Capture-based targeted sequencing using a T-cell control in myeloid malignancies and idiopathic cytopenias.

We report on a study of next-generation sequencing in 257 patients undergoing investigations for cytopenias. We sequenced bone marrow aspirates using a target enrichment panel comprising 82 genes and used T cells from paired blood as a control. One hundred and sixty patients had idiopathic cytopenias, 81 had myeloid malignancies and 16 had lymphoid malignancies or other diagnoses. Forty-seven of the 160 patients with idiopathic cytopenias had evidence of somatic pathogenic variants consistent with clonal cytopenias. Only 39 genes of the 82 tested were mutated in the 241 patients with either idiopathic cytopenias or myeloid neoplasms. We confirm that T cells can be used as a control to distinguish between germline and somatic variants. The use of paired analysis with a T-cell control significantly reduced the time molecular scientists spent reporting compared to unpaired analysis. We identified somatic variants of uncertain significance (VUS) in a higher proportion (24%) of patients with myeloid malignancies or clonal cytopenias compared to less than 2% of patients with non-clonal cytopenias. This suggests that somatic VUS are indicators of a clonal process. Lastly, we show that blood depleted of lymphocytes can be used in place of bone marrow as a source of material for sequencing.

Tamoxifen for the treatment of myeloproliferative neoplasms: A Phase II clinical trial and exploratory analysis.

Current therapies for myeloproliferative neoplasms (MPNs) improve symptoms but have limited effect on tumor size. In preclinical studies, tamoxifen restored normal apoptosis in mutated hematopoietic stem/progenitor cells (HSPCs). TAMARIN Phase-II, multicenter, single-arm clinical trial assessed tamoxifen's safety and activity in patients with stable MPNs, no prior thrombotic events and mutated JAK2V617F, CALRins5 or CALRdel52 peripheral blood allele burden ≥20% (EudraCT 2015-005497-38). 38 patients were recruited over 112w and 32 completed 24w-treatment. The study's A'herns success criteria were met as the primary outcome ( ≥ 50% reduction in mutant allele burden at 24w) was observed in 3/38 patients. Secondary outcomes included ≥25% reduction at 24w (5/38), ≥50% reduction at 12w (0/38), thrombotic events (2/38), toxicities, hematological response, proportion of patients in each IWG-MRT response category and ELN response criteria. As exploratory outcomes, baseline analysis of HSPC transcriptome segregates responders and non-responders, suggesting a predictive signature. In responder HSPCs, longitudinal analysis shows high baseline expression of JAK-STAT signaling and oxidative phosphorylation genes, which are downregulated by tamoxifen. We further demonstrate in preclinical studies that in JAK2V617F+ cells, 4-hydroxytamoxifen inhibits mitochondrial complex-I, activates integrated stress response and decreases pathogenic JAK2-signaling. These results warrant further investigation of tamoxifen in MPN, with careful consideration of thrombotic risk.

Tumor endothelial cell autophagy is a key vascular-immune checkpoint in melanoma.

Tumor endothelial cells (TECs) actively repress inflammatory responses and maintain an immune-excluded tumor phenotype. However, the molecular mechanisms that sustain TEC-mediated immunosuppression remain largely elusive. Here, we show that autophagy ablation in TECs boosts antitumor immunity by supporting infiltration and effector function of T-cells, thereby restricting melanoma growth. In melanoma-bearing mice, loss of TEC autophagy leads to the transcriptional expression of an immunostimulatory/inflammatory TEC phenotype driven by heightened NF-kB and STING signaling. In line, single-cell transcriptomic datasets from melanoma patients disclose an enriched InflammatoryHigh /AutophagyLow TEC phenotype in correlation with clinical responses to immunotherapy, and responders exhibit an increased presence of inflamed vessels interfacing with infiltrating CD8+ T-cells. Mechanistically, STING-dependent immunity in TECs is not critical for the immunomodulatory effects of autophagy ablation, since NF-kB-driven inflammation remains functional in STING/ATG5 double knockout TECs. Hence, our study identifies autophagy as a principal tumor vascular anti-inflammatory mechanism dampening melanoma antitumor immunity.

CNL and aCML should be considered as a single entity based on molecular profiles and outcomes.

Chronic neutrophilic leukemia (CNL) and atypical chronic myeloid leukemia (aCML) are rare myeloid disorders that are challenging with regard to diagnosis and clinical management. To study the similarities and differences between these disorders, we undertook a multicenter international study of one of the largest case series (CNL, n = 24; aCML, n = 37 cases, respectively), focusing on the clinical and mutational profiles (n = 53 with molecular data) of these diseases. We found no differences in clinical presentations or outcomes of both entities. As previously described, both CNL and aCML share a complex mutational profile with mutations in genes involved in epigenetic regulation, splicing, and signaling pathways. Apart from CSF3R, only EZH2 and TET2 were differentially mutated between them. The molecular profiles support the notion of CNL and aCML being a continuum of the same disease that may fit best within the myelodysplastic/myeloproliferative neoplasms. We identified 4 high-risk mutated genes, specifically CEBPA (ß = 2.26, hazard ratio [HR] = 9.54, P = .003), EZH2 (ß = 1.12, HR = 3.062, P = .009), NRAS (ß = 1.29, HR = 3.63, P = .048), and U2AF1 (ß = 1.75, HR = 5.74, P = .013) using multivariate analysis. Our findings underscore the relevance of molecular-risk classification in CNL/aCML as well as the importance of CSF3R mutations in these diseases.

Prediction of Tensile Strength in Friction Welding Joins Made of SA213 Tube to SA387 Tube Plate through Optimization Techniques.

In modern times, the Industry X.0 has emerged as the paradigm that has become the core of digital technology-driven business organizations. Further, this paper establishes a tube to tube plate friction welding technology with the help of deploying an external tool, also known referred to as the FWTPET scheme. Besides, the SA213 tube and SA387 tube plate were combined by employing a unique interference fit technique. Also, the strength of this combined portion was assessed with and without the aid of a holding block. Subsequently, the analytic optimization approaches like genetic algorithm, analysis of variance, and Taguchi L9 orthogonal array design were deployed in the prediction of the optimum joining strength. Moreover, the input parameters include the projection of the tube (mm), the rotational speed of the tool (rpm), and depth of cut (mm); besides, the tensile strength is considered as the output parameter. Also, the grain size distribution around the weld zone and the presence of base metal were measured through an optical microscope as per ASTM linear intercept method. Further, it is evident that grain refinement had occurred in the weld zone, which in turn increases the tensile strength. The exceptional weld strength (tensile strength) was obtained when joining of SA213 tube and SA387 tube plate through interference fit using a holding block without a hole in the tube. Experimentally, it was found that the achieved tensile strengths were 836.8 MPa (without a hole) and 789.35 MPa (with hole) using the holding block, respectively. Additionally, it was found that in the absence of a holding block, the achieved tensile strength is 762.2 MPa (without a hole), and 700.8 MPa (with a hole), correspondingly. The deviation of tensile strength between the predicted (genetic algorithm) and experimental was found minimal. Therefore, for achieving this strength, the suitable operating parameters set include the rotational speed of the tool (1300 rpm), projection of the tube (1 mm), and depth of cut (0.5 mm) with backing block configuration.

Sensor-Assisted Assessment of the Tribological Behavioral Patterns of Al-SiCp Composites under Various Environmental Temperature Conditions.

Currently, the use of sensors and supporting technologies has become indispensable in the assessment of tribological behavioral patterns of composites. Furthermore, the current investigation focused on the assessment of the tribological behavior of the Al-SiCp composite for high-temperature applications. Moreover, the Al-SiCp composite was fabricated by adapting the liquid metallurgy route with varying weight percentages of SiCp (x = 3, 6, and 9). Density, hardness, and high-temperature wear tests were performed to evaluate the hardness and tribological characteristics and properties of modern-day advanced composites. Moreover, the inclusion of SiCp enhanced the advanced composite materials hardness from 60 HV to 110 HV due to a high degree of refinement of the α-phase. Subsequently, the fabricated samples' wear behavior was assessed by varying the wear parameter viz. the applied load (20 N and 30 N) and sliding distance (250 m, 500 m, 750 m, and 1000 m) with the constant sliding velocity (0.45 m/s) for various temperatures (40 °C, 150 °C, and 250 °C). Moreover, the results revealed that the enhancement in the reinforcement percentage improves the wear resistance. Consequently, the wear rate decreased at 250 °C, possibly owing to the development of the oxide layers. Therefore, the occurrence of delamination and plastic deformation were evidenced in the wear-out surface, thereby depicting the prevalence of delamination and the abrasive wear-mechanism.

Managing anaemia in bone marrow failure syndromes.

PURPOSE OF REVIEW: Anaemia is a common haematological presentation in patients with bone marrow failure, yet a challenging condition to treat. As anaemia has a direct impact on the patient's symptoms, managing anaemia in the common bone marrow failure conditions, such as myelodysplastic syndrome will help to improve the quality of life. This review discusses the available treatment options and the benefit of improving the haemoglobin level. RECENT FINDINGS: Managing anaemia effectively has shown to improve the patient outcome, yet treatment option remain limited. Recently, activin inhibitors such as Luspatercept have shown to be effective in patients' refractory to ESAs and further clinical trials are ongoing to explore this further. SUMMARY: Transfusion still remains the mainstay of treatment in patients not suitable, lost response or refractory to erythropoiesis-stimulating agents (ESAs). Majority of these patients are not suitable for definite treatment options such as bone marrow transplantation. The aim of treatment remains improving the quality of life and newer therapeutic options may offer better and more sustained response.

Predictors of short-term outcomes in patients undergoing percutaneous coronary intervention in cardiogenic shock complicating STEMI-A tertiary care center experience.

BACKGROUND: Studying the outcomes in patients presenting with cardiogenic shock with ST-segment elevation myocardial infarction (CS-STEMI) and undergoing primary or rescue percutaneous coronary intervention (PCI) may give an insight to the unmet needs in STEMI-care in our region and may help in future recommendations in improving survival. MATERIALS AND METHODOLGY: During the period from January 2001- June 2017, there were 114 patients included in the study. The demographic, clinical and angiographic characteristics were compared between the survivors and non-survivors. All these variables were also compared between two-time frames (Phase 1- January 2001 to June 2007; Phase 2- July 2007 to June 2017). RESULTS: Among patients undergoing PCI for STEMI, 7.5% were in cardiogenic shock. In-hospital mortality for the patients included in the study was 53.5%. Total ischemic time (OR=0.99, 0.99-1; p=0.02), left ventricular ejection fraction (LVEF) (OR=0.90, 0.82-0.98; p=0.02), need for cardio-pulmonary resuscitation (OR=0.12, 0.24-0.66; p=0.01), and post PCI TIMI flows (OR=0.08, 0.02-0.29; p<0.001) were the significant determinants of in-hospital mortality in the regression analysis. There was no significant change in mortality between the two phases of the study, though there was a reduction in total ischemic and door-to-balloon times, transfer admissions, use of thrombolytics, glycoprotein IIb/IIIa inhibitors, intra-aortic balloon pump, and mechanical ventilation in phase 2. CONCLUSION: Patients presenting in CS-STEMI and undergoing PCI continue to experience high mortality rates, despite improvements in total ischemic times. Further improvement in the systems-of-care are required to bring about reduction in mortality in this high-risk subset.

Ischemic Preconditioning Protects Astrocytes against Oxygen Glucose Deprivation Via the Nuclear Erythroid 2-Related Factor 2 Pathway.

Induction of ischemic preconditioning (IPC) represents a potential therapy against cerebral ischemia by activation of adaptive pathways and modulation of mitochondria to induce ischemic tolerance to various cells and tissues. Mitochondrial dysfunction has been ascribed to contribute to numerous neurodegenerative conditions and cerebral ischemia. Nuclear erythroid 2-related factor 2 (Nrf2) is a transcription factor that has traditionally been involved in upregulating cellular antioxidant systems to combat oxidative stress in the brain; however, the association of Nrf2 with mitochondria in the brain remains unclear. In the present study, we investigated the effects of Nrf2 on (i) IPC-induced protection of astrocytes; (ii) OXPHOS protein expression; and (iii) mitochondrial supercomplex formation.Oxygen-glucose deprivation (OGD) was used as an in vitro model of cerebral ischemia and IPC in cultured rodent astrocytes derived from WT C57Bl/6J and Nrf2-/- mice. OXPHOS proteins were probed via western blotting, and supercomplexes were determined by blue native gel electrophoresis.IPC-induced cytoprotection in wild-type, but not Nrf2-/- mouse astrocyte cultures following a lethal duration of OGD. In addition, our results suggest that Nrf2 localizes to the outer membrane in non-synaptic brain mitochondria, and that a lack of Nrf2 in vivo produces altered supercomplex formation in mitochondria.Our findings support a role of Nrf2 in mediating IPC-induced protection in astrocytes, which can profoundly impact the ischemic tolerance of neurons. In addition, we provide novel evidence for the association of Nrf2 to brain mitochondria and supercomplex formation. These studies offer new targets and pathways of Nrf2, which may be heavily implicated following cerebral ischemia.

Ischemic preconditioning treatment of astrocytes transfers ischemic tolerance to neurons.

Ischemic preconditioning (IPC) represents a potential therapy against cerebral ischemia. While our group has previously shown IPC to induce neuroprotection through various pathways, the role of astrocytes in supporting IPC-induced neuroprotection has not been extensively studied. Astrocyte-derived lactate has gained attention as a potential soluble mediator through which astrocytes could impart ischemic tolerance to neurons. Therefore, the goal of this study was to determine if i) IPC-treatment of astrocytes alone could transfer ischemic tolerance to neurons; ii) if IPC-treatment of astrocytes increases lactate production; and if iii) exogenous lactate administration to neurons could induce neuroprotection against lethal ischemia in vitro. For this purpose, a co-culture system was used and modified from a previous method. This system allows astrocytes and neurons to be separated by a physical barrier, while allowing secreted substances from either cell type to interact with each other. Oxygen-glucose deprivation was used as a model of cerebral ischemia and IPC in cultured rodent astrocytes and neurons. Neurons incubated with IPC-treated astrocytes were significantly protected against lethal ischemic injury compared to neurons incubated with sham-treated astrocytes. In addition, IPC-treatment of astrocytes significantly increased lactate secretion into the extracellular media. Finally, exogenous lactate administration can significantly attenuate cell death in neuronal cultures following exposure to lethal OGD. Our results suggest that IPC-treatment of astrocytes alone can transfer ischemic tolerance to neurons. In addition, the ability of IPC to increase lactate production in astrocytes suggest that lactate could represent a neuroprotective agent to protect neurons against lethal ischemic injury.

Resveratrol Preconditioning Protects Against Cerebral Ischemic Injury via Nuclear Erythroid 2-Related Factor 2.

BACKGROUND AND PURPOSE: Nuclear erythroid 2 related factor 2 (Nrf2) is an astrocyte-enriched transcription factor that has previously been shown to upregulate cellular antioxidant systems in response to ischemia. Although resveratrol preconditioning (RPC) has emerged as a potential neuroprotective therapy, the involvement of Nrf2 in RPC-induced neuroprotection and mitochondrial reactive oxygen species production after cerebral ischemia remains unclear. The goal of our study was to study the contribution of Nrf2 to RPC and its effects on mitochondrial function. METHODS: We used rodent astrocyte cultures and an in vivo stroke model with RPC. An Nrf2 DNA binding ELISA and protein analysis via Western blotting of downstream Nrf2 targets were performed to determine RPC-induced activation of Nrf2 in rat and mouse astrocytes. After RPC, mitochondrial function was determined by measuring reactive oxygen species production and mitochondrial respiration in both wild-type and Nrf2-/- mice. Infarct volume was measured to determine neuroprotection, whereas protein levels were measured by immunoblotting. RESULTS: We report that Nrf2 is activated by RPC in rodent astrocyte cultures, and that loss of Nrf2 reduced RPC-mediated neuroprotection in a mouse model of focal cerebral ischemia. In addition, we observed that wild-type and Nrf2-/- cortical mitochondria exhibited increased uncoupling and reactive oxygen species production after RPC treatments. Finally, Nrf2-/- astrocytes exhibited decreased mitochondrial antioxidant expression and were unable to upregulate cellular antioxidants after RPC treatment. CONCLUSIONS: Nrf2 contributes to RPC-induced neuroprotection through maintaining mitochondrial coupling and antioxidant protein expression.

Signaling pathways leading to ischemic mitochondrial neuroprotection.

There is extensive evidence that ischemic/reperfusion mediated mitochondrial dysfunction is a major contributor to ischemic damage. However data also indicates that mild ischemic stress induces mitochondrial dependent activation of ischemic preconditioning. Ischemic preconditioning is a neuroprotective mechanism which is activated upon a brief sub-injurious ischemic exposure and is sufficient to provide protection against a subsequent lethal ischemic insult. Current research demonstrates that mitochondria are not only the inducers of but are also an important target of ischemic preconditioning mediated protection. Numerous proteins and signaling pathways are activated by ischemic preconditioning which protect the mitochondria against ischemic damage. In this review we examine some of the proteins activated by ischemic precondition which counteracts the deleterious effects of ischemia/reperfusion thereby maintaining normal mitochondrial activity and lead to ischemic tolerance.

A prospective, randomized study to evaluate the efficacy of various diuretic strategies in acute decompensated heart failure.

AIM: To evaluate the safety and efficacy of various initial strategies of loop diuretic administration in patients with acute decompensated heart failure (ADHF) on diuresis, renal function, electrolyte balance and clinical outcomes. METHODS: Consecutive patients admitted with ADHF were randomized into three groups - intravenous furosemide infusion + intravenous dopamine, intravenous furosemide bolus in two divided doses and intravenous furosemide continuous infusion alone. At 48 h, the treating physician could adjust the diuretic strategy. Primary endpoint was negative fluid balance at 24 h after admission. Secondary end points were duration of hospital stay, negative fluid balance at 48, 72, 96 h, the trend of serum electrolytes, and renal function and 30 day clinical outcome (death and emergency department visits). RESULTS: Overall ninety patients (thirty in each group) were included in the study. There was a greater diuresis in first 24 h (p = 0.002) and a shorter hospital stay (p = 0.023) with the bolus group. There was no significant difference in renal function and serum sodium and serum potassium levels. There was no difference in the number of emergency department visits among the three groups. CONCLUSION: All three modes of diuretic therapies can be practiced with no difference in worsening of renal function and electrolyte levels. Bolus dose administration with its rapid volume loss and shorter hospital stay might be a more effective diuretic strategy.

Perfluorocarbon emulsion improves oxygen transport of normal and sickle cell human blood in vitro.

Perfluorocarbons (PFC) are compounds with high gas solubility that could help deliver O2 to tissues and have been suggested as adjunct therapy to ischemia. Using a newly designed in vitro system, we tested the hypothesis that a third generation PFC emulsion (Oxycyte) increased O2 transport of blood by measuring changes in O2 extraction ratio. The system included a computer-controlled pump and blood-gas exchange chambers to oxygenate and deoxygenate the blood from nine sickle cell disease (SCD) patients and five healthy donors. The flowing blood reached various levels of hemoglobin O2 saturation and O2 partial pressures (PO2), measured using a CO-oximeter and a blood gas analyzer. The mixtures were kept at physiological blood pressure and temperature, constant flow, normobaric conditions, and FiO2 = 0.30. After adding PFC, the measurements suggested an increase in the transport of O2 and CO. Addition of PFC resulted in larger PO2 difference from 15 ± 2 mmHg to 23 ± 2 mmHg. Using normal blood and blood from SCD patients, the average O2 extraction ratio (O2ER) after PFC was significantly higher than baseline. Addition of saline did not cause statistically significant changes. The data suggest increased (facilitated) O2 transport by this PFC emulsion in both normal and SCD blood.

Epsilon PKC increases brain mitochondrial SIRT1 protein levels via heat shock protein 90 following ischemic preconditioning in rats.

Ischemic preconditioning is a neuroprotective mechanism whereby a sublethal ischemic exposure is protective against a subsequent lethal ischemic attack. We previously demonstrated that SIRT1, a nuclear localized stress-activated deacetylase, is vital for ischemic preconditioning neuroprotection. However, a recent study demonstrated that SIRT1 can also localize to the mitochondria. Mitochondrial localized SIRT1 may allow for a direct protection of mitochondria following ischemic preconditioning. The objective of this study was to determine whether ischemic preconditioning increases brain mitochondrial SIRT1 protein levels and to determine the role of PKCɛ and HSP90 in targeting SIRT1 to the mitochondria. Here we report that preconditioning rats, with 2 min of global cerebral ischemia, induces a delayed increase in non-synaptic mitochondrial SIRT1 protein levels which was not observed in synaptic mitochondria. This increase in mitochondrial SIRT1 protein was found to occur only in neuronal cells and was mediated by PKCε activation. Inhibition of HSP90, a protein chaperone involved in mitochondrial protein import, prevented preconditioning induced increases in mitochondrial SIRT1 and PKCε protein. Our work provides new insights into a possible direct role of SIRT1 in modulating mitochondrial function under both normal and stress conditions, and to a possible role of mitochondrial SIRT1 in activating preconditioning induced ischemic tolerance.

Ischemic preconditioning and clinical scenarios.

PURPOSE OF REVIEW: Ischemic preconditioning (IPC) is gaining attention as a novel neuroprotective therapy and could provide an improved mechanistic understanding of tolerance to cerebral ischemia. The purpose of this article is to review the recent work in the field of IPC and its applications to clinical scenarios. RECENT FINDINGS: The cellular signaling pathways that are activated following IPC are now better understood and have enabled investigators to identify several IPC mimetics. Most of these studies were performed in rodents, and efficacy of these mimetics remains to be evaluated in human patients. Additionally, remote ischemic preconditioning (RIPC) may have higher translational value than IPC. Repeated cycles of temporary ischemia in a remote organ can activate protective pathways in the target organ, including the heart and brain. Clinical trials are underway to test the efficacy of RIPC in protecting brain against subarachnoid hemorrhage. SUMMARY: IPC, RIPC, and IPC mimetics have the potential to be therapeutic in various clinical scenarios. Further understanding of IPC-induced neuroprotection pathways and utilization of clinically relevant animal models are necessary to increase the translational potential of IPC in the near future.