significant weight, but were uniformly protected from lethal disease, whereas all control mice succumbed to infection. Cytokine activity in the infected MAVS-deficient mice was markedly blunted. Subsequent investigation revealed that CCHFV infected mice lacking TNF-α receptor signaling (TNFA-R-deficient), but not IL-6 or IL-1 activity, had more limited liver injury and were largely protected from lethal outcomes. Treatment of mice with an anti-TNF-α neutralizing antibody also conferred partial protection in a post-virus exposure setting. Additionally, we found that a disease causing, but non-lethal strain of CCHFV produced more blunted inflammatory cytokine responses compared to a lethal strain in mice. Our work reveals that MAVS activation and cytokine production both contribute to CCHFV pathogenesis, potentially identifying new therapeutic targets to treat this disease.

Pain management on a trauma service: a crisis reveals opportunities.

Objectives: The opioid crisis has forced an examination of opioid prescribing and usage patterns. Multimodal pain management and limited, procedure-specific prescribing guidelines have been proposed in general surgery but are less well studied in trauma, where multisystem injuries and multispecialty caregivers are the norm. We hypothesized that opioid requirements would differ by primary type of injury and by age, and we sought to identify factors affecting opioid prescribing at discharge (DC). Methods: Retrospective analysis of pain management at a level II trauma center for January-November 2018. Consecutive patients with exploratory laparotomy (LAP); 3 or more rib fractures (fxs) (RIB); or pelvic (PEL), femoral (FEM), or tibial (TIB) fxs were included, and assigned to cohorts based on the predominant injury. Patients who died or had head Abbreviated Injury Scale >2 and Glasgow Coma Scale <15 were excluded. All pain medications were recorded daily; doses were converted to oral morphine equivalents (OMEs). The primary outcomes of interest were OMEs administered over the final 72 hours of hospitalization (OME72) and prescribed at DC (OMEDC). Multimodal pain therapy defined as 3 or more drugs used. Categorical variables and continuous variables were analyzed with appropriate statistical analyses. Results: 208 patients were included: 17 LAP, 106 RIB, 31 PEL, 26 FEM, and 28 TIB. 74% were male and 8% were using opiates prior to admission. Injury cohorts varied by age but not Injury Severity Score (ISS) or length of stay (LOS). 64% of patients received multimodal pain therapy. There was an overall difference in OME72 between the five injury groups (p<0.0001) and OME72 was lower for RIB compared with all other cohorts. Compared with younger (age <65) patients, older (≥65 years) patients had similar ISS and LOS, but lower OME72 (45 vs 135*) and OMEDC. Median OME72 differed significantly between older and younger patients with PEL (p=0.02) and RIB (p=0.01) injuries. No relationship existed between OMEDC across injury groups, by sex or injury severity. Patients were discharged almost exclusively by trauma service advanced practice clinicians (APCs). There was no difference among APCs in number of pills or OMEs prescribed. 81% of patients received opioids at DC, of whom 69% were prescribed an opioid/acetaminophen combination drug; and only 13% were prescribed non-steroidal anti-inflammatory drugs, 19% acetaminophen, and 31% gabapentin. Conclusions: Opioid usage varied among patients with different injury types. Opioid DC prescribing appears rote and does not correlate with actual opioid usage during the 72 hours prior to DC. Paradoxically, OMEDC tends to be higher among females, patients with ISS <16, and those with rib fxs, despite a tendency toward lower OME72 usage among these groups. There was apparent underutilization of non-opioid agents. These findings highlight opportunities for improvement and further study. Level of evidence: IV.