Stereocontrolled synthesis of the aconitine D ring from D-glucose.

The synthesis of a fully oxygenated aconitine D ring precursor from (D)-(+)-glucose is described. The route features a highly diastereoselective alkynyl Grignard ketone addition and a base-mediated enelactone to 1,3-diketone rearrangement.

Development of a hybrid bio-purification process of lactic acid solutions employing an engineered E. coli strain in a membrane bioreactor.

BACKGROUND: A potential alternative to lactic acid production through sugar fermentation is its recovery from grass silage leachate. The separation and purification of lactic acid from fermentation broths remain a key issue, as it amounts to up to 80% of its industrial production cost. In this study, a genetically engineered E. coli strain (A1:ldhA), that cannot catabolize lactic acid, has been used to selectively remove impurities from a synthetic medium comprising typical components (i.e., glucose and acetic acid) of green grass silage leachate. A systematic approach has been followed to provide a proof-of-concept for a bio-purification process of lactic acid solutions in a membrane bioreactor operating in semi-continuous mode. RESULTS: The synthetic medium composition was initially optimized in shake-flasks experiments, followed by scale-up in bench-scale bioreactor. Complete (i.e., 100%) and 60.4% removal for glucose and acetic acid, respectively, has been achieved in batch bioreactor experiments with a synthetic medium comprising 0.5 g/L glucose and 0.5 g/L acetic acid as carbon sources, and 10 g/L lactic acid; no lactic acid catabolism was observed in all batch fermentation tests. Afterwards, a hybrid biotechnological process combining semi-continuous bioreactor fermentation and ultrafiltration membrane separation (membrane bioreactor) was applied to in-situ separate purified medium from the active cells. The process was assessed under different semi-continuous operating conditions, resulting in a bacteria-free effluent and 100% glucose and acetic acid depletion, with no lactic acid catabolism, thus increasing the purity of the synthetic lactic acid solution. CONCLUSIONS: The study clearly demonstrated that a bio-purification process for lactic acid employing the engineered E. coli strain cultivated in a membrane bioreactor is a technically feasible concept, paving the way for further technological advancement.

Cannula Placement for Cerebral Protection Without Circulatory Arrest in Patients Undergoing Hemiarch Aortic Aneurysm Repair.

BACKGROUND: Aortic aneurysms involving the proximal aortic arch, which require hemiarch-type repair, typically require circulatory arrest with antegrade cerebral perfusion. Left carotid antegrade cerebral perfusion (LCP) via distal arch cannulation without circulatory arrest was used in this study's patient population. The goal was to assess the operative efficiency and clinical outcomes of using a distal arch cannulation technique that would not require any hypothermic circulatory arrest (HCA) time compared with more traditional brachiocephalic artery cannulation with right-sided unilateral antegrade cerebral perfusion (RCP) and HCA. METHODS: A single-center retrospective review of patients with replacement of the distal ascending aorta involving the proximal arch was performed. Patients with an intramural hematoma or dissection were excluded. Between January 2015 and December 2019, 68 adult patients had undergone a hemiarch repair because of aneurysmal disease. Analysis of baseline demographics, operative data, and clinical outcomes was performed. RESULTS: Comparing the 68 patients: 21 patients were treated with RCP (via brachiocephalic artery graft with HCA), and 47 patients were treated with LCP (via distal aortic arch cannulation with cross-clamp between the brachiocephalic and left common carotid arteries without HCA). Baseline characteristics and outcomes were evaluated for both groups. The LCP group was younger (LCP median [IQR] age, 60 [53-65] years vs RCP median [IQR] age, 67 [59-71] years]. Sex, race, body mass index, comorbidities, and ejection fraction were similar between the groups. Cardiopulmonary bypass time (LCP, 123 minutes vs RCP, 149 minutes) and unilateral cerebral perfusion time (LCP, 17 minutes vs RCP, 22 minutes) were longer in the RCP group. Bleeding, prolonged ventilatory support, kidney failure, and length of stay were similar. In-hospital mortality was 2% in the LCP group vs 0% in the RCP group. Stroke occurred in 2 patients (4.2%) in the LCP group and in 0% of the RCP group. Mortality at 6 months in the LCP and RCP groups was 3% and 10%, respectively. CONCLUSION: Distal arch cannulation with LCP without HCA is a reasonable and safe alternative strategy for patients requiring hemiarch replacement for aneurysmal disease. This technique may provide additional benefits by avoiding circulatory arrest in these complex cases.

Durable left ventricular assist device implant-how I teach it.

Left ventricular assist devices (LVADs) have become a mainstay of advanced heart failure therapy. The technical aspects of performing a device implant are nuanced and attention to these details allows for successful therapy with good outcomes. As more patient with heart failure are expected to benefit from mechanical circulatory support, the need for a concise and consistent technique for LVAD implantation is needed. Teaching this procedure is most comprehensible when broken down into separate steps, as with many other procedures. Here, we describe our standard protocol for LVAD implantation, as well as rudimentary outcomes of 6-year experience in our center. We hope this will provide some insight and guidance to centers who are expanding into the field of mechanical circulatory support and can help them form a foundation with which to build their own experience and success.

Interaction Between Platelet and Fibrinogen on Clot Strength in Healthy Patients.

OBJECTIVE: The purpose of this study was to explore the relationship between platelet concentration (PLT) (× 109/L) and clot strength measured by thromboelastography maximum amplitude (TEG-MA) in healthy volunteers without a history of coagulation abnormalities. Secondarily, the relationship between fibrinogen (mg/dL) and TEG-MA was analyzed. DESIGN: A prospective study. SETTING: At a university's tertiary-care center. MEASUREMENTS AND MAIN RESULTS: Using whole blood, PLT was reduced in the first part, and hematocrit was reduced in the second part of the study by hemodilution with platelet-rich and -poor plasma. Thromboelastography (TEG 5000 Haemonetics) was performed to measure clot formation and strength. Spearman correlation coefficients regression analyses and receiver-operating characteristics (ROC) were obtained to analyze the relationships among PLT, fibrinogen, and TEG-MA. Strong correlations were found in univariate analysis between PLT and TEG-MA (r = 0.88; p < 0.0001) and between Fibrinogen and TEG-MA (r = 0.70; p = 0.003). A biphasic relationship between PLT and TEG-MA was linear below a PLT 90 × 109/L, followed by a plateau above 100 × 109/L (p = 0.001). A linear relationship between fibrinogen (190-474 mg/dL) and TEG-MA (53-76 mm) was found (p = 0.0007). The ROC analysis found that PLT = 60 × 109/L was associated with a TEG-MA of 53.0 mm. The product of PLT and fibrinogen concentrations was more strongly correlated (r = 0.91) to TEG-MA than either PLT (r = 0.86) or fibrinogen (r = 0.71) alone. A ROC analysis revealed that a TEG-MA of 55 mm was associated with a PLT × fibrinogen of 16,720. CONCLUSION: In healthy patients, a PLT of 60 × 109/L was associated with normal clot strength (TEG-MA ≥53 mm), and there was little change in clot strength with PLT >90 × 109/L. Although prior analyses described the contributions of platelets and fibrinogen toward clot strength, they are presented and discussed independently. The data above described clot strength as an interaction among them. Future analyses and clinical care should evaluate and recognize the interplay.

Hereditary thrombocytopenia with platelet sialic acid deficiency and mutations in the GNE genes.

BACKGROUND: The inherited macrothrombocytopenias are rare disorders and the underlying cause can be identified in many cases but in some, this can remain enigmatic. Platelet transfusions are often administered during hemorrhagic events. METHODS: A patient with previously unexplained inherited macrothrombocytopenia with a platelet count between 3-20 × 109 /L is described in which studies were performed using exome sequencing (ES) and platelet flow cytometry. RESULTS: Both the hemoglobin and white cell counts were normal. ES revealed two suspicious variants, one likely pathogenic and one a variant of uncertain significance, in the UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) gene, and flow cytometry showed diminished expression of surface platelet sialic acid (about 5%) but normal red cell sialic acid. The Thrombopoietin (TPO) level was low, and the patient responded to TPO-mimetic treatment with an increase in the platelet count. CONCLUSION: Two variants in the GNE gene were able to be upgraded to pathogenic with apparently restricted expression to the megakaryocyte lineage. Platelet transfusion may be avoided in these patients with TPO-mimetic treatment.

Discordance in tests used to detect inhibition of the P2Y12 receptor in patients undergoing interventional neuroradiology procedures.

BACKGROUND: Clopidogrel is an inhibitor of the P2Y12 platelet receptor but testing to demonstrate a drug effect is controversial since there are often discordant results between different tests methods. METHODS: Samples from patients taking clopidogrel prior to intracranial flow-diversion procedures were tested using light transmission aggregometry (LTA), whole blood impedance aggregometry (WBIA) and the VerifyNow device (VND). Samples were classified as concordant if all test results were either responsive (inhibition) or resistant. Discordant results were separated using the VND into those with a responsive versus a resistant test result. RESULTS: Samples from 96 patients were studied. Concordance for all three tests was seen in 53/96 (55%) of samples, of which 41 (43%) were responsive and 12 (12%) were resistant. Discordance was observed in 43 samples (45%), 37 (28%) of which were caused by responsive VND and either a resistant WBIA or LTA and 6 (7%) of which were caused by a resistant VND but a responsive test result using either WBIA or LTA. These two discordant groups differed in both platelet count and hematocrit, but no such difference was present between the two concordant groups. CONCLUSION: Discordance in P2Y12 inhibition testing may be partly explained by sample platelet count and hematocrit.

Dual antiplatelet use in the management of COVID-19 associated acute ischemic stroke reocclusion.

INTRO: SARS-CoV-2 (COVID-19) infection is associated with acute ischemic stroke (AIS), which may be due to a prothrombotic state. Early reports have suggested high rates of reocclusion following mechanical thrombectomy (MT) with poor radiographic and clinical outcomes. We report our early experience using intra-procedural antithrombotics to address SARS-CoV-2 reocclusion. METHODS: We identified 6 patients that experienced early reocclusion after MT for COVID-19-associated AIS through retrospective chart review abstracting their basic demographics, COVID-19 status, and stroke management. All these patients were treated after reocclusion with aspirin and cangrelor intra-procedurally, the latter of which was converted to ticagrelor post-procedurally. Some patients additionally received argatroban infusion intraprocedurally. RESULTS: Mean age was 54. There were 3 post-procedural and 3 intra-procedural re-occlusions. After repeat thrombectomy and treatment with aspirin and cangrelor, those with post-procedure reocclusion did not show further reocclusion, while those with intra-procedural reocclusion showed radiographic improvement with intraprocedural cangrelor administration. Outcomes for these patients were poor, with a median mRS of 4. Two patients developed petechial hemorrhage of their stroke which was managed conservatively, and one developed a retroperitoneal hemorrhage from femoral access requiring transfusion. There were no patients who developed new parenchymal hematomas. CONCLUSION: COVID-19 AIS may be associated with a hypercoagulable state which risks malignant reocclusion complicating MT. We found antithrombotic treatment periprocedural cangrelor with or without argatroban transitioned to oral aspirin with ticagrelor to be a viable method for management of these patients.

In vitro comparison of CPD whole blood with conventional blood components.

INTRODUCTION: Resuscitation of severely injured trauma patients is commonly performed using red blood cells in additive solution supplemented with plasma and platelet concentrates. There is an increasing interest in the use of low anti-A titer Group O whole blood (LTOWB) in the early management of the resuscitation. It is unclear whether clinical outcome is improved using this approach. METHODS: Expired units of CPD-LTOWB were studied on Day 22 and expired units of thawed plasma on Day 6 and Day 7. LTOWB was assessed for hemoglobin content, clotting factor levels and platelet numbers and function using thromboelastography (TEG) and impedance aggregation. Assays of fibrinogen and FV, FVIII, FVII and FX were performed on the expired plasma. The LTOWB hemoglobin was compared to red cells in additive solution (AS-RBCs) and the clotting factor levels to those of expired thawed plasma. Platelet function was compared to fresh whole blood samples from healthy subjects. RESULTS: LTOWB contained slightly more hemoglobin than the AS-RBCs (Medians, 66 v 59 G), and the plasma content of fibrinogen was similar. Other clotting factors were reduced by approximately 15% except for FVIII which was 30% less. Both TEG and impedance aggregometry showed evidence of residual platelet function despite the prolonged period of refrigerator storage. CONCLUSION: LTOWB contains higher hemoglobin and adequate clotting factors, and residual platelet function is demonstrated indicating that this product would be expected to be at least equivalent to a single unit of each of the conventional components commonly used in trauma resuscitation.

A Pilot Study to Assess the Clinical Onset of IV Heparin in Interventional Cardiology and Cardiac Surgery.

OBJECTIVES: To determine the onset of heparin anticoagulation, using 2 different measures of activated clotting times (ACT), thromboelastography (TEG; R-time), and anti-Xa levels, after administering low- (100 U/kg) and high- (300 U/kg) dose intravenous (IV) heparin to patients undergoing transcatheter aortic valve replacement (TAVR) and cardiac surgery, respectively. DESIGN: Prospective study. SETTING: Single academic institution. PARTICIPANTS: Patients with normal baseline coagulation presenting for TAVR or cardiac valve surgery. INTERVENTIONS: Coagulation studies were performed at baseline, 30 seconds, 90 seconds, and 180 seconds after IV heparin administration. The tests included iSTAT (iACT) and Hemochron ACT (hACT), TEG R-Time, and anti-Xa levels. At the authors' institution, anti-Xa is the preferred measure of heparin anticoagulation when time permits. ACT, a rapid point- of-care test, is used to assess intraprocedural anticoagulation. MEASUREMENTS AND MAIN RESULTS: After both low- and high-dose heparin, there are peak increases in ACT and anti-Xa at 30 seconds, followed by a decline at 90 seconds and plateau at 180 seconds. The TEG R-time remained elevated (>80 minutes) throughout. For TAVR cases, all anti-Xa was >1.5 IU/mL, and was associated with an iACT >180 seconds and an hACT >200 seconds. For cardiac valve surgery cases, all anti-Xa was >2.4 and associated with an iACT >420 seconds and and hACT >340 seconds. Compared with hACT, iACTs were significantly lower at all time points after low-dose heparin, but not after high-dose heparin. CONCLUSIONS: In this pilot study, heparin anticoagulation was detected as early as 30 seconds after IV administration, based on ACT, anti-Xa levels, and TEG R-time.

Remote Dispensing of Emergency Release Red Blood Cells.

OBJECTIVES: Patients with acute bleeding are frequently transfused with emergency release (ER) group O RBCs. This practice has been reported to be safe with a low rate of acute hemolytic transfusion reactions (AHRs). METHODS: Records of patients who received ER RBCs over a 30-month period were examined at our hospitals. During this period, satellite refrigerators were on site in the emergency department (ED), which were electronically connected to the blood bank (electronically connected satellite refrigerator [ECSR]). Nurses accessing the refrigerator were required to give patient identification information, when known, prior to removal of the ER RBCs, allowing technologists the opportunity to check for previous serologic records and communicate directly with the ED if a serologic incompatibility was potentially present. RESULTS: In total, 935 patients were transfused with 1,847 units of ER RBCs. Thirty of these patients had a current (22/30) or historic (8/30) antibody. In 15 cases, incompatible RBCs were interdicted. In six cases, the transfusion was considered urgent, and an AHR occurred in four of these six (overall 0.4%), including one fatal AHR due to anti-KEL1. CONCLUSIONS: Use of KEL1-negative RBCs and ECSR merits consideration as approaches to mitigate the occurrence of ER RBC-associated AHRs.

Improving the specificity of E. coli acetate/propionate exclusion biosensors via iterative engineering.

Real-time monitoring of key performance indicator analytes such as acetate and propionate within anaerobic digestors (AD) is required for optimum biogas production. In this paper the further refinement of acetate and propionate whole cell (E. coli) exclusion biosensors is reported following an iterative process in which key metabolites that might interfere with O2-uptake measurements are identified and genes required for their catabolism are knocked out (exclusion). Analysis of biological leachate from an AD reactor treating lignocellulosic material revealed the presence of formate, which was subsequently shown to elicit a response in previously developed E. coli biosensor strains. P1 phage transduction was employed to delete two genes encoding formate dehydrogenase, fdoH and fdnH, to eliminate formate catabolism. Deletion of these genes from the propionate biosensor strain W:ldgyepak abolished interference from formate and enabled accurate determination of propionate concentrations in biological leachate. However, the acetate biosensing strain E1/pGDR11-acs, despite not having any response to formate, responded to propionate. It was likely that this was a result of the promiscuity of the wild type acetyl CoA synthetase, which was replaced with Acs2 from Saccharomyces cerevisiae, resolving the problem and enabling acetate determination with the biosensor. Acetate and propionate concentrations in authentic leachate influent were estimated to be 26.5 mM and 65.5 mM, respectively, using the biosensor, and 26.6 and 70 mM, respectively, by HPLC, demonstrating the accuracy and specificity of the refined biosensor.

Acute ischemic stroke on anti-Xa inhibitors: Pharmacokinetics and outcomes.

BACKGROUND AND PURPOSE: Direct oral anticoagulant (DOAC) ingestion within 48 h is an exclusion for thrombolysis in acute ischemic stroke (AIS) patients. We aim to shed light on pharmacokinetic correlates and outcomes in patients with AIS excluded from thrombolysis due to DOAC use. METHODS: This is a single center retrospective study of consecutive patients with AIS within 4.5 h from last known normal and excluded from thrombolytic therapy due to confirmed Xa inhibitor DOAC (DOACXa) intake within the prior 48 h. We used linear regression to test the correlation between time from last DOACXa ingestion and anti-Xa level. RESULTS: Over a period of 2.5 years, we identified 44 patients who did not receive thrombolysis because of presumed DOAC intake within 48 h. In adjusted linear regression, there was an association between time from last DOAC ingestion and Xa level (beta = -0.69, p < 0.001). Among the 37 patients with known atrial fibrillation not receiving alteplase due to DOAC use, the 90-day mortality was 35.1% (13/37) and 77% (10/13) of deaths were stroke related. CONCLUSIONS: Patients with AIS on DOAC therapy face a heightened risk of mortality. Studies are needed to investigate the safety and efficacy of thrombolysis in such patients based on time of last DOAC ingestion and/or anti-Xa/drug level.

Influenza-associated thrombotic thrombocytopenic purpura: A report of two cases and a brief review of the literature.

BACKGROUND AND OBJECTIVES: Thrombotic thrombocytopenic purpura (TTP) is often preceded by a recent history of an acute infection and influenza is the most implicated virus. MATERIALS AND METHODS: We identified two cases of TTP, which were preceded by influenza between 2010 and 2021. In one patient, we epitope mapped the binding specificity of antibodies using an overlapping peptide approach of the stalk protein of Influenza B and the cysteine-rich spacer domain (CRSD) of ADAMTS13. A literature search was performed for reports of influenza-associated TTP over the period 1980-2021. RESULTS: Two patients were identified in which TTP was preceded by influenza, one Influenza A and the other Influenza B. Epitope mapping of the latter's plasma identified target epitopes in both the stalk protein of Influenza B and CRSD of ADAMTS13. The literature review revealed only seven case reports, all but one from Europe or Asia and associated with Influenza A. Severe ADAMTS13 deficiency was demonstrated in only four cases. CONCLUSION: We report the first small case series of influenza-associated TTP. Moreover, it is the first case implicating Influenza B and a mechanism favouring polyclonal B-cell proliferation rather than molecular mimicry as the stimulus to form anti-ADAMTS13 auto-antibodies is suggested.

Red Cell Exchange as Adjunctive Therapy for Babesiosis: Is it Really Effective?

Human babesiosis is a parasitic disease prevalent in the Northeastern and Midwestern United States (US). Treatment with antibiotics is the standard of care but red cell exchange (RCE) has been used as an adjunctive treatment in more severe disease. Data for the efficacy of RCE in the treatment of babesiosis has been based on case reports and case series. An English language literature search was conducted for cases of babesiosis treated with RCE since 1980 and relevant laboratory and clinical outcome data were extracted. Similar data were obtained on severe cases of babesiosis referred for RCE in our hospitals in the time period 2000 to 2020. Fifty reports including forty-one individual case reports and nine case series were retrieved. There were 108 patients that underwent RCE with an overall mortality rate of 20%. Some patients had more than one RCE. The patients varied in the level of anemia and evidence of compromise of renal or pulmonary function. The pre-RCE level of parasitemia varied between 1.7% to 85% with the vast majority >10%. The post-RCE level of parasitemia varied between 1% to 10%. Since 2000, 32 patients were referred for RCE in our hospitals and RCE was performed on 23 of 32. There were more patients treated with RCE in the second decade as compared to the first decade, 19 versus 4 respectively. The overall mortality was 22% similar to the national data. Comparing the cohort treated with RCE to the 9 patients who were treated only with antibiotics, there were similar levels of parasitemia and laboratory parameters. The overall number of days needed to achieve a parasite count <1% was similar between the two cohorts and mortality for the antibiotics only cohort was 0%. More than 40 years after the first reported case of RCE in severe babesiosis it cannot be concluded that this adjunctive therapy favorably influences the clinical outcome. Since there is largely equipoise, a registry of severe patients treated with or without RCE could identify a benefit or otherwise.

Aortic root replacement with Medtronic Freestyle bioprosthesis: 25-year experience.

BACKGROUND: Stentless porcine bioprothesis is a surgical strategy to treat aortic root disease. Use has been limited due to the concern for long-term valve degeneration. This study evaluated the perioperative and late outcomes of patients with aortic root disease requiring root replacement. METHODS: A total of 409 patients underwent aortic root replacement by a single surgeon using a stentless porcine bioroot between February 1996 and May 2020. The cohort was divided into two groups (age ≤65 and >65 years). Descriptive statistics were used to analyze the data and Kaplan-Meier curves used to evaluate long-term outcomes. RESULTS: Patients age >65 years were more likely to be female (p = .01), have hypertension (p = .01), require circulatory arrest (p = .01), and have concomitant coronary artery bypass grafting (CABG) (p = .04). Baseline creatinine >1.8 (p = .20), diabetes (p = .06), and ejection fraction (p = .20) were similar between groups. The 1-, 5-, and 10-year survival for patients age ≤65 years were 92%, 87%, and 69%, respectively, significantly better than patients age >65 (88%, 73%, and 43%, respectively) (p < .01, Figure 1). The 1-, 5-, and 10-year freedom from reoperation for patients ≤65 years were 99%, 97%, and 93% versus 99%, 98%, and 96% in patients age >65 years, respectively (p = .24). CONCLUSION: Patients with aortic root disease can be treated with acceptable perioperative outcomes, long-term survival, and low reoperation rates using a stentless porcine bioprothesis. It should be considered irrespective of age due to its excellent durability and freedom from anti-coagulation requirement.

Laboratory Methods in the Assessment of Hereditary Hemostatic Disorders.

In patients presenting with a suspect hereditary bleeding disorder a detailed bleeding history is first obtained. Testing proceeds in a tiered manner with platelet count, platelet morphology, platelet histogram, PFA-100, fibrinogen, prothrombin time, and activated partial thromboplastin time. More detailed testing includes von Willebrand factor, individual clotting factor assays, and platelet function testing. Next, testing for a dysfibrinogenemia, FXIII, or a fibrinolytic defect is considered. Hemostatic abnormality is not demonstrated in a fraction of patients. An approach to management in these patients, such as desmopressin or antifibrinolytic therapy, may be required and empiric use of blood component therapy is discouraged.