Identification of Spatial Proteomic Signatures of Colon Tumor Metastasis: A Digital Spatial Profiling Approach.

Over 150,000 Americans are diagnosed with colorectal cancer (CRC) every year, and annually >50,000 individuals are estimated to die of CRC, necessitating improvements in screening, prognostication, disease management, and therapeutic options. CRC tumors are removed en bloc with surrounding vasculature and lymphatics. Examination of regional lymph nodes at the time of surgical resection is essential for prognostication. Developing alternative approaches to indirectly assess recurrence risk would have utility in cases where lymph node yield is incomplete or inadequate. Spatially dependent, immune cell-specific (eg, tumor-infiltrating lymphocytes), proteomic, and transcriptomic expression patterns inside and around the tumor-the tumor immune microenvironment-can predict nodal/distant metastasis and probe the coordinated immune response from the primary tumor site. The comprehensive characterization of tumor-infiltrating lymphocytes and other immune infiltrates is possible using highly multiplexed spatial omics technologies, such as the GeoMX Digital Spatial Profiler. In this study, machine learning and differential co-expression analyses helped identify biomarkers from Digital Spatial Profiler-assayed protein expression patterns inside, at the invasive margin, and away from the tumor, associated with extracellular matrix remodeling (eg, granzyme B and fibronectin), immune suppression (eg, forkhead box P3), exhaustion and cytotoxicity (eg, CD8), Programmed death ligand 1-expressing dendritic cells, and neutrophil proliferation, among other concomitant alterations. Further investigation of these biomarkers may reveal independent risk factors of CRC metastasis that can be formulated into low-cost, widely available assays.

A deep learning algorithm to detect cutaneous squamous cell carcinoma on frozen sections in Mohs micrographic surgery: A retrospective assessment.

Intraoperative margin analysis is crucial for the successful removal of cutaneous squamous cell carcinomas (cSCC). Artificial intelligence technologies (AI) have previously demonstrated potential for facilitating rapid and complete tumour removal using intraoperative margin assessment for basal cell carcinoma. However, the varied morphologies of cSCC present challenges for AI margin assessment. The aim of this study was to develop and evaluate the accuracy of an AI algorithm for real-time histologic margin analysis of cSCC. To do this, a retrospective cohort study was conducted using frozen cSCC section slides. These slides were scanned and annotated, delineating benign tissue structures, inflammation and tumour to develop an AI algorithm for real-time margin analysis. A convolutional neural network workflow was used to extract histomorphological features predictive of cSCC. This algorithm demonstrated proof of concept for identifying cSCC with high accuracy, highlighting the potential for integration of AI into the surgical workflow. Incorporation of AI algorithms may improve efficiency and completeness of real-time margin assessment for cSCC removal, particularly in cases of moderately and poorly differentiated tumours/neoplasms. Further algorithmic improvement incorporating surrounding tissue context is necessary to remain sensitive to the unique epidermal landscape of well-differentiated tumours, and to map tumours to their original anatomical position/orientation.

Role of gestational age at time of placental laser surgery in outcomes of twin-to-twin transfusion syndrome.

OBJECTIVE: There are conflicting data on whether fetoscopic laser photocoagulation of placental anastomoses (FLP) for treating twin-to-twin transfusion syndrome (TTTS) is associated with lower rates of overall survival. The objective of this study is to characterize survival and other associated morbidity after FLP across gestational ages of FLP. METHODS: This is a secondary analysis of prospectively collected data on patients with monochorionic-diamniotic twins that had FLP for TTTS at two centers between 2011 and 2022. Patients were divided into gestational age epochs for FLP before 18 wks, 18 0/7 - 19 6/7 wks, 20 0/7 - 21 6/7 wks, 22 0/7 - 23 6/7 wks, 24 0/7 - 25 6/7 wks and after 26 wks. Demographic characteristics, sonographic characteristics of TTTS and operative characteristics were compared across the gestational age epochs. Outcomes including overall survival, preterm delivery, preterm prelabor rupture of membranes (PPROM), intrauterine fetal demise (IUFD) and neonatal demise (NND) were also compared across gestational age epochs. Multivariate analysis was performed by fitting logistic regression models for these outcomes. Kaplan-Mejer curves were constructed to compare the interval from PPROM to delivery for each gestational age epoch. RESULTS: There were 768 patients that met inclusion criteria. The dual survival rate was 61.3% for FLP performed prior to 18 weeks compared to 78.0% - 86.7% across later gestational age epochs. This appears to be related to increased rates of donor IUFD following FLP performed before, versus after 18 weeks (28.0% vs. 9.3% - 14.1%). Rates of recipient IUFD/NND and donor NND were similar regardless of gestational age of FLP. Rates of PPROM were higher for earlier FLP, ranging from 45.6% for FLP before 18 weeks to 11.9% for FLP at 24 - 26 weeks gestational age. However, the gestational age of delivery was similar across gestational age epochs with a median of 31.7 weeks. In multivariate analysis, donor loss was independently associated with FLP before 18 weeks after adjusting for selective fetal growth restriction, Quintero stage and other covariates. PPROM and PTD were also associated with FLP before 18 weeks after adjusting for cervical length, placental location, trocar size, laser energy and amnioinfusion. CONCLUSION: FLP performed at earlier gestational ages is associated with lower overall survival, which is driven by higher risk of donor IUFD, as opposed to differences in PPROM or PTD. Counseling regarding survival should account for gestational age of presentation. This article is protected by copyright. All rights reserved.

Maternal-Infant Factors in Relation to Extracellular Vesicle and Particle miRNA in Prenatal Plasma and in Postpartum Human Milk.

MicroRNAs (miRNA) in extracellular vesicles and particles (EVPs) in maternal circulation during pregnancy and in human milk postpartum are hypothesized to facilitate maternal-offspring communication via epigenetic regulation. However, factors influencing maternal EVP miRNA profiles during these two critical developmental windows remain largely unknown. In a pilot study of 54 mother-child dyads in the New Hampshire Birth Cohort Study, we profiled 798 EVP miRNAs, using the NanoString nCounter platform, in paired maternal second-trimester plasma and mature (6-week) milk samples. In adjusted models, total EVP miRNA counts were lower for plasma samples collected in the afternoon compared with the morning (p = 0.024). Infant age at sample collection was inversely associated with total miRNA counts in human milk EVPs (p = 0.040). Milk EVP miRNA counts were also lower among participants who were multiparous after delivery (p = 0.047), had a pre-pregnancy BMI > 25 kg/m2 (p = 0.037), or delivered their baby via cesarean section (p = 0.021). In post hoc analyses, we also identified 22 specific EVP miRNA that were lower among participants who delivered their baby via cesarean section (Q < 0.05). Target genes of delivery mode-associated miRNAs were over-represented in pathways related to satiety signaling in infants (e.g., CCKR signaling) and mammary gland development and lactation (e.g., FGF signaling, EGF receptor signaling). In conclusion, we identified several key factors that may influence maternal EVP miRNA composition during two critical developmental windows, which should be considered in future studies investigating EVP miRNA roles in maternal and child health.

Lineage plasticity enables low-ER luminal tumors to evolve and gain basal-like traits.

Stratifying breast cancer into specific molecular or histologic subtypes aids in therapeutic decision-making and predicting outcomes; however, these subtypes may not be as distinct as previously thought. Patients with luminal-like, estrogen receptor (ER)-expressing tumors have better prognosis than patients with more aggressive, triple-negative or basal-like tumors. There is, however, a subset of luminal-like tumors that express lower levels of ER, which exhibit more basal-like features. We have found that breast tumors expressing lower levels of ER, traditionally considered to be luminal-like, represent a distinct subset of breast cancer characterized by the emergence of basal-like features. Lineage tracing of low-ER tumors in the MMTV-PyMT mouse mammary tumor model revealed that basal marker-expressing cells arose from normal luminal epithelial cells, suggesting that luminal-to-basal plasticity is responsible for the evolution and emergence of basal-like characteristics. This plasticity allows tumor cells to gain a new lumino-basal phenotype, thus leading to intratumoral lumino-basal heterogeneity. Single-cell RNA sequencing revealed SOX10 as a potential driver for this plasticity, which is known among breast tumors to be almost exclusively expressed in triple-negative breast cancer (TNBC) and was also found to be highly expressed in low-ER tumors. These findings suggest that basal-like tumors may result from the evolutionary progression of luminal tumors with low ER expression.

DNA methylation biomarkers for noninvasive detection of triple-negative breast cancer using liquid biopsy.

Noninvasive detection of aberrant DNA methylation could provide invaluable biomarkers for earlier detection of triple-negative breast cancer (TNBC) which could help clinicians with easier and more efficient treatment options. We evaluated genome-wide DNA methylation data derived from TNBC and normal breast tissues, peripheral blood of TNBC cases and controls and reference samples of sorted blood and mammary cells. Differentially methylated regions (DMRs) between TNBC and normal breast tissues were stringently selected, verified and externally validated. A machine-learning algorithm was applied to select the top DMRs, which then were evaluated on plasma-derived circulating cell-free DNA (cfDNA) samples of TNBC patients and healthy controls. We identified 23 DMRs accounting for the methylation profile of blood cells and reference mammary cells and then selected six top DMRs for cfDNA analysis. We quantified un-/methylated copies of these DMRs by droplet digital PCR analysis in a plasma test set from TNBC patients and healthy controls and confirmed our findings obtained on tissues. Differential cfDNA methylation was confirmed in an independent validation set of plasma samples. A methylation score combining signatures of the top three DMRs overlapping with the SPAG6, LINC10606 and TBCD/ZNF750 genes had the best capability to discriminate TNBC patients from controls (AUC = 0.78 in the test set and AUC = 0.74 in validation set). Our findings demonstrate the usefulness of cfDNA-based methylation signatures as noninvasive liquid biopsy markers for the diagnosis of TNBC.

Reduced prescription of TNF-inhibitors in chronic arthritis based on therapeutic drug monitoring: A randomized controlled trial.

OBJECTIVE: Dosing of tumour necrosis factor-α inhibitors (TNFis) is not personalized causing interindividual variation in serum drug levels; however, dose optimization is not widely implemented. We hypothesized that some patients are overdosed; thus, drug prescription could be reduced by therapeutic drug monitoring (TDM). METHOD: Independent of disease activity, 239 adults treated for rheumatoid arthritis (n = 99), psoriatic arthritis 15 (n = 48), or spondyloarthritis (n = 92) were recruited for a 48-week prospective, randomized open-label trial. Standard care alone or plus TDM was applied in chronic arthritis patients treated with infliximab (IFX), (n = 81), etanercept (ETN) (n = 79), or adalimumab (ADA) (n = 79). Serum TNFi trough levels assessed at inclusion and every 4 months determined patients within/outside predefined therapeutic intervals, supporting change in prescription or drug switch. The primary endpoint was reduced drug prescription. RESULTS: Compared to standard care, TDM reduced prescribed IFX [-12% (95% confidence interval -20, -3); p = 0.001] and ETN (-15% (-29, 1); p = 0.01], and prolonged the interdosing intervals of ETN [+235% (38, 432); p = 0.02] and ADA [+28% (6, 51); p = 0.04]. Time to drug switch was accelerated (χ2 = 6.03, p = 0.01). No group differences in adverse events, disease activity, or self-reported outcomes were shown, indicating equally sustained remission. CONCLUSIONS: TDM reduced prescription of IFX, ETN, and ADA and identified patients benefiting from accelerated drug switch, thereby minimizing treatment failure, risk of toxicity, and unnecessary adverse events.

Video-Based Deep Learning to Detect Dyssynergic Defecation with 3D High-Definition Anorectal Manometry.

BACKGROUND: We developed a deep learning algorithm to evaluate defecatory patterns to identify dyssynergic defecation using 3-dimensional high definition anal manometry (3D-HDAM). AIMS: We developed a 3D-HDAM deep learning algorithm to evaluate for dyssynergia. METHODS: Spatial-temporal data were extracted from consecutive 3D-HDAM studies performed between 2018 and 2020 at Dartmouth-Hitchcock Health. The technical procedure and gold standard definition of dyssynergia were based on the London consensus, adapted to the needs of 3D-HDAM technology. Three machine learning models were generated: (1) traditional machine learning informed by conventional anorectal function metrics, (2) deep learning, and (3) a hybrid approach. Diagnostic accuracy was evaluated using bootstrap sampling to calculate area-under-the-curve (AUC). To evaluate overfitting, models were validated by adding 502 simulated defecation maneuvers with diagnostic ambiguity. RESULTS: 302 3D-HDAM studies representing 1208 simulated defecation maneuvers were included (average age 55.2 years; 80.5% women). The deep learning model had comparable diagnostic accuracy [AUC 0.91 (95% confidence interval 0.89-0.93)] to traditional [AUC 0.93(0.92-0.95)] and hybrid [AUC 0.96(0.94-0.97)] predictive models in training cohorts. However, the deep learning model handled ambiguous tests more cautiously than other models; the deep learning model was more likely to designate an ambiguous test as inconclusive [odds ratio 4.21(2.78-6.38)] versus traditional/hybrid approaches. CONCLUSIONS: Deep learning is capable of considering complex spatial-temporal information on 3D-HDAM technology. Future studies are needed to evaluate the clinical context of these preliminary findings.

Human cytomegalovirus alters immune cell profile with potential implications for patient survival in head and neck cancer.

Cytomegalovirus (CMV) is a highly prevalent human herpes virus that exerts a strong influence on immune repertoire which may influence cancer risk. We have tested whether CMV immunoglobulin G (IgG) serostatus is associated with immune cell proportions (n = 132 population controls), human papillomavirus (HPV) co-infection and head and neck cancer risk (n = 184 cancer cases and 188 controls) and patient survival. CMV status was not associated with the proportion of Natural Killer cells, B cells or the neutrophil-to-lymphocyte ratio. However, CD8+ T cells increased with increasing categories of IgG titers (P =1.7 × 10-10), and titers were inversely associated with the CD4:CD8 ratio (P = 5.6 × 10-5). Despite these differences in T cell proportions, CMV was not associated with HPV16 co-infection. CMV seropositivity was similar in cases (52%) and controls (47%) and was not associated with patient survival (hazard ratio [HR] 1.14, 95% confidence interval [CI]: 0.70 to 1.86). However, those patients with the highest titers had the worst survival (HR 1.91, 95% CI: 1.13 to 3.23). Tumor-based data from The Cancer Genome Atlas demonstrated that the presence of CMV transcripts was associated with worse patient survival (HR 1.79, 95% CI: 0.96 to 2.78). These findings confirm that a history of CMV infection alters T cell proportions, but this does not translate to HPV16 co-infection or head and neck cancer risk. Our data suggest that high titers and active CMV virus in the tumor environment may confer worse survival.

Prediagnostic breast milk DNA methylation alterations in women who develop breast cancer.

Prior candidate gene studies have shown tumor suppressor DNA methylation in breast milk related with history of breast biopsy, an established risk factor for breast cancer. To further establish the utility of breast milk as a tissue-specific biospecimen for investigations of breast carcinogenesis, we measured genome-wide DNA methylation in breast milk from women with and without a diagnosis of breast cancer in two independent cohorts. DNA methylation was assessed using Illumina HumanMethylation450k in 87 breast milk samples. Through an epigenome-wide association study we explored CpG sites associated with a breast cancer diagnosis in the prospectively collected milk samples from the breast that would develop cancer compared with women without a diagnosis of breast cancer using linear mixed effects models adjusted for history of breast biopsy, age, RefFreeCellMix cell estimates, time of delivery, array chip and subject as random effect. We identified 58 differentially methylated CpG sites associated with a subsequent breast cancer diagnosis (q-value <0.05). Nearly all CpG sites associated with a breast cancer diagnosis were hypomethylated in cases compared with controls and were enriched for CpG islands. In addition, inferred repeat element methylation was lower in breast milk DNA from cases compared to controls, and cases exhibited increased estimated epigenetic mitotic tick rate as well as DNA methylation age compared with controls. Breast milk has utility as a biospecimen for prospective assessment of disease risk, for understanding the underlying molecular basis of breast cancer risk factors and improving primary and secondary prevention of breast cancer.

HiTIMED: hierarchical tumor immune microenvironment epigenetic deconvolution for accurate cell type resolution in the tumor microenvironment using tumor-type-specific DNA methylation data.

BACKGROUND: Cellular compositions of solid tumor microenvironments are heterogeneous, varying across patients and tumor types. High-resolution profiling of the tumor microenvironment cell composition is crucial to understanding its biological and clinical implications. Previously, tumor microenvironment gene expression and DNA methylation-based deconvolution approaches have been shown to deconvolve major cell types. However, existing methods lack accuracy and specificity to tumor type and include limited identification of individual cell types. RESULTS: We employed a novel tumor-type-specific hierarchical model using DNA methylation data to deconvolve the tumor microenvironment with high resolution, accuracy, and specificity. The deconvolution algorithm is named HiTIMED. Seventeen cell types from three major tumor microenvironment components can be profiled (tumor, immune, angiogenic) by HiTIMED, and it provides tumor-type-specific models for twenty carcinoma types. We demonstrate the prognostic significance of cell types that other tumor microenvironment deconvolution methods do not capture. CONCLUSION: We developed HiTIMED, a DNA methylation-based algorithm, to estimate cell proportions in the tumor microenvironment with high resolution and accuracy. HiTIMED deconvolution is amenable to archival biospecimens providing high-resolution profiles enabling to study of clinical and biological implications of variation and composition of the tumor microenvironment.

Elevated levels of interleukin-12/23p40 may serve as a potential indicator of dysfunctional heart rate variability in type 2 diabetes.

BACKGROUND: Systemic inflammatory processes plausibly contribute to the development of cardiovascular complications, causing increased morbidity and mortality in type 2 diabetes. Circulating inflammatory markers, i.e., interleukin (IL)-6 and tumour necrosis factor-α, are associated with neurocardiac measures. We examined a broad panel of various inflammatory and inflammation-related serum markers to obtain more detailed insight into the possible neuro-immune interaction between cardiovascular regulation and systemic level of inflammation. METHODS: Serum samples from 100 participants with type 2 diabetes were analysed. Heart rate variability, cardiovascular autonomic reflex tests, and cardiac vagal tone tests were performed based on electrocardiographic readings. Data regarding covariates (demographic-, diabetes-, and cardiovascular risk factors) were registered. RESULTS: Increased serum levels of IL-12/IL-23p40 (p < 0.01) and intercellular adhesion molecule (ICAM)-1 (p < 0.007) were associated with diminished heart rate variability measures. After all adjustments, the associations between IL-12/23p40, SDANN and VLF persisted (p = 0.001). Additionally, serum levels of vascular endothelial growth factor (VEGF)-C were associated with response to standing (p = 0.005). DISCUSSION: The few but robust associations between neurocardiac regulation and serum markers found in this study suggest systemic changes in proinflammatory, endothelial, and lymphatic function, which collectively impacts the systemic cardiovascular function. Our results warrant further exploration of IL-12/IL-23p40, ICAM-1, and VEGF-C as possible cardiovascular biomarkers in T2D that may support future decisions regarding treatment strategies for improved patient care.

Interventions to prevent preterm delivery in women with short cervix before fetoscopic laser surgery for twin-twin transfusion syndrome.

OBJECTIVE: Preoperative short cervical length (CL) remains a major risk factor for preterm birth after laser surgery for twin-twin transfusion syndrome (TTTS), but the optimal intervention to prolong pregnancy remains elusive. The objective of this study was to compare secondary methods for the prevention of preterm birth in twin pregnancies with TTTS undergoing fetoscopic laser photocoagulation (FLP), in the setting of a short cervix at the time of FLP, in five North American Fetal Treatment Network (NAFTNet) centers. METHODS: This was a secondary analysis of data collected prospectively at five NAFTNet centers, conducted from January 2013 to March 2020. Inclusion criteria were a monochorionic diamniotic twin pregnancy complicated by TTTS, undergoing FLP, with preoperative CL < 30 mm. Management options for a short cervix included expectant management, vaginal progesterone, pessary (Arabin, incontinence or Bioteque cup), cervical cerclage or a combination of two or more treatments. Patients were not included if the intervention was initiated solely on the basis of having a twin gestation rather than at the diagnosis of a short cervix. Demographics, ultrasound characteristics, operative data and outcomes were compared. The primary outcome was FLP-to-delivery interval. Propensity-score matching was performed, with each treatment group matched (1:1) to the expectant-management group for CL, in order to estimate the effect of each treatment on the FLP-to-delivery interval. RESULTS: A total of 255 women with a twin pregnancy complicated by TTTS and a short cervix undergoing FLP were included in the study. Of these, 151 (59%) were managed expectantly, 32 (13%) had vaginal progesterone only, 21 (8%) had pessary only, 21 (8%) had cervical cerclage only and 30 (12%) had a combination of treatments. A greater proportion of patients in the combined-treatment group had had a prior preterm birth compared with those in the expectant-management group (33% vs 9%; P = 0.01). Mean preoperative CL was shorter in the pessary, cervical-cerclage and combined-treatment groups (14-16 mm) than in the expectant-management and vaginal-progesterone groups (22 mm for both) (P < 0.001). There was no significant difference in FLP-to-delivery interval between the groups, nor in gestational age at delivery or the rate of live birth or neonatal survival. Vaginal progesterone was associated with a decrease in the risk of delivery before 28 weeks' gestation compared with cervical cerclage and combined treatment (P = 0.03). Using propensity-score matching for CL, cervical cerclage was associated with a reduction in FLP-to-delivery interval of 13 days, as compared with expectant management. CONCLUSIONS: A large proportion of pregnancies with TTTS and a short maternal cervix undergoing FLP were managed expectantly for a short cervix, establishing a high (62%) risk of delivery before 32 weeks in this condition. No treatment that significantly improved outcome was identified; however, there were significant differences in potential confounders and there were also likely to be unmeasured confounders. Cervical cerclage should not be offered as a secondary prevention for preterm birth in twin pregnancies with TTTS and a short cervix undergoing FLP. A large randomized controlled trial is urgently needed to determine the effects of treatments for the prevention of preterm birth in these pregnancies. © 2021 International Society of Ultrasound in Obstetrics and Gynecology.

Managing organic dairy herd health: Current roles and possible future roles for veterinarians with organic dairy clientele.

This study aimed to understand the veterinary-client relationship and perspectives in organic dairy production systems. Twenty-three organic dairy producers and 12 veterinarians were interviewed using semi-structured, one-on-one interviews. Consultation with local veterinarians was typically infrequent, as there was greater reliance on other producers and organic industry professionals for advice on dairy herd health management, perhaps due to a shared focus on organic farming principles. Organic producers generally consulted local veterinarians most frequently to develop vaccine protocols, address animal health emergencies, and gain reproductive services. Common reasons organic producers cited for not regularly consulting a local veterinarian included the cost of veterinary services and a perception that local veterinarians have limited experience with organic systems and products. Some producers also expressed the idea that infrequent veterinarian usage is indicative of a healthy herd. Meanwhile, local veterinarians reported that organic dairy producers often delayed consulting them about individual cases until animal health problems were severe. Local veterinarians also identified challenges in servicing organic herds, such as the lack of standardized regulations between different organic certifiers and limited safety and efficacy data for many herd health products used by organic producers. In addition, their formal training generally did not include organic herd health strategies. Organic dairy clients were typically a small fraction of the overall workload for local veterinarians, which precluded their ability to accumulate experience. In contrast, a handful of specialized veterinarians in the organic milk processing industry work more frequently with organic producers and have published most of the available resource materials on organic dairy herd health. Veterinary-client partnerships could be improved through more open communication and discussion of the challenges identified in this study and through participatory research and outreach engagement that includes local veterinarians, organic certifiers, organic industry veterinarians, and producers.

Tracing human stem cell lineage during development using DNA methylation.

Stem cell maturation is a fundamental, yet poorly understood aspect of human development. We devised a DNA methylation signature deeply reminiscent of embryonic stem cells (a fetal cell origin signature, FCO) to interrogate the evolving character of multiple human tissues. The cell fraction displaying this FCO signature was highly dependent upon developmental stage (fetal versus adult), and in leukocytes, it described a dynamic transition during the first 5 yr of life. Significant individual variation in the FCO signature of leukocytes was evident at birth, in childhood, and throughout adult life. The genes characterizing the signature included transcription factors and proteins intimately involved in embryonic development. We defined and applied a DNA methylation signature common among human fetal hematopoietic progenitor cells and have shown that this signature traces the lineage of cells and informs the study of stem cell heterogeneity in humans under homeostatic conditions.

A large-scale internal validation study of unsupervised virtual trichrome staining technologies on nonalcoholic steatohepatitis liver biopsies.

Non-alcoholic steatohepatitis (NASH) is a fatty liver disease characterized by accumulation of fat in hepatocytes with concurrent inflammation and is associated with morbidity, cirrhosis and liver failure. After extraction of a liver core biopsy, tissue sections are stained with hematoxylin and eosin (H&E) to grade NASH activity, and stained with trichrome to stage fibrosis. Methods to computationally transform one stain into another on digital whole slide images (WSI) can lessen the need for additional physical staining besides H&E, reducing personnel, equipment, and time costs. Generative adversarial networks (GAN) have shown promise for virtual staining of tissue. We conducted a large-scale validation study of the viability of GANs for H&E to trichrome conversion on WSI (n = 574). Pathologists were largely unable to distinguish real images from virtual/synthetic images given a set of twelve Turing Tests. We report high correlation between staging of real and virtual stains ([Formula: see text]; 95% CI: 0.84-0.88). Stages assigned to both virtual and real stains correlated similarly with a number of clinical biomarkers and progression to End Stage Liver Disease (Hazard Ratio HR = 2.06, 95% CI: 1.36-3.12, p < 0.001 for real stains; HR = 2.02, 95% CI: 1.40-2.92, p < 0.001 for virtual stains). Our results demonstrate that virtual trichrome technologies may offer a software solution that can be employed in the clinical setting as a diagnostic decision aid.

Short-term transcutaneous non-invasive vagus nerve stimulation may reduce disease activity and pro-inflammatory cytokines in rheumatoid arthritis: results of a pilot study.

Objective: Rheumatoid arthritis (RA) is a chronic, autoimmune, inflammatory disease. Studies suggest that pro-inflammatory cytokines may be attenuated by the vagus nerve through the cholinergic anti-inflammatory pathway. We aimed to evaluate the anti-inflammatory effects of short-term transcutaneous non-invasive vagus nerve stimulation (n-VNS) applied to the cervical vagus nerve in patients with RA. Method: We conducted a single-centre, open-label, preliminary proof-of-concept study of n-VNS in two cohorts of participants with RA: one with high disease activity (n = 16) and one with low disease activity (n = 20). Disease Activity Score based on 28-joint count-C-reactive protein (DAS28-CRP), cardiac vagal tone, and pro-inflammatory cytokines were measured at baseline and after 1 and 4 days of n-VNS. Results: In the high disease activity group, n-VNS resulted in reductions in DAS28-CRP (4.1 to 3.8, p = 0.02), CRP (8.2 to 6 mg/mL, p = 0.01), and interferon-γ (29.8 to 22.5 pg/mL, p = 0.02). In the low disease activity group, there was no effect on DAS28-CRP, and n-VNS was associated with a decrease in cardiac vagal tone (p = 0.03) and a reduction in interleukin-10 (0.8 to 0.6 pg/mL, p = 0.02). Participants with high disease activity had lower baseline cardiac vagal tone than those with low disease activity (3.6 ± 2 vs 4.9 ± 3 linear vagal scale, p = 0.03). Cardiac vagal tone was negatively associated with DAS28-CRP (r = -0.37, p = 0.03). Overall, n-VNS was well tolerated. Conclusion: This study provides preliminary support for an anti-inflammatory effect of n-VNS in patients with RA. These findings warrant further investigation in larger placebo-controlled trials.

Vagal Nerve Stimulation-Modulation of the Anti-Inflammatory Response and Clinical Outcome in Psoriatic Arthritis or Ankylosing Spondylitis.

OBJECTIVES: The vagal nerve exerts an essential pathway in controlling the cholinergic anti-inflammatory reflex. Thus, the study is aimed at investigating the acute effect of a noninvasive transcutaneous vagus nerve stimulation on clinical disease activity and systemic levels of inflammation in patients with psoriatic arthritis or ankylosing spondylitis. METHODS: Twenty patients with psoriatic arthritis (PsA) and 20 patients with ankylosing spondylitis (AS) were included and stimulated bilaterally with a handheld vagal nerve stimulator for 120 seconds 3 times a day for 5 consecutive days. All patients were in remission. Cardiac vagal tone, clinical scores, CRP, and cytokine levels were assessed. RESULTS: In PsA and AS, decreased heart rate was observed, confirming compliance. Furthermore, in PsA, a clear reduction of clinical disease activity associated with a 20% reduction in CRP was shown. In AS, a reduction in interferon-γ, interleukin- (IL-) 8, and 10 was shown. No side effects were described. CONCLUSION: This open-label study provides support for an anti-inflammatory effect of transcutaneous vagus nerve stimulation in patients with psoriatic arthritis and ankylosing spondylitis. The modulated immune response and reduced disease activity and CRP-levels raise the fascinating possibility of using neuromodulation as an add-on to existing pharmacological treatments.

Organic dairy producer experiences and decisions related to disease prevention and treatment.

Organic dairy producers have distinct perspectives, approaches, challenges, and experiences when managing herd health, but little research has been conducted to develop a deeper understanding of producer decision-making related to disease-prevention and treatment strategies. We used 23 semi-structured interviews with organic dairy producers to better understand their perceived herd health challenges and overall disease-prevention strategies; decision-making and attitudes related to vaccination; and approaches to the treatment of infectious diseases using alternative or conventional antimicrobial therapies. Organic dairy producers reported improvements in overall animal health after they transitioned from a conventional to an organic system. Outdoor access, a balanced forage-based diet, and animal and environmental cleanliness were reported as the predominant disease-prevention measures. Over half of producers vaccinated their animals, and almost all preferred to use vaccines sparingly. Among producers who did not use vaccines, reasons cited were low perceived risk for infectious disease and minor vaccine safety concerns, yet many were not opposed to using vaccines in the future. Non-antimicrobial therapies were viewed as alternative effective treatment strategies for most common infectious diseases. Producers used antimicrobials very rarely and generally reserved them for severe cases when alternative treatments failed. Because US organic standards prohibit selling milk from cows that have ever been treated with antimicrobials, producers described the decision to use antimicrobials as extremely challenging and involved balancing considerations of economics, convenience, and animal welfare. This research fosters a better understanding of organic dairy producers' disease-prevention and treatment strategies and could be used by veterinarians and other animal health specialists to facilitate services for organic clients.

MethylNet: an automated and modular deep learning approach for DNA methylation analysis.

BACKGROUND: DNA methylation (DNAm) is an epigenetic regulator of gene expression programs that can be altered by environmental exposures, aging, and in pathogenesis. Traditional analyses that associate DNAm alterations with phenotypes suffer from multiple hypothesis testing and multi-collinearity due to the high-dimensional, continuous, interacting and non-linear nature of the data. Deep learning analyses have shown much promise to study disease heterogeneity. DNAm deep learning approaches have not yet been formalized into user-friendly frameworks for execution, training, and interpreting models. Here, we describe MethylNet, a DNAm deep learning method that can construct embeddings, make predictions, generate new data, and uncover unknown heterogeneity with minimal user supervision. RESULTS: The results of our experiments indicate that MethylNet can study cellular differences, grasp higher order information of cancer sub-types, estimate age and capture factors associated with smoking in concordance with known differences. CONCLUSION: The ability of MethylNet to capture nonlinear interactions presents an opportunity for further study of unknown disease, cellular heterogeneity and aging processes.