Identification of BoLA Alleles Associated with BLV Proviral Load in US Beef Cows.

Bovine leukemia virus (BLV) causes enzootic bovine leukosis, the most common neoplastic disease in cattle. Previous work estimates that 78% of US beef operations and 38% of US beef cattle are seropositive for BLV. Infection by BLV in a herd is an economic concern for producers as evidence suggests that it causes an increase in cost and a subsequent decrease in profit to producers. Studies investigating BLV in dairy cattle have noted disease resistance or susceptibility, measured by a proviral load (PVL) associated with specific alleles of the bovine leukocyte antigen (BoLA) DRB3 gene. This study aims to investigate the associations between BoLA DRB3 alleles and BLV PVL in beef cattle. Samples were collected from 157 Midwest beef cows. BoLA DRB3 alleles were identified and compared with BLV PVL. One BoLA DRB3 allele, *026:01, was found to be associated with high PVL in relation to the average of the sampled population. In contrast, two alleles, *033:01 and *002:01, were found to be associated with low PVL. This study provides evidence of a relationship between BoLA DRB3 alleles and BLV PVL in US beef cows.

Phenotypic Selection of Dairy Cattle Infected with Bovine Leukemia Virus Demonstrates Immunogenetic Resilience through NGS-Based Genotyping of BoLA MHC Class II Genes.

Characterization of the bovine leukocyte antigen (BoLA) DRB3 gene has shown that specific alleles associate with susceptibility or resilience to the progression of bovine leukemia virus (BLV), measured by proviral load (PVL). Through surveillance of multi-farm BLV eradication field trials, we observed differential phenotypes within seropositive cows that persist from months to years. We sought to develop a multiplex next-generation sequencing workflow (NGS-SBT) capable of genotyping 384 samples per run to assess the relationship between BLV phenotype and two BoLA genes. We utilized longitudinal results from milk ELISA screening and subsequent blood collections on seropositive cows for PVL determination using a novel BLV proviral load multiplex qPCR assay to phenotype the cows. Repeated diagnostic observations defined two distinct phenotypes in our study population, ELISA-positive cows that do not harbor detectable levels of provirus and those who do have persistent proviral loads. In total, 565 cows from nine Midwest dairy farms were selected for NGS-SBT, with 558 cows: 168 BLV susceptible (ELISA-positive/PVL-positive) and 390 BLV resilient (ELISA-positive/PVL-negative) successfully genotyped. Three BoLA-DRB3 alleles, including one novel allele, were shown to associate with disease resilience, *009:02, *044:01, and *048:02 were found at rates of 97.5%, 86.5%, and 90.3%, respectively, within the phenotypically resilient population. Alternatively, DRB3*015:01 and *027:03, both known to associate with disease progression, were found at rates of 81.1% and 92.3%, respectively, within the susceptible population. This study helps solidify the immunogenetic relationship between BoLA-DRB3 alleles and BLV infection status of these two phenotypic groupings of US dairy cattle.

Single-dose del Nido Cardioplegia in Minimally Invasive Aortic Valve Surgery.

del Nido cardioplegia (DC) offers prolonged cardiac protection with single-dose administration and has been shown to be safe in adult CABG surgery. We set out to evaluate the efficacy of cardiac protection and clinical outcomes of DC versus standard blood cardioplegia (BC) in minimally invasive aortic valve surgery. From August 2011 to May 2016, 178 patients underwent minimally invasive aortic valve replacement (mini-AVR) with BC (n = 101) or DC (n = 77). Ministernotomy or right minithoracotomy was utilized for surgical access. Clinical patient characteristics and data were extracted from our local Society of Thoracic Surgeons (STS) database and the electronic medical record. Patients were propensity matched for age, gender, body mass index, valve size and type, STS score, surgical access, preop creatinine, diabetes, and chronic obstructive pulmonary disease, yielding 63 well-matched pairs. There was no difference in patient age, preoperative creatinine, body mass index, diabetes, chronic obstructive pulmonary disease, or STS score between BC and DC before or after propensity matching. BC patients received both anterograde and retrograde cardioplegias in multiple doses, whereas DC was delivered almost entirely anterograde with 95% of the patients (73/77) receiving a single dose only. DC was associated with decreased cardiopulmonary bypass time (108 ± 24 vs 135 ± 43 minutes, P = 0.001) and aortic cross-clamp time (80 ± 16 vs 102 ± 30 min, P = 0.001) and maximal glucose levels during cardiopulmonary bypass (165 ± 39 vs 202 ± 49 mg/dL, P = 0.001), whereas troponin T level did not differ between DC and BC (0.3 ± 0.29 vs 0.44 ± 1.7 ng/mL, P = 0.7). Preoperative ejection fraction did not change in either BC (64% ± 12% vs 61% ± 10%, P = 0.09) or DC (58% ± 14% vs 57% ± 14%, P = 0.4) after AVR. In minimally invasive AVR surgery, DC provided equivalent myocardial protection and clinical outcomes to BC while simplifying cardioprotective regimen and reducing aortic cross-clamp time. DC was associated with lower cardiopulmonary bypass glucose levels and demonstrated the feasibility of a single-dose administration.

WITHDRAWN: Single-dose del Nido cardioplegia in minimally invasive aortic valve surgery.

This article has been withdrawn at the request of the author(s) and/or editor. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Impact of robotics and a suspended lead suit on physician radiation exposure during percutaneous coronary intervention.

BACKGROUND: Reports of left-sided brain malignancies among interventional cardiologists have heightened concerns regarding physician radiation exposure. This study evaluated the impact of a suspended lead suit and robotic system on physician radiation exposure during percutaneous coronary intervention (PCI). METHODS: Real-time radiation exposure data were prospectively collected from dosimeters worn by operating physicians at the head- and chest-level during consecutive PCI cases. Exposures were compared in three study groups: 1) manual PCI performed with traditional lead apparel; 2) manual PCI performed using suspended lead; and 3) robotic PCI performed in combination with suspended lead. RESULTS: Among 336 cases (86.6% manual, 13.4% robotic) performed over 30weeks, use of suspended lead during manual PCI was associated with significantly less radiation exposure to the chest and head of operating physicians than traditional lead apparel (chest: 0.0 [0.1] µSv vs 0.4 [4.0] µSv, p<0.001; head: 0.5 [1.9] µSv vs 14.9 [51.5] µSv, p<0.001). Chest-level radiation exposure during robotic PCI performed in combination with suspended lead was 0.0 [0.0] µSv, which was significantly less chest exposure than manual PCI performed with traditional lead (p<0.001) or suspended lead (p=0.046). In robotic PCI the median head-level exposure was 0.1 [0.2] µSv, which was 99.3% less than manual PCI performed with traditional lead (p<0.001) and 80.0% less than manual PCI performed with suspended lead (p<0.001). CONCLUSIONS: Utilization of suspended lead and robotics were observed to result in significantly less radiation exposure to the chest and head of operating physicians during PCI.

Percutaneous coronary intervention using a combination of robotics and telecommunications by an operator in a separate physical location from the patient: an early exploration into the feasibility of telestenting (the REMOTE-PCI study).

AIMS: The present study explores the feasibility of telestenting, wherein a physician operator performs stenting on a patient in a separate physical location using a combination of robotics and telecommunications. METHODS AND RESULTS: Patients undergoing robotic stenting were eligible for inclusion. All manipulations of guidewires, balloons, and stents were performed robotically by a physician operator located in an isolated separate room outside the procedure room housing the patient. Communication between the operating physician and laboratory personnel was via telecommunication devices providing real-time audio and video connectivity. Among 20 patients who consented to participate, technical success, defined as successful advancement and retraction of guidewires, balloons, and stents by the robotic system without conversion to manual operation, was achieved in 19 of 22 lesions (86.4%). Procedural success, defined as <30% residual stenosis upon completion of the procedure in the absence of death or repeat revascularisation prior to hospital discharge, was achieved in 19 of 20 patients (95.0%). There were no deaths or repeat revascularisations prior to hospital discharge. CONCLUSIONS: To the best of our knowledge, the present study is the first to explore the feasibility of telestenting. Additional studies are required to determine if future advancements in robotics will facilitate telestenting over greater geographic distances.

Propensity Matched Analysis of del Nido Cardioplegia in Adult Coronary Artery Bypass Grafting: Initial Experience With 100 Consecutive Patients.

BACKGROUND: Del Nido cardioplegia (DC) offers prolonged cardiac protection with single-dose administration and has had a long safety record in pediatric cardiac surgery. However, its application in the adult population has thus far been limited. We evaluated the efficacy of cardiac protection and clinical outcomes of DC vs blood cardioplegia (BC) in adult coronary artery bypass graft (CABG) patients. METHODS: Clinical outcomes of 100 consecutive isolated CABG patients who received DC (May to September 2014) were compared with the previous 100 consecutive isolated CABG patients receiving BC (December 2013 to April 2014). Propensity matching yielded 82 pairs. The same surgeons operated on all patients. Clinical patient characteristics and data were extracted from our local The Society of Thoracic Surgeons database and the electronic medical record. RESULTS: Preoperative characteristics were similar between BC and DC patients before and after propensity matching. BC patients received anterograde and retrograde cardioplegia, whereas DC was delivered anterograde, with 92 of 100 patients receiving a single dose only. Inotropic support upon arrival to the recovery unit did not differ between BC and DC (0.28 ± 0.11 vs 0.27 ± 0.11 µg/kg/min milrinone [p = 0.8] and 0.05 ± 0.03 vs 0.05 ± 0.03 µg/kg/min norepinephrine [p = 0.7]), nor did postoperative troponin T levels (0.56 ± 0.48 vs 0.70 ± 1.27 ng/mL; p = 0.3). The peak intraoperative glucose level was higher in BC (209.8 ± 40.4 mg/dL) than in DC (161.4 ± 42.3 mg/dL) patients (p < 0.001). No patients died in either group, and the postoperative incidence of atrial fibrillation, stroke, reoperation for bleeding, and prolonged intubation did not differ between the groups before and after matching. There was also no difference in the postoperative ejection fraction between the groups (0.51 ± 0.13 vs 0.47 ± 0.13 for BC and DC, respectively; p = 0.17). CONCLUSIONS: In our initial experience, DC provided equivalent myocardial protection and clinical outcomes to BC in adult isolated CABG patients. DC was associated with lower cardiopulmonary bypass glucose levels than BC and demonstrated the feasibility of single-dose administration for routine coronary operations.

Combined Near-Infrared Spectroscopy and Intravascular Ultrasound Imaging of Pre-Existing Coronary Artery Stents: Can Near-Infrared Spectroscopy Reliably Detect Neoatherosclerosis?

BACKGROUND: Neoatherosclerosis is an emerging phenomenon in which lipid-rich plaques (LRPs) develop within pre-existing stents. This study was undertaken to describe near-infrared spectroscopy (NIRS) and intravascular ultrasound findings in pre-existing stents and to compare NIRS findings in pre-existing stents, in which an increased lipid signal has been speculated to indicate neoatherosclerosis, and NIRS findings in a control group of freshly implanted stents, in which any lipid signal originates from fibroatheroma under the stent. METHODS AND RESULTS: At the site of LRP detected by NIRS in a cohort of pre-existing stents, intravascular ultrasound was used to determine the presence of neointimal tissue. The lipid-core burden index and maximum lipid-core burden index in 4 mm were measured within stented segments. Findings were compared between pre-existing stents and a control group of freshly implanted stents. Among 60 pre-existing stents implanted 5.5±4.0 years earlier, NIRS detected LRP in 33%. At the site of LRP, intravascular ultrasound found no neointimal tissue in 35% of cases. NIRS findings in pre-existing stents were indistinguishable from those of freshly implanted stents (lipid-core burden index: 50±72 versus 42±58; P=0.40 and maximum lipid-core burden index in 4 mm: 156±184 versus 155±203; P=0.69). CONCLUSIONS: The detection of LRP in a pre-existing stent by NIRS alone is not reliable evidence of neoatherosclerosis, as the lipid signal may originate from fibroatheroma underlying the stent. By identifying the presence or absence of neointimal tissue at the site of LRP detected by NIRS, intravascular ultrasound may provide some insight into the potential source of the lipid signal in pre-existing stents. REGISTRATION INFORMATION: URL: http://www.clinicaltrials.gov. Unique identifier: NCT01694368.

Cabozantinib (XL184) Inhibits Growth and Invasion of Preclinical TNBC Models.

PURPOSE: Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype that is associated with poor clinical outcome. There is a vital need for effective targeted therapeutics for TNBC patients, yet treatment strategies are challenged by the significant intertumoral heterogeneity within the TNBC subtype and its surrounding microenvironment. Receptor tyrosine kinases (RTK) are highly expressed in several TNBC subtypes and are promising therapeutic targets. In this study, we targeted the MET receptor, which is highly expressed across several TNBC subtypes. EXPERIMENTAL DESIGN: Using the small-molecule inhibitor cabozantinib (XL184), we examined the efficacy of MET inhibition in preclinical models that recapitulate human TNBC and its microenvironment. To analyze the dynamic interactions between TNBC cells and fibroblasts over time, we utilized a 3D model referred to as MAME (Mammary Architecture and Microenvironment Engineering) with quantitative image analysis. To investigate cabozantinib inhibition in vivo, we used a novel xenograft model that expresses human HGF and supports paracrine MET signaling. RESULTS: XL184 treatment of MAME cultures of MDA-MB-231 and HCC70 cells (± HGF-expressing fibroblasts) was cytotoxic and significantly reduced multicellular invasive outgrowths, even in cultures with HGF-expressing fibroblasts. Treatment with XL184 had no significant effects on MET(neg) breast cancer cell growth. In vivo assays demonstrated that cabozantinib treatment significantly inhibited TNBC growth and metastasis. CONCLUSIONS: Using preclinical TNBC models that recapitulate the breast tumor microenvironment, we demonstrate that cabozantinib inhibition is an effective therapeutic strategy in several TNBC subtypes.

Integrin Based Isolation Enables Purification of Murine Lineage Committed Cardiomyocytes.

In contrast to mature cardiomyocytes which have limited regenerative capacity, pluripotent stem cells represent a promising source for the generation of new cardiomyocytes. The tendency of pluripotent stem cells to form teratomas and the heterogeneity from various differentiation stages and cardiomyocyte cell sub-types, however, are major obstacles to overcome before this type of therapy could be applied in a clinical setting. Thus, the identification of extracellular markers for specific cardiomyocyte progenitors and mature subpopulations is of particular importance. The delineation of cardiomyocyte surface marker patterns not only serves as a means to derive homogeneous cell populations by FACS, but is also an essential tool to understand cardiac development. By using single-cell expression profiling in early mouse embryonic hearts, we found that a combination of integrin alpha-1, alpha-5, alpha-6 and N-cadherin enables isolation of lineage committed murine cardiomyocytes. Additionally, we were able to separate trabecular cardiomyocytes from solid ventricular myocardium and atrial murine cells. These cells exhibit expected subtype specific phenotype confirmed by electrophysiological analysis. We show that integrin expression can be used for the isolation of living, functional and lineage-specific murine cardiomyocytes.

Melanoma patient derived xenografts acquire distinct Vemurafenib resistance mechanisms.

Variable clinical responses, tumor heterogeneity, and drug resistance reduce long-term survival outcomes for metastatic melanoma patients. To guide and accelerate drug development, we characterized tumor responses for five melanoma patient derived xenograft models treated with Vemurafenib. Three BRAF(V600E) models showed acquired drug resistance, one BRAF(V600E) model had a complete and durable response, and a BRAF(V600V) model was expectedly unresponsive. In progressing tumors, a variety of resistance mechanisms to BRAF inhibition were uncovered, including mutant BRAF alternative splicing, NRAS mutation, COT (MAP3K8) overexpression, and increased mutant BRAF gene amplification and copy number. The resistance mechanisms among the patient derived xenograft models were similar to the resistance pathways identified in clinical specimens from patients progressing on BRAF inhibitor therapy. In addition, there was both inter- and intra-patient heterogeneity in resistance mechanisms, accompanied by heterogeneous pERK expression immunostaining profiles. MEK monotherapy of Vemurafenib-resistant tumors caused toxicity and acquired drug resistance. However, tumors were eradicated when Vemurafenib was combined the MEK inhibitor. The diversity of drug responses among the xenograft models; the distinct mechanisms of resistance; and the ability to overcome resistance by the addition of a MEK inhibitor provide a scheduling rationale for clinical trials of next-generation drug combinations.

Oculoectodermal syndrome is a mosaic RASopathy associated with KRAS alterations.

Oculoectodermal syndrome (OES) is a rare disease characterized by a combination of congenital scalp lesions and ocular dermoids, with additional manifestations including non-ossifying fibromas and giant cell granulomas of the jaw occurring during the first decade of life. To identify the genetic etiology of OES, we conducted whole-genome sequencing of several tissues in an affected individual. Comparison of DNA from a non-ossifying fibroma to blood-derived DNA allowed identification of a somatic missense alteration in KRAS NM_033360.3(KRAS):c.38G>A, resulting in p.Gly13Asp. This alteration was also observed in the patient's other affected tissues including the skin and muscle. Targeted sequencing in a second, unrelated OES patient identified an NM_033360.3(KRAS):c.57G>C, p.Leu19Phe alteration. Allelic frequencies fell below 40% in all tissues examined in both patients, suggesting that OES is a mosaic RAS-related disorder, or RASopathy. The characteristic findings in OES, including scalp lesions, ocular dermoids, and benign tumors, are found in other mosaic and germline RASopathies. This discovery also broadens our understanding of the spectrum of phenotypes resulting from KRAS alterations. Future research into disease progression with regard to malignancy risk and investigation of RAS-targeted therapies in OES is warranted. KRAS sequencing is clinically available and may also now improve OES diagnostic criteria.

Partial deletion of the sulfate transporter SLC13A1 is associated with an osteochondrodysplasia in the Miniature Poodle breed.

A crippling dwarfism was first described in the Miniature Poodle in Great Britain in 1956. Here, we resolve the genetic basis of this recessively inherited disorder. A case-control analysis (8:8) of genotype data from 173 k SNPs revealed a single associated locus on CFA14 (P(raw) <10(-8)). All affected dogs were homozygous for an ancestral haplotype consistent with a founder effect and an identical-by-descent mutation. Systematic failure of nine, nearly contiguous SNPs, was observed solely in affected dogs, suggesting a deletion was the causal mutation. A 130-kb deletion was confirmed both by fluorescence in situ hybridization (FISH) analysis and by cloning the physical breakpoints. The mutation was perfectly associated in all cases and obligate heterozygotes. The deletion ablated all but the first exon of SLC13A1, a sodium/sulfate symporter responsible for regulating serum levels of inorganic sulfate. Our results corroborate earlier findings from an Slc13a1 mouse knockout, which resulted in hyposulfatemia and syndromic defects. Interestingly, the metabolic disorder in Miniature Poodles appears to share more clinical signs with a spectrum of human disorders caused by SLC26A2 than with the mouse Slc13a1 model. SLC26A2 is the primary sodium-independent sulfate transporter in cartilage and bone and is important for the sulfation of proteoglycans such as aggregan. We propose that disruption of SLC13A1 in the dog similarly causes undersulfation of proteoglycans in the extracellular matrix (ECM), which impacts the conversion of cartilage to bone. A co-dominant DNA test of the deletion was developed to enable breeders to avoid producing affected dogs and to selectively eliminate the mutation from the gene pool.