Prehospital Tirofiban Increases the Rate of Disrupted Myocardial Infarction in Patients with ST-Segment Elevation Myocardial Infarction: Insights From the On-TIME 2 Trial.

BACKGROUND: In patients with ST-segment elevation myocardial infarction (STEMI), prehospital tirofiban significantly improved myocardial reperfusion. However, its impact on the rate of disrupted myocardial infarction (MI), particularly in the context of high-sensitive cardiac troponin (hs-cTn) assays, is still unclear. METHODS: The On-TIME 2 (The Ongoing Tirofiban In Myocardial infarction Evaluation 2) trial randomly assigned STEMI patients to prehospital tirofiban or placebo before transportation to a percutaneous coronary intervention (PCI) centre. In this post hoc analysis, we evaluated STEMI patients that underwent primary PCI and had measured hs-cTn levels. Troponin T levels were collected at 18-24 h and 72-96 h after PCI. Disrupted MI was defined as peak hs-cTn T levels ≤10 times the upper limit of normal (≤140 ng/L). RESULTS: Out of 786 STEMI patients, 47 (6%) had a disrupted MI. Disrupted MI occurred in 31 of 386 patients (8.0%) in the tirofiban arm and in 16 of 400 patients (4.0%) in the placebo arm (p=0.026). After multivariate adjustment, prehospital tirofiban remained independently associated with disrupted MI (OR 2.03; 95% CI 1.10 to 3.87; P= 0.027). None of the patients with disrupted MI died during the one-year follow-up, compared to a mortality rate of 2.6% among those without disrupted MI. CONCLUSION: Among STEMI patients undergoing primary PCI, the use of prehospital tirofiban was independently associated with a higher rate of disrupted MI. These results, highlighting a potential benefit, underscore the need for future research focusing on innovative pretreatment approaches which may increase the rate of disrupted MI.

Robotic Right Hepatectomy with En Bloc Caudatectomy for Bismuth IIIa Hilar Cholangiocarcinoma: A Video Demonstration of Left-Liver-First Anterior Radical Modular Orthotopic Right Hemihepatectomy.

BACKGROUND: Minimally invasive resection for perihilar cholangiocarcinoma is a complicated and technically demanding surgical procedure. Radical surgical resection is regarded as the best treatment for hepatic hilar cholangiocarcinoma.1,2 Right hepatectomy with caudate lobe resection is necessary as the treatment for bismuth IIIa hilar cholangiocarcinoma.3 The left-liver-first anterior radical modular orthotopic right hemihepatectomy (LARMORH), which can simplify surgical steps and decrease procedural difficulty, may be a better choice for Bismuth IIIa hilar cholangiocarcinoma.4 However, there are no reports of this approach using robotic technique for this operation. We will provide a detailed introduction to this method through this video. METHODS: A 45-year-old female patient was diagnosed with a hilar cholangiocarcinoma. Following a 7-day percutaneous biliary drainage of the left intrahepatic bile duct and obtaining informed consent, we performed a robotic radical resection of the HCCA using the LARMORH approach. The patient was positioned supine with the entire bed elevated 20° and tilted 15° to the left. Trocars were placed in position (Fig. 1). After entering the abdominal cavity, it was explored for tumor metastasis. The surgery adopted a left approach, initially exploring the left hepatic artery and vein to further assess resectability. After confirming resectability, the right hepatic artery and gastroduodenal artery (GDA) were dissected. The common bile duct was dissected and transected at its distal end, ensuring R0 surgical margins. Lymph nodes were cleared from the foot side to the head side, confirming the metastasis to the lymph node group 13a, so we further cleared the group 16 and 9 lymph nodes.5 Subsequently, we approached the resection of the right half and the entire caudate lobe with the reverse thinking of left hepatic resection mode, preserving only the left branch of the portal vein and left hepatic artery, and dissecting the liver tissue along the resection plane of the left liver. After transection of the left hepatic duct, the activity space of the left liver was larger and the caudate lobe could be better exposed. The Spiegel lobe was lifted to the right in a "turn the page" fashion for in situ resection of the entire caudate lobe and the right half of the liver. Finally, a bilioenteric anastomosis was performed using the Roux-en-Y method. RESULTS: Robotic right hepatectomy with caudate lobectomy was successfully performed in 450 min, with an estimated blood loss of 200 ml. The histological grading was determined as T1aN1M0 (stage IIIB) on the basis of postoperative pathological biopsy results. The patient achieved a satisfactory postoperative recovery and was discharged on the 14th postoperative day without any major complications. Following the operation, the patient received capecitabine chemotherapy according to the Chinese Society of Clinical Oncology (CSCO) criteria. Since September 2022, our team has completed three radical resections for Bismuth IIIa HCCA using this technique. All patients achieved a satisfactory postoperative recovery without any further complications. CONCLUSIONS: Robotic left-liver-first anterior radical modular orthotopic right hemihepatectomy for Bismuth IIIa HCCA is both safe and feasible. This method may provide a new surgical approach for patients with type IIIA HCCA or liver diseases requiring right hemihepatectomy combined with total caudate lobectomy.

Assessment of platelet functional activity in healthy individuals and patients receiving antiplatelet therapy. Possible inconsistencies between aggregation and flow cytometry tests.

Platelet functional activity was assessed in healthy volunteers (HV, n=92), patients with stable angina pectoris (SA, n=42) and acute coronary syndrome (ACS, n=73), treated with acetylsalicylic acid (ASA) + clopidogrel and ASA + ticagrelor, respectively. In all HV and patients we have compared parameters of platelet aggregation (maximum light transmission and velocity, Tmax and Vmax) and parameters, characterizing exposure of platelet activation markers, evaluated by flow cytometry. HV platelets were activated by 10 µM, 1 µM TRAP, and 20 µM, 5 µM, 2.5 µM ADP; patient platelets were activated by 10 µM TRAP and by 20 µM and 5 µM ADP. Strong and significant correlations between the aggregation and flow cytometry parameters (the r correlation coefficient from 0.4 up to >0.6) most frequently were registered in HV platelet during activation by 1 µM TRAP and in SA patients during platelet activation by 20 µM and 5 µM ADP. However, in many other cases these correlations were rather weak (r < 0.3) and sometimes statistically insignificant. In HV the differences in PAC-1 binding parameters between platelets activated by 10 µM TRAP (the strongest agonist) and all ADP concentrations were negligible (≤ 10%), while CD62P binding (at all ADP concentrations) and LTA parameters for (5 µM and 2.5 µM ADP) were significantly lower (by 40-60%). Antiplatelet therapy in patients decreased all parameters as compared to HV, but to varying extents. For 10 µM TRAP the MFI index for PAC-1 binding (40-50% decrease) and for both ADP concentrations the Tmax values (60-85% decrease) appeared to be the most sensitive in comparison with the other parameters that decreased to a lesser extent. The data obtained indicate a possibility of inconsistency between different LTA and flow cytometry parameters in assessing platelet activity and efficacy of antiplatelet drugs.

Long-term toxicity evaluation of aristolochic acid-IIIa in mice.

Aristolochic acid (AA)-IIIa is an AA analog present in Aristolochiaceae plants. To evaluate the chronic toxicity of AA-IIIa, mice were intragastrically administered with media control, 1 mg/kg AA-IIIa, and 10 mg/kg AA-IIIa, and designated as the control (CTL), AA-IIIa low dose (AA-IIIa-L), and AA-IIIa high dose (AA-IIIa-H) groups, respectively. AA-IIIa was administered three times a week, every other day, for 24 weeks (24-week time point). Thereafter, some mice were sacrificed immediately, while others were sacrificed 29 or 50 weeks after AA-IIIa withdrawal (53- or 74-week time point). Serum and organs were collected for biochemical and pathological analyses, respectively. Whole-genome sequencing was performed on the kidney, liver, and stomach tissues of AA-IIIa-treated mice for single-nucleotide polymorphism (SNP) detection. AA-IIIa-H mice died at 66 weeks, and the remaining mice showed moribund conditions at the 69 weeks. AA-IIIa induced minor kidney tubule injury, fibroblast hyperplasia, and forestomach carcinoma in mice. Bladder, intestine, liver, heart, spleen, lung, and testis tissues were not pathologically altered by AA-IIIa. In addition, AA-IIIa increased the C:G > A:T mutation in the kidney; however, no SNP mutation changes were observed in the liver and forestomach tissues of AA-IIIa-H mice at the 24-week time point compared with control mice. Therefore, we suspect that AA-IIIa is potentially mutagenic for mice after overdose and long-term administration. On the other hand, the forestomach is a unique organ in mice, but it does not exist in humans; thus, we hypothesize that the stomach toxicity induced by AA-IIIa is not a suitable reference for toxicological evaluation in humans. We recommend that Aristolochiaceae plants containing AA-IIIa should be properly supervised, and overdosing and long-term administration of drugs containing AA-IIIa should be avoided.

[Incidence of intrahospitalary major cardiac events with the use of mechanic thromboaspiration versus only use of IIB/IIIA glucoprotein inhibitors on patients with acute ST elevation myocardial infarction]. / Incidencia de eventos cardiovasculares mayores intrahospitalarios en pacientes sometidos a tromboaspiración mecánica más IIb/IIIa contra solo inhibidores de la glucoproteína IIb/IIIa en infarto agudo al miocardio con elevación del segmento ST.

Objective: To identify the incidence of in-hospital major adverse cardiac events (MACE) with the use of mechanic thromboaspiration plus IIb/IIIa glycoprotein inhibitors versus only use of IIb/IIIa glycoprotein inhibitors on patients with acute ST elevation myocardial infarction. Method: Retrospective, observational, cohort analytic study, on patients with acute ST elevation myocardial infarction that had angiography thrombus TIMI 5 grade, treated between October 2021 and December 2022. Results: A total of 237 patients were included. In 113 patients thromboaspiration were used, 124 patients didn't used. 81.6% were men. In-hospital MACE occurred on 31.9% of patients with thromboaspiration use vs. 30.6% on patients with no use (RR: 1.05; IC95%: 0.61-1.93; p = 0.840). Incidence of malignant arrhythmias were of 8% with thromboaspiration use vs. 1.6% on patients with no use (RR: 5.27; IC95%: 1.11-24.97; p = 0.020). Conclusions: The use of thromboaspiration on concomitant treatment with IIb/IIIa glycoprotein inhibitors was similar with only IIb/IIIa glycoprotein inhibitors in reducing incidence of in-hospital MACE on patients with ST elevation acute myocardial infarction and high thrombus burden. The study has several limitations, so results should be taken with caution.

Objetivo: Identificar la incidencia de eventos cardiovasculares adversos mayores (ECAM) intrahospitalarios con el uso de tromboaspiración mecánica más inhibidores de la glucoproteína IIb/IIIa contra solo inhibidores de la glucoproteína IIb/IIIa en pacientes con infarto agudo al miocardio con elevación del segmento ST (IAMCEST). Método: Estudio retrospectivo, observacional, analítico, de cohorte, en pacientes con IAMCEST con trombo angiográfico de grado TIMI 5, tratados entre octubre de 2021 y diciembre de 2022. Resultados: Cumplieron los criterios de inclusión 237 pacientes. En 113 se usó tromboaspirador más inhibidores IIb/IIIa y en 124 solo inhibidores IIb/IIIa. El 81.6% fueron hombres. La incidencia de ECAM intrahospitalarios fue del 31.9% en los pacientes con tromboaspiración y del 30.6% en los pacientes con solo inhibidores IIb/IIIa (RR: 1.05; IC95%: 0.61-1.93; p = 0.840). La incidencia de arritmias graves fue del 8% en los pacientes con tromboaspiración y del 1.6% en los pacientes con solo inhibidores IIb/IIIa (RR: 5.27; IC95%: 1.11-24.97; p = 0.020). Conclusiones: La frecuencia de ECAM asociados al uso de tromboaspiración mecánica como coadyuvante a los inhibidores de la glucoproteína IIb/IIIa en pacientes con IAMCEST y trombo angiográfico de grado TIMI 5 no es diferente de la de aquellos pacientes en las que solo se utilizan inhibidores de la glucoproteína IIb/IIa. El estudio tiene varias limitaciones, por lo que los resultados deben tomarse con cautela.

A Young Patient With Acute Ostial Right Coronary Artery Aneurysm Presenting As ST Elevation Myocardial Infarction.

A coronary artery aneurysm (CAA) is a localized dilatation of a coronary artery segment >1.5 times the diameter of the adjacent normal segment. CAA is more common in men than women and has multiple etiologies, including genetic causes, infections, and atherosclerotic diseases. Kawasaki disease is the most common cause of CAA in children, whereas atherosclerosis is the most common etiology in adults. We present the case of a male in his 30s who presented with sudden-onset chest pain and inferior ST segment elevation on an ECG. Echocardiography revealed preserved left ventricular function and mild hypokinesia. The patient underwent an emergency coronary angiogram that showed an ostial right CAA with thrombi. He was initially managed with a glycoprotein IIb/IIIa inhibitor tirofiban infusion, followed by triple therapy with aspirin, clopidogrel, and rivaroxaban. The patient underwent magnetic resonance imaging of his head, which was normal, and he did not attend outpatient computed tomography coronary angiography. The patient was discharged with lifelong rivaroxaban 20 mg once daily.

Distinct CD16a features on human NK cells observed by flow cytometry correlate with increased ADCC.

Natural killer (NK) cells destroy tissue that have been opsonized with antibodies. Strategies to generate or identify cells with increased potency are expected to enhance NK cell-based immunotherapies. We previously generated NK cells with increased antibody-dependent cell mediated cytotoxicity (ADCC) following treatment with kifunensine, an inhibitor targeting mannosidases early in the N-glycan processing pathway. Kifunensine treatment also increased the antibody-binding affinity of Fc γ receptor IIIa/CD16a. Here we demonstrate that inhibiting NK cell N-glycan processing increased ADCC. We reduced N-glycan processing with the CRIPSR-CAS9 knockdown of MGAT1, another early-stage N-glycan processing enzyme, and showed that these cells likewise increased antibody binding affinity and ADCC. These experiments led to the observation that NK cells with diminished N-glycan processing capability also revealed a clear phenotype in flow cytometry experiments using the B73.1 and 3G8 antibodies binding two distinct CD16a epitopes. We evaluated this "affinity profiling" approach using primary NK cells and identified a distinct shift and differentiated populations by flow cytometry that correlated with increased ADCC.

Safety and effectiveness of glycoprotein IIb/IIIa inhibitors in acute coronary syndromes: insights from the SPUM-ACS study.

AIMS: Data on Glycoprotein IIb/IIIa inhibitors (GPI) use in real world ACS patients following the introduction of potent P2Y12 inhibitors and newer generation stents are scant. Here, we aimed to assess the utilization, effectiveness, and safety of GPI in a large prospective multi-centre cohort of contemporary ACS patients. METHODS AND RESULTS: SPUM-ACS prospectively recruited patients presenting with ACS between 2009 and 2017. The primary endpoint of the present study was major adverse cardiovascular events (MACE), a composite of all-cause death, non-fatal myocardial infarction (MI) and non-fatal stroke at one year. Secondary endpoints were defined as any bleeding events, BARC 3-5 bleeding, and net adverse cardiovascular events (NACE). A total of 4395 ACS patients were included in the analysis. GPI-treated patients had more total coronary artery occlusion (56% vs 35%, p<0.001) and thrombus (60% vs 35%, p<0.001) at angiography. Among the propensity score matched (PSM) population (1992 patients equally split into two groups), GPI-treated patients showed lower risk of MACE (PSM adjusted HR 0.70, 95% CI 0.49-0.99), but a higher risk of any (PSM adj HR 1.46, 95% CI 1.06-1.99) and major bleedings (PSM adj HR 1.73, 95% CI 1.09-2.76), resulting in a neutral effect on NACE (PSM adj HR 0.87, 95% CI 0.65-1.17). These results remained consistent across all subgroups. CONCLUSION: In patients with ACS undergoing PCI and receiving potent P2Y12 inhibitors, we observed a reduced risk of MACE and an increased risk of major bleedings at 1 year in patients treated with GPI. Although the routine use of GPI is currently not recommended, they might be considered in selected patients following a personalized balancing between ischaemic and bleeding risks.

On Decorating a Honeycomb AlN Monolayer with Hydrogen and Fluorine Atoms: Ab Initio and Experimental Aspects.

Mono- and few-layer hexagonal AlN (h-AlN) has emerged as an alternative "beyond graphene" and "beyond h-BN" 2D material, especially in the context of its verification in ultra-high vacuum Scanning Tunneling Microscopy and Molecular-beam Epitaxy (MBE) experiments. However, graphitic-like AlN has only been recently obtained using a scalable and semiconductor-technology-related synthesis techniques, such as metal-organic chemical vapor deposition (MOCVD), which involves a hydrogen-rich environment. Motivated by these recent experimental findings, in the present work, we carried out ab initio calculations to investigate the hydrogenation of h-AlN monolayers in a variety of functionalization configurations. We also investigated the fluorination of h-AlN monolayers in different decoration configurations. We find that a remarkable span of bandgap variation in h-AlN, from metallic properties to nar-row-bandgap semiconductor, and to wide-bandgap semiconductor can be achieved by its hy-drogenation and fluorination. Exciting application prospects may also arise from the findings that H and F decoration of h-AlN can render some such configurations magnetic. We complemented this modelling picture by disclosing a viable experimental strategy for the fluorination of h-AlN.

Factor VIII moiety of recombinant Factor VIII Fc fusion protein impacts Fc effector function and CD16+ NK cell activation.

Recombinant Factor VIII-Fc fusion protein (rFVIIIFc) is an enhanced half-life therapeutic protein product used for the management of hemophilia A. Recent studies have demonstrated that rFVIIIFc interacts with Fc gamma receptors (FcγR) resulting in the activation or inhibition of various FcγR-expressing immune cells. We previously demonstrated that rFVIIIFc, unlike recombinant Factor IX-Fc (rFIXFc), activates natural killer (NK) cells via Fc-mediated interactions with FcγRIIIA (CD16). Additionally, we showed that rFVIIIFc activated CD16+ NK cells to lyse a FVIII-specific B cell clone. Here, we used human NK cell lines and primary NK cells enriched from peripheral blood leukocytes to study the role of the FVIII moiety in rFVIIIFc-mediated NK cell activation. Following overnight incubation of NK cells with rFVIIIFc, cellular activation was assessed by measuring secretion of the inflammatory cytokine IFNγ by ELISA or by cellular degranulation. We show that anti-FVIII, anti-Fc, and anti-CD16 all inhibited indicating that these molecules were involved in rFVIIIFc-mediated NK cell activation. To define which domains of FVIII were involved, we used antibodies that are FVIII domain-specific and demonstrated that blocking FVIII C1 or C2 domain-mediated membrane binding potently inhibited rFVIIIFc-mediated CD16+ NK cell activation, while targeting the FVIII heavy chain domains did not. We also show that rFVIIIFc binds CD16 with about five-fold higher affinity than rFIXFc. Based on our results we propose that FVIII light chain-mediated membrane binding results in tethering of the fusion protein to the cell surface, and this, together with increased binding affinity for CD16, allows for Fc-CD16 interactions to proceed, resulting in NK cellular activation. Our working model may explain our previous results where we observed that rFVIIIFc activated NK cells via CD16, whereas rFIXFc did not despite having identical IgG1 Fc domains.

Long-term personalized high-protein, high-fat diet in pediatric patients with glycogen storage disease type IIIa: Evaluation of myopathy, metabolic control, physical activity, growth, and dietary compliance.

Dietary lipid manipulation has recently been proposed for managing glycogen storage disease (GSD) type IIIa. This study aimed to evaluate the myopathic, cardiac, and metabolic status, physical activity, growth, and dietary compliance of a personalized diet high in protein and fat for 24 months. Of 31 patients with type IIIa GSD, 12 met the inclusion criteria. Of these, 10 patients (mean age 11.2 ± 7.4 years) completed the study. Patients were prescribed a personalized high-protein, high-fat diet, comprising 3.0-3.5 g/kg/day of protein and 3.0-4.5 g/kg/day of fat, constituting 18.5%-28% and 70.5%-75.7% of daily energy, respectively. Dietary compliance was ensured and assessed via the regular administration of questionnaires. Our results revealed consistent and significant decreases of 22%, 54%, and 30% in the creatinine kinase, creatine kinase-myocardial band, and lactate dehydrogenase levels, respectively. Echocardiography revealed improvements in the Z-scores of the left ventricular mass and interventricular septum thickness. A significant increase in body muscle mass was observed, and a higher score was achieved using the Daily Activity Questionnaire. Growth monitoring revealed an arrest in the height-SDS at the 6th and 12th months, followed by subsequent improvement at the end of the second year. A gradual and persistent decline in the periods of hypo- and hyperglycemia has been reported. Biotinidase activity decreased, whereas hepatosteatosis increased and then decreased by the end of the study. Implementing a high-protein, high-fat diet and monitoring key parameters in patients with type IIIa GSD can lead to myopathic and cardiac improvements and increased physical activity.

Survival paradox between stage IIB/C and stage IIIA colon cancer: is it time to revise the American Joint Committee on Cancer TNM system?

INTRODUCTION: A survival paradox between T4N0 (Stage IIB/IIC) and Stage IIIA colon cancer exists, even after adjusting for adequate lymph node (LN) retrieval and receipt of adjuvant chemotherapy (C). We conducted a large hospital-based study to re-evaluate this survival paradox based on the newest 8th edition staging system. METHODS: The National Cancer Data Base was queried to evaluate 35,606 patients diagnosed with Stage IIB, IIC, and IIIA colon cancer between 2010 and 2017. The Kaplan-Meier method and log-rank test were used to compare unadjusted overall survival (OS). Multivariable Cox proportional hazards model was used to determine the association of stage with hazard ratios adjusted for relevant demographic and clinical variables including ≥ 12 LNs retrieved and receipt of adjuvant chemotherapy. P value < 0.05 was considered statistically significant. RESULTS: The 5-year OS for optimally treated stage IIIA colon cancer (receipt of C) was 84.3%, which was significantly higher than stage IIB/C (≥ 12 LNs retrieved + C) (72.8%; P < 0.0001). Stage was an independent predictor of OS. Among optimally treated Stage IIIA patients, T1N1 had the best survival (90.6%) while stage T4bN0 (stage IIC) had the worst (70.9%) (P < 0.0001). Compared to stage IIB, stage IIC had a 17% increased risk of overall death while stage IIIA had a 21% reduction in death (P < 0.0001). CONCLUSION: Stage IIB/C and Stage IIIA survival paradox persists even after accounting for receipt of adjuvant chemotherapy and adequate lymph node retrieval. Future iteration of the TNM system should take this paradox into consideration.

Potential Mechanism of Platelet GPIIb/IIIa and Fibrinogen on Retinal Vein Occlusion.

PURPOSE: To explore the role of coagulation and fibrinolytic factors, and the potential mechanism of platelet aggregation in the pathogenesis of retinal vein occlusion. METHODS: Coagulation and fibrinolytic parameters in patients with retinal vein occlusion were determined using hemagglutinin and HISCL-5000. Relationships between these elevated parameters and factors representing typical clinical manifestations of retinal vein occlusion were examined, and these parameters were analyzed using a STRING database to indicate the potential role of platelet aggregation. Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) levels were evaluated by flow cytometry after antiplatelet treatment in patients and mouse models. Furthermore, the GPIIb/IIIa ligand fibrinogen in peripheral blood and retina of mouse models was assessed by the turbidimetric method and real-time PCR, respectively. RESULTS: In patients, significant increases in peripheral blood fibrinogen and GPIIb/IIIa levels were observed (p = 0.0040, p < 0.0001, respectively). A positive correlation was observed between macular thickness (MT) and both fibrinogen and GPIIb/IIIa (r = 0.4528, p = 0.0063; r = 0.3789, p = 0.0427, respectively). After intravitreal injections of anti-vascular endothelial growth factor drugs, a significant reduction in fibrinogen levels was observed (p = 0.0072). In addition, the use of antiplatelet drugs resulted in a significant decrease in GPIIb/IIIa (p < 0.0001). In a mouse model, antiplatelet therapy significantly reduced both peripheral blood and retina fibrinogen levels and the overall rate of vein occlusion 3 days after occlusion (p < 0.0005). In addition, the reduction in GPIIb/IIIa levels after antiplatelet therapy was remarkable. CONCLUSION: Fibrinogen and GPIIb/IIIa may be involved in retinal vein occlusion and blocking platelet aggregation may be a new therapeutic approach for retinal vein occlusion.

A novel ITGA2B double cytosine frameshift variant (c.1986_1987insCC) leads to Glanzmann's thrombasthenia in a cat.

BACKGROUND: Glanzmann's thrombasthenia (GT) is a congenital platelet disorder affecting approximately 1:1 000 000 people globally and characterized by impaired platelet aggregation and clot retraction. Autosomal recessive, loss-of-function, variants in ITGA2B or ITGB3 of the αIIbß3 receptor cause the disease in humans. A cat affected by Glanzmann's and macrothrombocytopenia was presented to the UC Davis VMTH. HYPOTHESIS/OBJECTIVES: Severe thrombopathia in this cat has an underlying genetic etiology. ANIMALS: A single affected patient, 2 age-matched clinically healthy controls, and a geriatric population (n = 20) of normal cats. METHODS: Physical examination and clinical pathology tests were performed on the patient. Flow cytometry and platelet aggregometry analyses for patient phenotyping were performed. Patient and validation cohort gDNA samples were extracted for Sanger sequencing of a previously identified ITGA2B (c.1986delC) variant. Reverse transcriptase PCR was performed on patient and healthy control PRP samples to verify ITGA2B variant consequence. RESULTS: A novel c.1986_1987insCC autosomal recessive variant in ITGA2B was identified. This variant was absent in a population of 194 unrelated cats spanning 44 different breeds. Complete loss of ITGA2B transcript and protein expression was verified by RT-PCR and flow cytometry, explaining the underlying etiology of GT, and likely macrothrombocytopenia, in this cat. CONCLUSIONS AND CLINICAL IMPORTANCE: This study emphasizes the role of precision medicine in cardiovascular disease of cats and identified yet another variant that may be of utility for screening in the feline population. This study provides a small-volume, standardized, successful protocol for adequate platelet RNA isolation and subsequent molecular assessment of gene expression in cats.

Development of the Integrated Computer Simulation Model of the Intracellular, Transmembrane, and Extracellular Domain of Platelet Integrin α IIb ß 3 (Platelet Membrane Glycoprotein: GPIIb-IIIa).

Background The structure and functions of the extracellular domain of platelet integrin α IIb ß 3 (platelet membrane glycoprotein: GPIIb-IIIa) change substantially upon platelet activation. However, the stability of the integrated model of extracellular/transmembrane/intracellular domains of integrin α IIb ß 3 with the inactive state of the extracellular domain has not been clarified. Methods The integrated model of integrin α IIb ß 3 was developed by combining the extracellular domain adopted from the crystal structure and the transmembrane and intracellular domain obtained by Nuclear Magnetic Resonace (NMR). The transmembrane domain was settled into the phosphatidylcholine (2-oleoyl-1-palmitoyl-sn-glycerol-3-phosphocholine (POPC)) lipid bilayer model. The position coordinates and velocity vectors of all atoms and water molecules around them were calculated by molecular dynamic (MD) simulation with the use of Chemistry at Harvard Macromolecular Mechanics force field in every 2 × 10 -15 seconds. Results The root-mean-square deviations (RMSDs) of atoms constructing the integrated α IIb ß 3 model apparently stabilized at approximately 23 Å after 200 ns of calculation. However, minor fluctuation persisted during the entire calculation period of 650 ns. The RMSDs of both α IIb and ß 3 showed similar trends before 200 ns. The RMSD of ß 3 apparently stabilized approximately at 15 Å at 400 ns with persisting minor fluctuation afterward, while the structural fluctuation in α IIb persisted throughout the 650 ns calculation period. Conclusion In conclusion, the integrated model of the intracellular, transmembrane, and extracellular domain of integrin α IIb ß 3 suggested persisting fluctuation even after convergence of MD calculation.

Analysis of the binding mechanism for a water-soluble Pd(II) complex containing ß-amino alcohols with HSA applying experimental and computational methods.

In the study ahead, the binding interactions of the [Pd (HEAC) Cl2] complex with human serum albumin (HSA) protein have been assayed in vitro (pH= 7.40) utilizing computational and experimental procedures. The mentioned complex was synthesized as a water-soluble complex from {2-((2-((2-hydroxyethyl)amino)ethyl)amino) cyclohexanol} ligand = HEAC. The results of electronic absorption and circular dichroism investigations illustrated that the hydrophobicity of the Tryptophan microenvironment in HSA undergoes the changes by binding to the Pd(II) complex without substantial perturbations on the protein secondary structure. The fluorescence emission spectroscopy analysis revealed that with rising temperature, the quenching constant (Ksv) in the Stern-Volmer's relation decreases; so, it can be said that the interaction process is along with a static quenching mechanism. The values of 2.88 × 105 M-1, and 1.26 represent the binding constant (Kb) and the number of the binding sites (n), respectively. The Job graph showed the maximum point at χ = 0.5, which means organizing a new set with 1:1 stoichiometry. Thermodynamic profile (ΔH < 0, ΔS < 0, and ΔG < 0) has affirmed that van der Waals forces and hydrogen bonds have a basic function in the Pd(II) complex-albumin bindings. The ligand-competitive displacement studies utilizing warfarin and ibuprofen have represented that Pd(II) complex interacts with albumin by site II (subdomain IIIA). The computational molecular docking theory approved the results of the site-competitive tests; also, it indicated the existence of hydrogen bonds and van der Waals forces in Pd(II) complex-albumin interactions.Communicated by Ramaswamy H. Sarma.

Late-onset mucopolysaccharidosis type IIIA mimicking Usher syndrome.

Mucopolysaccharidosis type IIIA (MPS IIIA or Sanfilippo syndrome type A) is an autosomal recessive lysosomal storage disorder caused by pathogenic variants in the SGSH gene encoding N-sulfoglucosamine sulfohydrolase, an enzyme involved in the degradation of heparan sulfate. MPS IIIA is typically characterized by neurocognitive decline and hepatosplenomegaly with childhood onset. Here, we report on a 53-year-old male subject initially diagnosed with Usher syndrome for the concurrence of retinitis pigmentosa and sensorineural hearing loss. Clinical exome sequencing identified biallelic missense variants in SGSH, and biochemical assays showed complete deficiency of sulfamidase activity and increased urinary glycosaminoglycan excretion. Reverse phenotyping revealed left ventricle pseudo-hypertrophy, hepatosplenomegaly, bilateral deep white matter hyperintensities upon brain MRI, and decreased cortical metabolic activity by PET-CT. On neuropsychological testing, the proband presented only partial and isolated verbal memory deficits. This case illustrates the power of unbiased, comprehensive genetic testing for the diagnosis of challenging mild or atypical forms of MPS IIIA.

Genetic Engineering of Therapeutic Phages Using Type III CRISPR-Cas Systems.

The functional characterization of "hypothetical" phage genes is a major bottleneck in basic and applied phage research. To compound this issue, the most suitable phages for therapeutic applications-the strictly lytic variety-are largely recalcitrant to classical genetic techniques due to low recombination rates and lack of selectable markers. Here we describe methods for fast and effective phage engineering that rely upon a Type III-A CRISPR-Cas system. In these methods, the CRISPR-Cas system is used as a powerful counterselection tool to isolate rare phage recombinants.

Ability change across multiple domains in mucopolysaccharidosis (Sanfilippo syndrome) type IIIA.

The objective of this paper is 1) to expand the scope of the domains previously published in a natural history study of Mucopolysaccharidosis IIIA (Sanfilippo syndrome type A) (MPS IIIA) and 2) to present evidence regarding the capacity of a new metric, Growth Scale Values (GSVs), in comparison with traditional metrics, to show changes in skills as assessed by the Bayley Scales of Infant Development -III (BSID-III) and the Vineland Adaptive Behavior Scales, Second Edition (VABS-II). We re-analyzed a cohort of 25 children, 20 with rapid progressing disease and 5 with slow progression, who had been followed over two years using the BSID-III, and the VABS-II. Previously findings were reported using age equivalent scores; now we are also presenting findings with GSVs. For the re-analysis, Language and Motor scores were added to the Cognitive scale on the BSID-III, and Domain- and Subdomain-level scores added to the Total VABS-II score (i.e., ABC Composite). We evaluated raw scores, age equivalent scores, and GSVs (and standard scores for the VABS-II only). Individual patient data can be found in the appendices to this publication. Results indicate that 1) Cognition as measured by GSVs was the most sensitive to decline; 2) GSVs showed significant decline in the range of 4 to 6 years of age; 3) For children under 4 years of age, positive growth occurs on most scales and most metrics, with the exception of language which slows somewhat earlier; 4) Other than the Cognitive scale, Receptive Language on the BSID-III and Receptive Communication on the VABS-II showed the most sensitivity to change; 5) Gross Motor skills showed the least decline over time and appeared to lack sensitivity to MPS IIIA motor concerns; and 6) No evidence for sensitivity to change for any metric was found in time intervals less than one year. We conclude that GSVs are a precise measurement of change to detect decline in function, and they are a valuable method for future clinical trials in MPS IIIA. Evidence continues to support cognition as a primary endpoint. Additional work is needed to identify sensitive measures of meaningful endpoints to families.

Repetitive, non-invasive imaging of neurodegeneration, and prevention of it with gene replacement, in mice with Sanfilippo syndrome.

Hampering assessment of treatment outcomes in gene therapy and other clinical trials in patients with childhood dementia is the lack of an objective, non-invasive measure of neurodegeneration. Optical coherence tomography (OCT) is a widely available, rapid, non-invasive, and quantitative method for examining the integrity of the neuroretina. Profound brain and retinal dysfunction occur in patients and animal models of childhood dementia, including Sanfilippo syndrome and we recently revealed a correlation between the age of onset and rate of progression of retinal and brain degeneration in sulfamidase-deficient Sanfilippo mice. The aim of the current study was to use OCT to visualise the discrete changes in retinal structure that occur during disease progression. A progressive decline in retinal thickness was readily observable in Sanfilippo mice using OCT, with differences seen in affected animals from 10-weeks of age. OCT applied to i.v. AAV9-sulfamidase-treated Sanfilippo mice enabled visualisation of improved retinal anatomy in living animals, an outcome confirmed via histology. Importantly, brain disease lesions were also ameliorated in treated Sanfilippo mice. The findings highlight the sensitivity, ease of repetitive use and quantitative capacity of OCT for detection of discrete changes in retinal structure and their prevention with a therapeutic. Combined with the knowledge that retinal and brain degeneration are correlated in Sanfilippo syndrome, OCT provides a window to the brain in this and potentially other childhood dementias.