Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

Defining the KRAS- and ERK-dependent transcriptome in KRAS-mutant cancers.

How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal-regulated kinase (ERK)-dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.

Metabolite profiling of human renal cell carcinoma reveals tissue-origin dominance in nutrient availability.

The tumor microenvironment is a determinant of cancer progression and therapeutic efficacy, with nutrient availability playing an important role. Although it is established that the local abundance of specific nutrients defines the metabolic parameters for tumor growth, the factors guiding nutrient availability in tumor compared to normal tissue and blood remain poorly understood. To define these factors in renal cell carcinoma (RCC), we performed quantitative metabolomic and comprehensive lipidomic analyses of tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples collected from patients. TIF nutrient composition closely resembles KIF, suggesting that tissue-specific factors unrelated to the presence of cancer exert a stronger influence on nutrient levels than tumor-driven alterations. Notably, select metabolite changes consistent with known features of RCC metabolism are found in RCC TIF, while glucose levels in TIF are not depleted to levels that are lower than those found in KIF. These findings inform tissue nutrient dynamics in RCC, highlighting a dominant role of non-cancer-driven tissue factors in shaping nutrient availability in these tumors.

Cancer cells convert nutrients into energy differently compared to healthy cells. This difference in metabolism allows them to grow and divide more quickly and sometimes to migrate to different areas of the body. The environment around cancer cells ­ known as the tumor microenvironment ­ contains a variety of different cells and blood vessels, which are bathed in interstitial fluid. This microenvironment provides nutrients for the cancer cells to metabolize, and therefore influences how well a tumor grows and how it might respond to treatment. Recent advances with techniques such as mass spectrometry, which can measure the chemical composition of a substance, have allowed scientists to measure nutrient levels in the tumor microenvironments of mice. However, it has been more difficult to conduct such studies in humans, as well as to compare the tumor microenvironment to the healthy tissue the tumors arose from. Abbott, Ali, Reinfeld et al. aimed to fill this gap in knowledge by using mass spectrometry to measure the nutrient levels in the tumor microenvironment of 55 patients undergoing surgery to remove kidney tumors. Comparing the type and levels of nutrients in the tumor interstitial fluid, the neighboring healthy kidney and the blood showed that nutrients in the tumor and healthy kidney were more similar to each other than those in the blood. For example, both the tumor and healthy kidney interstitial fluids contained less glucose than the blood. However, the difference between nutrient composition in the tumor and healthy kidney interstitial fluids was insignificant, suggesting that the healthy kidney and its tumor share a similar environment. Taken together, the findings indicate that kidney cancer cells must adapt to the nutrients available in the kidney, rather than changing what nutrients are available in the tissue. Future studies will be required to investigate whether this finding also applies to other types of cancer. A better understanding of how cancer cells adapt to their environments may aid the development of drugs that aim to disrupt the metabolism of tumors.

Level of BPA contamination in resin composites determines BPA release.

OBJECTIVES: Resin composites may release bisphenol A (BPA) due to impurities present in the monomers. However, there is a lack of knowledge regarding the leaching characteristics of BPA from resin composites. Therefore, experimental resin composites were prepared with known amounts of BPA. The objective of this study was (1) to determine which amount of BPA initially present in the material leaches out in the short term and, (2) how this release is influenced by the resin composition. METHODS: BPA (0, 0.001, 0.01, or 0.1 wt%) was added to experimental resin composites containing 60 mol% BisGMA, BisEMA(3), or UDMA, respectively, as base monomer and 40 mol% TEGDMA as diluent monomer. Polymerized samples (n = 5) were immersed at 37 °C for 7 days in 1 mL of water, which was collected and refreshed daily. BPA release was quantified with UPLC-MS/MS after derivatization with pyridine-3-sulfonyl chloride. RESULTS: Between 0.47 to 0.67 mol% of the originally added BPA eluted from the resin composites after 7 days. Similar elution trends were observed irrespective of the base monomer. Two-way ANOVA showed a significant effect of the base monomer on BPA release, but the differences were small and not consistent. SIGNIFICANCE: The released amount of BPA was directly proportional to the quantity of BPA present in the resin composite as an impurity. BPA release was mainly diffusion-based, while polymer composition seemed to play a minor role. Our results underscore the importance for manufacturers only to use monomers of the highest purity in dental resin composites to avoid unnecessary BPA exposure in patients.

Magnet-assisted endoscopic removal of ferromagnetic metallic gastric foreign bodies in 4 dogs.

OBJECTIVE: Describe presenting signs, diagnostic findings, and magnet-assisted endoscopic removal method of ferromagnetic gastric foreign bodies (FBs) in dogs. CLINICAL PRESENTATION: Four dogs presented with ingestion of sharp metallic FBs. The presence of gastric FBs was confirmed by abdominal radiography. RESULTS: In 3 cases, initial attempts at endoscopic removal were unsuccessful because of ingesta and fluid in the stomach. A magnet contained within a Roth net was introduced endoscopically. Magnet and attached objects were successfully removed from the stomach. In the fourth case, removal with a magnet was judged to be the most expedient method of removal because multiple metallic objects were present. CLINICAL RELEVANCE: An endoscopic technique was used for the removal of difficult-to-visualize or multiple metallic FBs. The use of this technique allows the removal of ferromagnetic gastric FBs without surgery or risk of complications associated with the passage of sharp material through the gastrointestinal (GI) tract.

Long-term outcomes of revision total hip arthroplasty with the Zimmer Modular Revision hip system.

Aims: There are limited long-term studies reporting on outcomes of the Zimmer Modular Revision (ZMR) stem, and concerns remain regarding failure. Our primary aim was to determine long-term survival free from all-cause revision and stem-related failure for this modular revision stem in revision total hip arthroplasty (THA). Secondary aims included evaluating radiological and functional outcomes. Methods: We retrospectively identified all patients in our institutional database who underwent revision THA using the ZMR system from January 2000 to December 2007. We included 106 patients (108 hips) with a mean follow-up of 14.5 years (2.3 to 22.3). Mean patient age was 69.2 years (37.0 to 89.4), and 51.9% were female (n = 55). Indications for index revision included aseptic loosening (73.1%), infection (16.7%), fracture (9.3%), and stem fracture (0.9%). Kaplan-Meier analysis was used to determine the all-cause and stem-related failure revision-free survival. At most recent follow-up, Oxford Hip Scores (OHS) were collected, and radiological stem stability was determined using the Engh classification. Results: A total of 17 hips (15.7%) underwent re-revision of any component. Indications for re-revision were stem failure (35.3%; n = 6), infection (29.4%; n = 5), instability (29.4%; n = 5), and acetabular aseptic loosening (5.9%; n = 1). The five- and 15-year all-cause survival was 89.7% (95% confidence interval (CI) 86.7 to 92.7) and 83.3% (95% CI 79.6 to 87.0), respectively. There were six re-revisions (5.6%) for stem failure; five for stem fracture and one for aseptic loosening. The five- and 15-year survival free from stem-related failure was 97.2% (95% CI 95.6 to 98.8) and 94.0% (95% CI 91.6 to 96.4), respectively. At final follow-up, the mean OHS was 36.9 (8.0 to 48.0) and 95.7% (n = 66) of surviving modular revision stems were well-fixed in available radiographs. Conclusion: Femoral revision with the ZMR offers satisfactory long-term all-cause revision-free survival, good survival free of stem-related failure, and favourable clinical outcomes. Stem fracture was the most common reason for stem-related failure and occurred both early and late. This highlights the importance of both early and long-term surveillance for stem-related failure.

FOXO1 enhances CAR T cell stemness, metabolic fitness and efficacy.

Chimeric antigen receptor (CAR) T cell therapy has transformed the treatment of haematological malignancies such as acute lymphoblastic leukaemia, B cell lymphoma and multiple myeloma1-4, but the efficacy of CAR T cell therapy in solid tumours has been limited5. This is owing to a number of factors, including the immunosuppressive tumour microenvironment that gives rise to poorly persisting and metabolically dysfunctional T cells. Analysis of anti-CD19 CAR T cells used clinically has shown that positive treatment outcomes are associated with a more 'stem-like' phenotype and increased mitochondrial mass6-8. We therefore sought to identify transcription factors that could enhance CAR T cell fitness and efficacy against solid tumours. Here we show that overexpression of FOXO1 promotes a stem-like phenotype in CAR T cells derived from either healthy human donors or patients, which correlates with improved mitochondrial fitness, persistence and therapeutic efficacy in vivo. This work thus reveals an engineering approach to genetically enforce a favourable metabolic phenotype that has high translational potential to improve the efficacy of CAR T cells against solid tumours.

Diet and physical activity interventions to improve cardiovascular disease risk factors in liver transplant recipients: Systematic review and meta-analysis.

BACKGROUND AND AIMS: Cardiovascular disease, associated risk factors and obesity are prevalent after liver transplant and modifiable through lifestyle changes. Understanding what lifestyle interventions and their respective components are effective is essential for translation to clinical practice. We aimed to investigate the effects of diet and physical activity interventions on weight, body mass index and other cardiovascular disease risk factors in liver transplant recipients, and systematically describe the interventions. METHODS: We systematically searched Embase, MEDLINE, Psycho Info, CINAHL, Cochrane central register of controlled trials, PeDro, AMED, BNI, Web of Science, OpenGrey, ClinicalTrials.gov and the international clinical trials registry from inception to 31 May 2023. Search results were screened by two independent reviewers: randomised control trials with interventions that targeted diet and physical activity behaviours in liver transplant recipients were considered eligible. Two independent reviewers extracted and synthesised data for study, participant and intervention details and results. We used the Revised Cochrane Risk of Bias Tool for Randomised Trials to assess risk of bias for outcomes and the GRADE approach to rate the quality of the body of evidence. When two or more studies reported findings for an outcome, we pooled data using random-effects meta-analysis. RESULTS: Six studies were included, reporting three physical activity and three combined diet and physical activity interventions. Participants were 2 months-4 years post-transplant. Interventions lasted 12 weeks-10 months and were delivered remotely and/or in-person, most commonly delivered to individual participants by health care or sports professionals. Five studies described individual tailoring, e.g. exercise intensity. Adherence to interventions ranged from 51% to 94%. No studies reported fidelity. Intervention components were not consistently reported. In meta-analysis, diet and physical activity interventions did not significantly reduce weight or body mass index compared to control groups, however no studies targeted participants with obesity. Diet and physical activity interventions reduced percentage body fat and triglycerides compared to control groups but did not reduce total cholesterol or increase activity. The GRADE quality of evidence was low or very low. CONCLUSION: Diet and physical activity interventions reduced percentage body fat and triglycerides in liver transplant recipients. Further good quality research is needed to evaluate their effect on other cardiovascular disease risk factors, including weight and BMI. Interventions need to be better described and evaluated to improve evidence base and inform patient care.

ctDNA transiting into urine is ultrashort and facilitates noninvasive liquid biopsy of HPV+ oropharyngeal cancer.

BACKGROUNDTransrenal cell-free tumor DNA (TR-ctDNA), which transits from the bloodstream into urine, has the potential to enable noninvasive cancer detection for a wide variety of nonurologic cancer types.MethodsUsing whole-genome sequencing, we discovered that urine TR-ctDNA fragments across multiple cancer types are predominantly ultrashort (<50 bp) and, therefore, likely to be missed by conventional ctDNA assays. We developed an ultrashort droplet digital PCR assay to detect TR-ctDNA originating from HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC) and confirmed that assaying ultrashort DNA is critical for sensitive cancer detection from urine samples.ResultsTR-ctDNA was concordant with plasma ctDNA for cancer detection in patients with HPV+ OPSCC. As proof of concept for using urine TR-ctDNA for posttreatment surveillance, in a small longitudinal case series, TR-ctDNA showed promise for noninvasive detection of recurrence of HPV+ OPSCC.ConclusionOur data indicate that focusing on ultrashort fragments of TR-ctDNA will be important for realizing the full potential of urine-based cancer diagnostics. This has implications for urine-based detection of a wide variety of cancer types and for facilitating access to care through at-home specimen collections.FundingNIH grants R33 CA229023, R21 CA225493; NIH/National Cancer Institute grants U01 CA183848, R01 CA184153, and P30CA046592; American Cancer Society RSG-18-062-01-TBG; American Cancer Society Mission Boost grant MBGI-22-056-01-MBG; and the A. Alfred Taubman Medical Research Institute.

Twin study dissects CXCR3+ memory B cells as non-heritable feature in multiple sclerosis.

BACKGROUND: In multiple sclerosis (MS), B cells are considered main triggers of the disease, likely as the result of complex interaction between genetic and environmental risk factors. Studies on monozygotic twins discordant for MS offer a unique way to reduce this complexity and reveal discrepant subsets. METHODS: In this study, we analyzed B cell subsets in blood samples of monozygotic twins with and without MS using publicly available data. We verified functional characteristics by exploring the role of therapy and performed separate analyses in unrelated individuals. FINDINGS: The frequencies of CXCR3+ memory B cells were reduced in the blood of genetically identical twins with MS compared to their unaffected twin siblings. Natalizumab (anti-VLA-4 antibody) was the only treatment regimen under which these frequencies were reversed. The CNS-homing features of CXCR3+ memory B cells were supported by elevated CXCL10 levels in MS cerebrospinal fluid and their in vitro propensity to develop into antibody-secreting cells. CONCLUSIONS: Circulating CXCR3+ memory B cells are affected by non-heritable cues in people who develop MS. This underlines the requirement of environmental risk factors such as Epstein-Barr virus in triggering these B cells. We propose that after CXCL10-mediated entry into the CNS, CXCR3+ memory B cells mature into antibody-secreting cells to drive MS. FUNDING: This work was supported by Nationaal MS Fonds (OZ2021-016), Stichting MS Research (19-1057 MS, 20-490f MS, and 21-1142 MS), the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program grant agreement no. 882424, and the Swiss National Science Foundation (733 310030_170320, 310030_188450, and CRSII5_183478).

Kidney Function and Physical Performance Decline: The Brain in Kidney Disease (BRINK) Cohort Study.

Rationale & Objective: Patients with kidney failure have poor physical performance, but its trajectory is less clear. We examined physical function over the course of kidney disease, including the transition to dialysis. Study Design: Observational cohort. Setting & Participants: Community-dwelling adults aged ≥45 years in the Brain in Kidney Disease (BRINK) cohort study. Predictors: Estimated glomerular filtration rate (eGFR) and urine albumin to creatinine ratio (UACR). Outcomes: Change in physical performance using the Short Physical Performance Battery (SPPB) (primary) and gait speed (secondary). Analytical Approach: Linear mixed effects regression models. Results: The analytical cohort included 562 participants with mean age of 69.3 (SD, 9.8) years followed for up to 63 months. In total, 49.8% were women. In addition, 79.9% self-identified as White, and 15.3% self-identified as Black. In total, 48.8% had diabetes. Mean eGFR at baseline was 48.1 (SD, 24.3) mL/min/1.73 m2. In unadjusted analysis, lower eGFR was associated with greater decline in SPPB score (P trend < 0.001). The decline in SPPB score was larger among participants with lower eGFR, with a gradient from -0.15 (95% CI, -0.23 to -0.07) points per year for participants with eGFR ≥60 mL/min/1.73 m2 to -0.56 (95% CI, -0.84 to -0.27) for participants with eGFR <15 mL/min/1.73 m2 and -0.61 (95% CI, -0.90 to -0.33) after dialysis initiation. In covariate-adjusted models, SPPB did not continue to decline after dialysis initiation. In secondary analyses evaluating change in gait speed, gait speed continued to decline after dialysis initiation. Higher UACR was also associated with a greater decline in SPPB score and gait speed in unadjusted and adjusted models. Limitations: Small number of participants started dialysis. Conclusions: We found a graded association of chronic kidney disease stage and albuminuria with decline in physical performance. The decline in SPPB was not accelerated after dialysis initiation in covariate-adjusted models, whereas gait speed continued to decline.

Physical function is an important patient-centered outcome in chronic kidney disease (CKD), but whether physical performance changes as kidney disease progresses or when patients start dialysis is not well understood. We found that measures of physical performance, like strength and walking speed, worsened as kidney disease worsened. However, 1 combination of physical performance tests appeared stable (rather than getting worse) after starting dialysis compared to those with very advanced CKD who had not yet started dialysis, while gait speed continued to get worse. This information may help counsel patients who are learning about CKD and considering treatment options. It may also help guide research on interventions to improve physical function in patients with CKD.

Metabolite profiling of human renal cell carcinoma reveals tissue-origin dominance in nutrient availability.

The tumor microenvironment is a determinant of cancer progression and therapeutic efficacy, with nutrient availability playing an important role. Although it is established that the local abundance of specific nutrients defines the metabolic parameters for tumor growth, the factors guiding nutrient availability in tumor compared to normal tissue and blood remain poorly understood. To define these factors in renal cell carcinoma (RCC), we performed quantitative metabolomic and comprehensive lipidomic analyses of tumor interstitial fluid (TIF), adjacent normal kidney interstitial fluid (KIF), and plasma samples collected from patients. TIF nutrient composition closely resembles KIF, suggesting that tissue-specific factors unrelated to the presence of cancer exert a stronger influence on nutrient levels than tumor-driven alterations. Notably, select metabolite changes consistent with known features of RCC metabolism are found in RCC TIF, while glucose levels in TIF are not depleted to levels that are lower than those found in KIF. These findings inform tissue nutrient dynamics in RCC, highlighting a dominant role of non-cancer driven tissue factors in shaping nutrient availability in these tumors.

Management of rectal cancer in the era of total neoadjuvant therapy and watch and wait: A multidisciplinary team discussion at the Australasian Gastro-Intestinal Trials Group (AGITG) Annual Scientific Meeting 2022.

Rectal cancer is a common malignancy. The management of rectal cancer has recently evolved and has undergone a paradigm shift with the advent of treatment approaches such as total neoadjuvant therapy and the watch-and-wait approach. However, despite the recently available evidence, there is no consensus on the optimal management approach in the setting of locally advanced rectal cancer. To address some of the controversies, a joint multidisciplinary panel discussion was conducted at the Australasian Gastro-Intestinal Trials Group (AGITG) Annual Scientific Meeting in November 2022. Members from different subspecialties formed two panels and discussed three clinical cases in a debate format. Each case represented some of the complex issues faced by clinicians in this setting. The discussion is now presented in this manuscript, which depicts the different available management approaches and reiterates the importance of a multidisciplinary approach.

Effect of anterior quadratus lumborum block with ropivacaine on the immune response after laparoscopic surgery in colon cancer: a substudy of a randomized clinical trial.

BACKGROUND: Surgery induces a temporal change in the immune system, which might be modified by regional anesthesia. Applying a bilateral preoperative anterior quadratus lumborum block has proven to be a safe and effective technique in pain management after abdominal and retroperitoneal surgery, but the effect on the immune response is not thoroughly investigated. METHODS: This study is a substudy of a randomized, controlled, double-blinded trial of patients undergoing laparoscopic hemicolectomy due to colon cancer. Twenty-two patients were randomized to undergo either a bilateral anterior quadratus lumborum nerve block with a total of 60 mL ropivacaine 0.375% or placebo with corresponding isotonic saline injections. The main objective of this exploratory substudy was to investigate the systemic immune response in the first postoperative day by examining changes in blood transcript levels (n=750) and stimulated secretion of cytokines (n=17) on ex vivo activation with microbial ligands and anti-CD3/CD28. RESULTS: Using unsupervised data analysis tools, we observed no effect of the bilateral anterior quadratus lumborum nerve block on gene expression in immune cells (permutational multivariate analysis of variance using distance matrices: F=0.52, p=0.96), abundances of major immune cell populations (Wilcoxon rank-sum test: p>0.05), and stimulated cytokine secretion (Wilcoxon rank-sum test: p>0.05). CONCLUSIONS: Our study provides evidence that administration of bilateral anterior quadratus lumborum nerve block as a part of a multimodal analgesic regimen in an enhanced recovery after surgery for laparoscopic hemicolectomy in this cohort does not alter the systemic immune response.Trial registration number NCT03570541.

A2AR eGFP reporter mouse enables elucidation of A2AR expression dynamics during anti-tumor immune responses.

There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, enabling the expression of A2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4+ and CD8+ T lymphocytes and on a MHCIIhiCD86hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1+A2AR- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A2AR and synergizes with A2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.

Identification of a targetable JAK-STAT enriched androgen receptor and androgen receptor splice variant positive triple-negative breast cancer subtype.

Triple-negative breast cancer (TNBC) is an aggressive subtype with no targeted therapeutics. The luminal androgen receptor (LAR) subtype constitutes 15% of TNBC and is enriched for androgen receptor (AR) and AR target genes. Here, we show that a cohort of TNBC not only expresses AR at a much higher rate (∼80%) but also expresses AR splice variants (AR-SVs) (∼20%), further subclassifying LAR-TNBC. Higher AR and AR-SV expression and corresponding aggressive phenotypes are observed predominantly in specimens obtained from African American women. LAR TNBC specimens are enriched for interferon, Janus kinase (JAK)-signal activator and transducer (STAT), and androgen signaling pathways, which are exclusive to AR-expressing epithelial cancer cells. AR- and AR-SV-expressing TNBC cell proliferation and xenograft and patient-tumor explant growth are inhibited by AR N-terminal domain-binding selective AR degrader or by a JAK inhibitor. Biochemical analysis suggests that STAT1 is an AR coactivator. Collectively, our work identifies pharmacologically targetable TNBC subtypes and identifies growth-promoting interaction between AR and JAK-STAT signaling.

Amyloid ß Proteoforms Elucidated by Quantitative LC/MS in the 5xFAD Mouse Model of Alzheimer's Disease.

Numerous Aß proteoforms, identified in the human brain, possess differential neurotoxic and aggregation propensities. These proteoforms contribute in unknown ways to the conformations and resultant pathogenicity of oligomers, protofibrils, and fibrils in Alzheimer's disease (AD) manifestation owing to the lack of molecular-level specificity to the exact chemical composition of underlying protein products with widespread interrogating techniques, like immunoassays. We evaluated Aß proteoform flux using quantitative top-down mass spectrometry (TDMS) in a well-studied 5xFAD mouse model of age-dependent Aß-amyloidosis. Though the brain-derived Aß proteoform landscape is largely occupied by Aß1-42, 25 different forms of Aß with differential solubility were identified. These proteoforms fall into three natural groups defined by hierarchical clustering of expression levels in the context of mouse age and proteoform solubility, with each group sharing physiochemical properties associated with either N/C-terminal truncations or both. Overall, the TDMS workflow outlined may hold tremendous potential for investigating proteoform-level relationships between insoluble fibrils and soluble Aß, including low-molecular-weight oligomers hypothesized to serve as the key drivers of neurotoxicity. Similarly, the workflow may also help to validate the utility of AD-relevant animal models to recapitulate amyloidosis mechanisms or possibly explain disconnects observed in therapeutic efficacy in animal models vs humans.

Laparoscopic vs. open adrenalectomy: perioperative data and survival analysis in 70 dogs with an adrenal tumor.

Adrenalectomy is the treatment of choice in case of functional adrenal tumors and malignant adrenal incidentalomas. Laparoscopic adrenalectomy (LA) in dogs has gained popularity in recent years, however, clinical studies on large patient populations are scarce. This retrospective study describes perioperative and recurrence data, survival, and prognostic factors in 70 dogs that underwent LA or open adrenalectomy (OA) in our hospital between 2008 and 2022. Diagnosis was based on history, clinical signs, endocrine function tests and advanced diagnostic imaging. Laparoscopic adrenalectomy was performed in 42 dogs (n = 27 naturally occurring hypercortisolism, n = 4 pheochromocytoma, n = 1 pheochromocytoma with concurrent hypercortisolism, n = 10 incidentaloma) and OA in 28 dogs (n = 22 hypercortisolism, n = 3 pheochromocytoma, n = 3 incidentaloma). Bilateral adrenalectomy was performed in 8/70 dogs. Surgical duration of LA and OA did not differ significantly in unilateral and bilateral procedures (P = 0.108 and P = 0.101, respectively). Systemic hypertension occurred in 7/41 and 1/28 dogs during LA and OA, respectively (P = 0.130). Hypotension occurred in 2/41 and 4/28 dogs during LA and OA, respectively (P = 0.214). A total of 40/42 dogs in the LA group and 27/28 in the OA group survived to discharge (P = 0.810). Mean hospital stay was significantly shorter (P = 0.006) after LA (1.5 days, range 1-3) than after OA (2.2 days, range 1-4). No significant differences were demonstrated between LA and OA groups in recurrence of adrenal-dependent endocrine disease (P = 0.332), disease-free period (P = 0.733) and survival time (P = 0.353). The disease-specific 1-, 2- and 3-year survival rates were 95, 89, and 89% after LA and 92, 88, and 81% after OA. Tumor size was significantly associated with the occurrence of a recurrence. In addition, tumor size had a negative effect on the disease-free period and survival time. This study shows a favorable outcome of both LA and OA in dogs. Based on low perioperative complication rate, short hospitalization time and long-term outcomes comparable to OA in selected cases, the less invasive laparoscopic approach is considered the preferred technique.

CRISPR-Cas9 screening identifies an IRF1-SOCS1-mediated negative feedback loop that limits CXCL9 expression and antitumor immunity.

CXCL9 expression is a strong predictor of response to immune checkpoint blockade therapy. Accordingly, we sought to develop therapeutic strategies to enhance the expression of CXCL9 and augment antitumor immunity. To perform whole-genome CRISPR-Cas9 screening for regulators of CXCL9 expression, a CXCL9-GFP reporter line is generated using a CRISPR knockin strategy. This approach finds that IRF1 limits CXCL9 expression in both tumor cells and primary myeloid cells through induction of SOCS1, which subsequently limits STAT1 signaling. Thus, we identify a subset of STAT1-dependent genes that do not require IRF1 for their transcription, including CXCL9. Targeting of either IRF1 or SOCS1 potently enhances CXCL9 expression by intratumoral macrophages, which is further enhanced in the context of immune checkpoint blockade therapy. We hence show a non-canonical role for IRF1 in limiting the expression of a subset of STAT1-dependent genes through induction of SOCS1.