Pulsed Photothermal Therapy of Solid Tumors as a Precondition for Immunotherapy.

Photothermal therapy (PTT) refers to the use of plasmonic nanoparticles to convert electromagnetic radiation in the near infrared region to heat and kill tumor cells. Continuous wave lasers have been used clinically to induce PTT, but the treatment is associated with heat-induced tissue damage that limits usability. Here, the engineering and validation of a novel long-pulsed laser device able to induce selective and localized mild hyperthermia in tumors while reducing the heat affected zone and unwanted damage to surrounding tissue are reported. Long-pulsed PTT induces acute necrotic cell death in heat affected areas and the release of tumor associated antigens. This antigen release triggers maturation and stimulation of CD80/CD86 in dendritic cells in vivo that primes a cytotoxic T cell response. Accordingly, long-pulsed PTT enhances the therapeutic effects of immune checkpoint inhibition and increases survival of mice with bladder cancer. Combined, the data promote long-pulsed PTT as a safe and effective strategy for enhancing therapeutic responses to immune checkpoint inhibitors while minimizing unwanted tissue damage.

Stage-Based Detection Methods and Recurrence Patterns for Cutaneous Melanoma.

Melanoma surveillance guidelines vary. Melanoma recurrence patterns and detection methods were examined. Resected melanoma patients were reviewed. Recurrence detection included patient complaint (PC), physical exam (PE), cross-sectional imaging (CSI), and ultrasound (US). 276 patients were included: 131 stage I, 83 stage II, and 62 stage III. Recurrence rates were 8%, 24%, and 27%, respectively. For stage I patients, 46% of recurrences were local, 18% regional, and 36% distant. Patient complaint identified 55% of recurrences, PE 36%, and CSI 9%. For stage II, 20% of recurrences were local, 20% regional, and 60% distant. Patient complaint identified 35% of recurrences, PE 20%, and CSI 45%. For stage III, 6% of recurrences were local, 53% regional, and 41% distant. Patient complaint identified 17% of recurrences, PE 12%, CSI 59%, and US 12%. Average time to recurrence by stage was 23.7, 24.6, and 17.7 months, respectively. H&P for all melanoma patients and CSI for higher stages are effective surveillance strategies.

A genome-wide CRISPR screen maps endogenous regulators of PPARG gene expression in bladder cancer.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor central in the regulation of key cellular processes including cell metabolism, tissue differentiation, and regulation of the immune system. PPARγ is required for normal differentiation of the urothelium and is thought to be an essential driver of the luminal subtype of bladder cancer. However, the molecular components that regulate PPARG gene expression in bladder cancer remain unclear. Here, we developed an endogenous PPARG reporter system in luminal bladder cancer cells and performed genome-wide CRISPR knockout screening to identify bona fide regulators of PPARG gene expression. Functional validation of the dataset confirmed GATA3, SPT6, and the cohesin complex components SMC1A, and RAD21, as permissive upstream positive regulators of PPARG gene expression in luminal bladder cancer. In summary, this work provides a resource and biological insights to aid our understanding of PPARG regulation in bladder cancer.

open-UST: An Open-Source Ultrasound Tomography Transducer Array System.

Fast imaging methods are needed to promote clinical adoption of ultrasound tomography (UST), and more widely available UST hardware could support the experimental validation of new measurement configurations. In this work, an open-source 256-element transducer ring array was developed (morganjroberts.github. io/open-UST) and manufactured using rapid prototyping, for only £2k. Novel manufacturing techniques were used, resulting in a 1.17° mean beam axis skew angle, a [Formula: see text] mean element position error, and a [Formula: see text] deviation in matching layer thickness. The nominal acoustic performance was measured using hydrophone scans and watershot data, and the 61.2 dB signal-to-noise ratio (SNR), 55.4° opening angle, 10.2 mm beamwidth, and 54% transmit-receive bandwidth (-12 dB) were found to be similar to existing systems and compatible with state-of-the-art full-waveform-inversion image reconstruction methods. The interelement variation in acoustic performance was typically < 10% without using normalization, meaning that the elements can be modeled identically during image reconstruction, removing the need for individual source definitions based on hydrophone measurements. Finally, data from a phantom experiment were successfully reconstructed. These results demonstrate that the open-UST system is accessible for users and is suitable for UST imaging research.

Effects of the COVID-19 Pandemic on the Trauma Population in a Level 1 Trauma Center.

In this study, we evaluated the effects of the pandemic on our trauma population. We performed a retrospective review of the trauma registry in the 2 years prior, and then 2 years during the pandemic. We evaluated age, race, gender, injury severity score (ISS), mechanism of trauma, rate of self-inflicted injury, rate of gunshot wounds (GSW), presence of EtOH, drug screen results, mortality, rate of burn traumas, and zip code of residence. Our query captured 5 054 patients before, and 5 731 during the pandemic. We found no statistical difference in age, gender, mechanism of trauma, rate of self-inflicted injuries, and mortality during the pandemic when compared to before. There were statistically significant differences in race, ISS, rate of GSWs, EtOH use, drug screen results, and burn traumas. Geospatial mapping found a rise in GSWs for zip code 36606. Gun violence and substance use rose in our trauma population during COVID-19.

A Multidisciplinary Approach for the Management of Portal Vein Thrombosis.

Portal venous thrombosis (PVT) is an uncommon disease associated with highly morbid conditions such as intestinal ischemia and portal hypertension. Patients at higher risk of developing PVT include those with cirrhosis, malignancy, or prothrombotic states. The mainstay of treatment is early initiation of anticoagulation. The first case is a 49-year-old female diagnosed with a cecal mass and PVT. She was started on anticoagulation and underwent a right hemicolectomy with several small bowel resections. She developed portal hypertension that required TIPS and mechanical thrombectomy. The second patient is a 65-year-old female found to have PVT. She was anticoagulated with heparin and given systemic TPA. She developed intestinal ischemia and portal hypertension requiring small bowel resection, TIPS, and mechanical thrombectomy. These cases give insight into the impact of a multidisciplinary team approach to PVT. The role and timing of endovascular treatment is not well established and needs to be further investigated.

Characterization of Growth Performance, Pork Quality, and Body Composition in Mangalica Pigs.

European heritage breeds, such as the Blonde (B), Red (R), and Swallow-bellied (SB) Mangalica pig, display an extreme propensity to fatten and are reputed to produce superior quality pork. This suggests that Mangalica pork should command a higher price, and the Mangalica is a candidate breed to target niche markets within the United States. Our objectives were to test this hypothesis by (1) directly comparing growth performance and carcass merit of purebred Yorkshire (Y), B, R, and SB Mangalica pigs to identify the best breed for adoption, and (2) comparing indices of pork quality in purebred R, Y, and crossbred (R × Y) pigs to determine if crossbreeding represented a viable alternative to the adoption of purebred Mangalica. Daily feed intake, average daily gain (ADG), and feed efficiency were highest in Y and lowest in SB pigs with B and R ranked intermediately (p < 0.001). Backfat thickness was greatest in B and lowest in Y with R and SB ranked intermediately (p < 0.001). Marbling score was greatest in R pigs and lowest in Y pigs with B and SB ranked intermediately (p < 0.01). In contrast, loin eye area (LEA) was greatest in Y pigs compared to B, R, and SB (p < 0.001). Indices of meat quality were then compared in R, R × Y, and Y pigs. Backfat thickness and marbling scores were greater in R than R × Y and Y pigs (p < 0.001) while LEA was greater in Y than R × Y and R pigs (p < 0.001). Loin and ham ultimate pH, color, and firmness scores were significantly greater in R than R × Y or Y pigs (p < 0.05). Meanwhile, cook loss was significantly less in R than Y pigs (p < 0.007) while Warner-Bratzler Shear Force (WBS) was not different in chops between groups (p < 0.11). These data indicate that though Mangalica exhibit poorer growth performance, Mangalica pork exhibits superior pork quality attributes, suggesting that higher price points for Mangalica pork in niche markets are justified.

Occupational safety and health guidelines in relation to COVID-19 risk, death risk, and case-fatality proportion: An international, ecological study.

Background: Coronavirus disease 2019 (COVID-19) began in 2019 with several unknown factors. The World Health Organization (WHO) subsequently developed COVID-19 occupational safety and health (OSH) guidelines to reduce occupational COVID-19 transmission. Many countries also developed their own COVID-19 OSH guidelines, but whether these guidelines included WHO's guidelines and whether including WHO's guidelines in countries' COVID-19 OSH guidelines reduced COVID-19 transmission is unknown. Objectives: The objectives of our study were to (1) compare the COVID-19 OSH guidelines of several countries to WHO's OSH guidelines, (2) estimate associations between characteristics of countries and their OSH guidelines and the number of WHO's OSH guidelines included in countries' OSH guidelines, and (3) estimate associations between WHO's OSH guidelines included in countries' OSH guidelines and COVID-19 risk, death risk, and case-fatality proportion. Methods: This study represents international, ecological research of 36 countries from all six world health regions. Countries' COVID-19 OSH guidelines were compared with WHO's OSH guidelines. Linear regression models adjusted for potential confounders were used to estimate associations of interest. Results: The median number of WHO's 15 COVID-19 OSH guidelines included in countries' COVID-19 OSH guidelines was eight. Countries' COVID-19 OSH guidelines focused on workers included significantly more of WHO's COVID-19 OSH guidelines than countries' COVID-19 OSH guidelines focused on general populations. Including "provide personal protective equipment for workers" and "create workplace policy for wearing personal protective equipment" in countries' COVID-19 OSH guidelines were significantly related to decreased COVID-19 risk, death risk, and/or case-fatality proportion. Conclusions: Countries' COVID-19 OSH guidelines should include WHO's guidelines, focus on workers, and include "provide personal protective equipment for workers" and "create workplace policy for wearing personal protective equipment."

Reducing Health Care Burden of Emergency General Surgery with a 24-Hour Dedicated Emergency General Surgery Service.

BACKGROUND: Emergency general surgery (EGS) diagnoses account for 11% of surgical admissions and 50% of surgical mortality. In this population, 7 specific operations are associated with 80.3% of deaths, 78.9% of complications, and 80.2% of hospital costs. In 2016, our institution established a comprehensive in-house EGS service. Herein, we hypothesize that formation of a dedicated EGS service is associated with a significant reduction in morbidity for patients undergoing the most common EGS procedures. METHODS: All patients undergoing one of the most common EGS procedures within 2 days of admission were identified from 1/1/2013 to 5/9/2019 via a retrospective chart review. Patients were cohorted as pre- and post-EGS implementation. The primary outcome measure was the overall complication rate. Secondary endpoints included mortality, individual complication rate, time to operation, overnight operation, and length of stay. Finally, both cohorts were benchmarked to national outcomes. RESULTS: 718 patients met inclusion criteria (pre-EGS = 409 and post-EGS = 309). Overall complication rate decreased significantly (19.8% vs 13.9%, P = .0387) and overnight operations increased significantly in the post-EGS group (7.8%-16.5%, P = .0003). Pre-EGS complications were higher than national data in all but 1 procedure group, whereas post-EGS complications rates were lower in all but 2 categories. DISCUSSION: Implementation of a dedicated EGS service line was associated with a significant decrease in complication rate among the most complication-prone EGS procedures. Number of operations within 24 hours did not increase significantly; however, overnight operations did increase. Our results indicate that establishing a service-specific EGS line is reasonable and beneficial.

Longitudinal Analysis of the Intestinal Microbiota in the Obese Mangalica Pig Reveals Alterations in Bacteria and Bacteriophage Populations Associated With Changes in Body Composition and Diet.

Due to its immunomodulatory potential, the intestinal microbiota has been implicated as a contributing factor in the development of the meta-inflammatory state that drives obesity-associated insulin resistance and type 2 diabetes. A better understanding of this link would facilitate the development of targeted treatments and therapies to treat the metabolic complications of obesity. To this end, we validated and utilized a novel swine model of obesity, the Mangalica pig, to characterize changes in the gut microbiota during the development of an obese phenotype, and in response to dietary differences. In the first study, we characterized the metabolic phenotype and gut microbiota in lean and obese adult Mangalica pigs. Obese or lean groups were created by allowing either ad libitum (obese) or restricted (lean) access to a standard diet for 54 weeks. Mature obese pigs were significantly heavier and exhibited 170% greater subcutaneous adipose tissue mass, with no differences in muscle mass compared to their lean counterparts. Obese pigs displayed impaired glucose tolerance and hyperinsulinemia following oral glucose challenge, indicating that a metabolic phenotype also manifested with changes in body composition. Consistent with observations in human obesity, the gut microbiota of obese pigs displayed altered bacterial composition. In the second study, we characterized the longitudinal changes in the gut microbiota in response to diet and aging in growing Mangalica pigs that were either limit fed a standard diet, allowed ad libitum access to a standard diet, or allowed ad libitum access to a high fat-supplemented diet over an 18-week period. As expected, weight gain was highest in pigs fed the high fat diet compared to ad libitum and limit fed groups. Furthermore, the ad libitum and high fat groups displayed significantly greater adiposity consistent with the development of obesity relative to the limit fed pigs. The intestinal microbiota was generally resilient to differences in dietary intake (limit fed vs ad libitum), though changes in the microbiota of pigs fed the high fat diet mirrored changes observed in mature obese pigs during the first study. This is consistent with the link observed between the microbiota and adiposity. In contrast to intestinal bacterial populations, bacteriophage populations within the gut microbiota responded rapidly to differences in diet, with significant compositional changes in bacteriophage genera observed between the dietary treatment groups as pigs aged. These studies are the first to describe the development of the intestinal microbiota in the Mangalica pig, and are the first to provide evidence that changes in body composition and dietary conditions are associated with changes in the microbiome of this novel porcine model of obesity.

Development of a bispecific immune engager using a recombinant malaria protein.

As an immune evasion and survival strategy, the Plasmodium falciparum malaria parasite has evolved a protein named VAR2CSA. This protein mediates sequestration of infected red blood cells in the placenta through the interaction with a unique carbohydrate abundantly and exclusively present in the placenta. Cancer cells were found to share the same expression of this distinct carbohydrate, termed oncofetal chondroitin sulfate on their surface. In this study we have used a protein conjugation system to produce a bispecific immune engager, V-aCD3, based on recombinant VAR2CSA as the cancer targeting moiety and an anti-CD3 single-chain variable fragment linked to a single-chain Fc as the immune engager. Conjugation of these two proteins resulted in a single functional moiety that induced immune mediated killing of a broad range of cancer cells in vitro and facilitated tumor arrest in an orthotopic bladder cancer xenograft model.

Measurement and simulation of steered acoustic fields generated by a multielement array for therapeutic ultrasound.

Modelling of fields generated by therapeutic ultrasound arrays can be prone to errors arising from differences from nominal transducer parameters, and variations in relative outputs of array elements when driven under different conditions, especially when simulating steered fields. Here, the effect of element size, element positions, relative source pressure variations, and electrical crosstalk on the accuracy of modelling pressure fields generated by a 555 kHz 32-element ultrasonic array were investigated. For this transducer, errors in pressure amplitude and focal position were respectively reduced from 20% to 4% and 3.3 mm to 1.5 mm using crosstalk prediction, and experimentally determined positions.

Notum deacylates octanoylated ghrelin.

OBJECTIVES: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined. METHODS: We used mass spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme-substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation. RESULTS: Mass spectrometry detected the removal of octanoyl from ghrelin by purified active Notum but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex showed that the octanoyl lipid was accommodated in the hydrophobic pocket of the Notum. The knock-in allele expressing HA-tagged Notum revealed that Notum was produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed a high-fat diet led to a small but significant increase in acylated ghrelin in the circulation, while no such increase was seen in wild-type animals on the same diet. CONCLUSIONS: Overall, our data demonstrate that Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high-fat diet conditions. Our study therefore adds Notum to the list of enzymes, including butyrylcholinesterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.

Deep Phenotyping by Mass Cytometry and Single-Cell RNA-Sequencing Reveals LYN-Regulated Signaling Profiles Underlying Monocyte Subset Heterogeneity and Lifespan.

RATIONALE: Monocytes are key effectors of the mononuclear phagocyte system, playing critical roles in regulating tissue homeostasis and coordinating inflammatory reactions, including those involved in chronic inflammatory diseases such as atherosclerosis. Monocytes have traditionally been divided into 2 major subsets termed conventional monocytes and patrolling monocytes (pMo) but recent systems immunology approaches have identified marked heterogeneity within these cells, and much of what regulates monocyte population homeostasis remains unknown. We and others have previously identified LYN tyrosine kinase as a key negative regulator of myeloid cell biology; however, LYN's role in regulating specific monocyte subset homeostasis has not been investigated. OBJECTIVE: We sought to comprehensively profile monocytes to elucidate the underlying heterogeneity within monocytes and dissect how Lyn deficiency affects monocyte subset composition, signaling, and gene expression. We further tested the biological significance of these findings in a model of atherosclerosis. METHODS AND RESULTS: Mass cytometric analysis of monocyte subsets and signaling pathway activation patterns in conventional monocytes and pMos revealed distinct baseline signaling profiles and far greater heterogeneity than previously described. Lyn deficiency led to a selective expansion of pMos and alterations in specific signaling pathways within these cells, revealing a critical role for LYN in pMo physiology. LYN's role in regulating pMos was cell-intrinsic and correlated with an increased circulating half-life of Lyn-deficient pMos. Furthermore, single-cell RNA sequencing revealed marked perturbations in the gene expression profiles of Lyn-/- monocytes with upregulation of genes involved in pMo development, survival, and function. Lyn deficiency also led to a significant increase in aorta-associated pMos and protected Ldlr-/- mice from high-fat diet-induced atherosclerosis. CONCLUSIONS: Together our data identify LYN as a key regulator of pMo development and a potential therapeutic target in inflammatory diseases regulated by pMos.

Y-box binding protein-1 is crucial in acquired drug resistance development in metastatic clear-cell renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is a highly vascular tumor and patients with low risk metastatic RCC of clear-cell histological sub-type (mccRCC) are treated with tyrosine-kinase inhibitors (TKIs), sunitinib, as the first-line of treatment. Unfortunately, TKI resistance eventually develops, and the underlying molecular mechanism is not well understood. METHODS: RCC cell-line with metastatic clear-cell histology (Caki-1), and patient samples were analysed to identify the role of Y-box binding protein 1 (YB-1) and ATP-binding cassette sub-family B member 1 (ABCB-1) in acquired sunitinib-resistance development. Caki-1 was conditioned with increasing sunitinib doses to recapitulate acquired resistance development in clinics. Sunitinib-conditioned and wild-type Caki-1 were subjected to cell viability assay, scratch assay, chicken embryo chorioallantoic membrane engraftment and proteomics analysis. Classical biochemical assays like flow cytometry, immunofluorescent staining, immunohistochemical staining, optical coherence tomography imaging, Western Blot and RT-PCR assays were applied to determine the possible mechanism of sunitinib-resistance development and the effect of drug treatments. Publicly available data was also used to determine the role of YB-1 upregulation in ccRCC and the patients' overall survival. RESULTS: We demonstrate that YB-1 and ABCB-1 are upregulated in sunitinib-resistant in vitro, ex vivo, in vivo and patient samples compared to the sensitive samples. This provides evidence to a mechanism of acquired sunitinib-resistance development in mccRCC. Furthermore, our results establish that inhibiting ABCB-1 with elacridar, in addition to sunitinib, has a positive impact on reverting sunitinib-resistance development in in vitro, ex vivo and in vivo models. CONCLUSION: This work proposes a targeted therapy (elacridar and sunitinib) to re-sensitize sunitinib-resistant mccRCC and, possibly, slow disease progression.

Author Correction: Evasion of immunosurveillance by genomic alterations of PPARγ/RXRα in bladder cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Natural Killer Cells Regulate Th17 Cells After Autologous Hematopoietic Stem Cell Transplantation for Relapsing Remitting Multiple Sclerosis.

In autoimmunity, the balance of different helper T (Th) cell subsets can influence the tissue damage caused by autoreactive T cells. Pro-inflammatory Th1 and Th17 T cells are implicated as mediators of several human autoimmune conditions such as multiple sclerosis (MS). Autologous hematopoietic stem cell transplantation (aHSCT) has been tested in phase 2 clinical trials for MS patients with aggressive disease. Abrogation of new clinical relapses and brain lesions can be seen after ablative aHSCT, accompanied by significant reductions in Th17, but not Th1, cell populations and activity. The cause of this selective decrease in Th17 cell responses following ablative aHSCT is not completely understood. We identified an increase in the kinetics of natural killer (NK) cell reconstitution, relative to CD4+ T cells, in MS patients post-aHSCT, resulting in an increased NK cell:CD4+ T cell ratio that correlated with the degree of decrease in Th17 responses. Ex vivo removal of NK cells from post-aHSCT peripheral blood mononuclear cells resulted in higher Th17 cell responses, indicating that NK cells can regulate Th17 activity. NK cells were also found to be cytotoxic to memory Th17 cells, and this toxicity is mediated through NKG2D-dependent necrosis. Surprisingly, NK cells induced memory T cells to secrete more IL-17A. This was preceded by an early rise in T cell expression of RORC and IL17A mRNA, and could be blocked with neutralizing antibodies against CD58, a costimulatory receptor expressed on NK cells. Thus, NK cells provide initial co-stimulation that supports the induction of a Th17 response, followed by NKG2D-dependent cytotoxicity that limits these cells. Together these data suggest that rapid reconstitution of NK cells following aHSCT contribute to the suppression of the re-emergence of Th17 cells. This highlights the importance of NK cells in shaping the reconstituting immune system following aHSCT in MS patients.