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The main goal of this study is to analyze the magnetic, dielectric, and magneto-dielectric characteristics of Pb1− x La x Ti1− x O 3 - (Ba1–3 x Nd2 x )4Co2Fe36O60 (where x = 0.25) composite material at various temperatures T1-T3 (i.e 1100°C ,1200°C, 1300°C respectively). The X-ray diffraction investigation has been introduced in order to pinpoint the creation of the U-type hexaferrite phase. SEM micrographs reveal that sample sintered at T2 reached the maximum value of grain size and the largest experimental density value of 6.14 g/cm3 due to the composite material's intensified grain growth. The magnetic investigations further indicate that the sample T2 achieved the highest remnant magnetization, measuring 1.550 emu/g, revealing the accuracy of the sintering temperature. The magneto-dielectric investigations demonstrate the presence of multiferroicity in all samples, and came to the conclusion that sample T2 exhibits the highest magneto-dielectric response of 41.99 at 1.2 Tesla and a magneto-dielectric coefficient (γ) of around 0.7609 g2/emu2. Numerous metrics, including nyquist plots, impedance, electrical modulus, dielectric constant, and conductivity, were carefully examined in order to determine the electrical properties of the proposed sample. It was found that sample T2 produced the enhanced results and had the right temperature for the substance to develop.

Objective: The aim of the research work to monitor impurities profiling and degradation kinetics of Carvedilol Pharmaceutical Dosage Form (Tablets) through stress degradation study. Methods: To study impurity profiling and degradation kinetics Chromatographic condition used as, Inertsil ODS 3V column (150 mm x 4.6 mm, 5μm) with mobile phase consisting Mobile phase-A (Water, Acetonitrile and Trifluroacetic acid in the ratio of 80:20:0.1 v/v/v respectively and pH adjusted to 2.0 with dilute potassium hydroxide solution) and Mobile phase-B (Water and acetonitrile in the ratio of 100:900 v/v respectively) delivered at flow rate of 1.0 ml min-1 and the detection wavelength 240 nm. The column compartment temperature maintained at 40 °C. Results: Stress degradation study conducted using Acid, Alkali, Oxidation, Humidity, Thermal and Photolytic stress degradation conditions. Known, unknown and degradant impurities nature and degradation kinetics in different stressed degradation conditions were monitored through stability indicated method reverse phase HPLC method. Carvedilol molecule found sensitive to Oxidation and Alkali conditions. Impurity-A significantly increased from its not detected level. Carvedilol molecule found stable in Acid, Humidity, Thermal and Photolytic stress degradation condition. In all stressed conditions, mass balance was found between 95% to 105% and peak purity of carvedilol peak was found pure. Conclusion: Stress degradation study is required to know the degradation pathway of product and to prove the stability indicating nature of the analytical method. Study provide information pertaining to the intrinsic stability of drug product.

Background: Vildagliptin is a drug for the treatment of diabetes. DPP-IV inhibitor represents a new class of oral antihyperglycemic agents to treat patients with type 2 diabetes. Several RP-HPLC methods have been reported to determine Vildagliptin alone. However, it has been noted that there are no available stability-indicating methods in pharmacopeias (USP/BP/EP/JP) nor in the available literature to quantify known and unknown impurity patterns for vildagliptin in vildagliptin tablets. Objective: The aim of this study is to develop a new single, sensitive, robust and specific gradient RP-HPLC method to quantify known and unknown impurities and degradants of Vildagliptin in Vildagliptin tablets. Methods: Chromatographic separation has been accomplished on the Hypersil ODS column (250 x 4.6) mm, 5 μm with a mobile phase consisting of a mixture of Perchloric acid Buffer, methanol, acetonitrile and Triethylamine delivered at a flow rate of 1.0 mL minute-1 and the detection wavelength 210 nm. The developed method was validated as per ICH guidelines. Results: Vildagliptin was found degraded significantly under oxidative and alkaline stress conditions. The degradation products were well resolved from Vildagliptin and its impurities. An analytical method was found linear, accurate and precise from LOQ (Limit of Quantification) level to 150% of impurity specification limit (0.5%). Conclusion: The method found sensitive, rapid and accurate quantification of known, unknown impurities and degradants. The peak purity results confirmed that the Vildagliptin peak was homogeneous and pure in all stress samples, thus proving the stability-indicating nature of the method.

With the advancement of technology, we are able to use Arduino in this project to digitally sense body temperature and heart rate. Arduino is primarily used because it has the ability to perceive its surroundings by receiving data from various sensors and change it by controlling lights, motors and other actuators. Arduino programming language is used to program the microcontroller of the board. The goal of this work is to develop a system that can remotely monitor body temperature and heart rate in real time with the measurement results displayed on a web server. This research involves a number of phases, including the generation of research hypotheses based on literature reviews and the design of a hardware and software system.

Objective: The aim of the research work to develop a simple, sensitive, rugged, robust and specific novel gradient stability indicating reverse phase HPLC method for quantitative determination of known and unknown impurities profiling of Carvedilol pharmaceutical dosage forms (Tablets). Methods: Chromatographic separation has been achieved on an Inertsil ODS 3V column (150 mm x 4.6 mm, 5μm) with mobile phase consisting Mobile phase-A (Water, Acetonitrile and Trifluroacetic acid in the ratio of 80:20:0.1 v/v/v respectively and pH adjusted to 2.0 with dilute potassium hydroxide solution) and Mobile phase-B (Water and acetonitrile in the ratio of 100:900 v/v respectively) delivered at flow rate of 1.0 ml min-1 and the detection wavelength 240 nm. The column compartment temperature maintained at 40 °C. Results: Resolution between Carvedilol and its impurities has been achieved greater than 1.5. The developed method was validated as per ICH guidelines. Analytical method found Precise, Linear, accurate, specific, rugged and robust. Conclusion: Developed and validated novel analytical method can be used to for impurity profile analysis of Carvedilol Pharmaceutical dosage form (Tablets).

Background A novel gradient, high-sensitive and specific stability-indicating reverse-phase HPLC method was developed and validated for quantitative purpose of known, unknown and degradant impurities profiling for diltiazem hydrochloride tablets. The impurities were separated on the Zorbax RX C8 column (150 mm × 4.6 mm, 5 μm) with mobile phase-A consisting of a mixture of 0.05 M sodium dihydrogen phosphate monohydrate buffer pH 3.0 and methanol in the ratio 800:200v/v and mobile phase-B consisting of acetonitrile with a flow rate of 1.0 mL min−1. The column compartment was maintained at 35 °C, and the detection wavelength was 240 nm. Diltiazem hydrochloride, its known impurities and unknown impurities have been well resolved from each other. Results The linearity of the method has been demonstrated across the concentration range of 0.18 to 5.65 µg mL−1 for EP impurity-F with correlation coefficient R2 greater than 0.99. Recovery of method was proved from LOQ to 150% for known and unknown impurities with respect to test concentration and found in between 80 and 120%. Forced degradation study and specificity experiment results with mass balance proved the stability-indicating nature of the method and separated all known, unknown impurities and degradants from each other as well as from main drug component (diltiazem hydrochloride). The mass balance for stress study was found in between 95 and 105%. Conclusion Newly developed analytical method was validated as per ICH Q2 (R1) guidelines “Validation of analytical procedure” and found linear, accurate, specific, robust and precise in the established working range.

Streptococcus pyogenes, or group A Streptococcus (GAS), a gram-positive bacterium, is implicated in a wide range of clinical manifestations and life-threatening diseases. One of the key virulence factors of GAS is streptopain, a C10 family cysteine peptidase. Since its discovery, various homologs of streptopain have been reported from other bacterial species. With the increased affordability of sequencing, a significant increase in the number of potential C10 family-like sequences in the public databases is anticipated, posing a challenge in classifying such sequences. Sequence-similarity-based tools are the methods of choice to identify such streptopain-like sequences. However, these methods depend on some level of sequence similarity between the existing C10 family and the target sequences. Therefore, in this work, we propose a novel predictor, C10Pred, for the prediction of C10 peptidases using sequence-derived optimal features. C10Pred is a support vector machine (SVM) based model which is efficient in predicting C10 enzymes with an overall accuracy of 92.7% and Matthews’ correlation coefficient (MCC) value of 0.855 when tested on an independent dataset. We anticipate that C10Pred will serve as a handy tool to classify novel streptopain-like proteins belonging to the C10 family and offer essential information.

In every developed and developing country, the major facing problem is heavy metals toxicity. Due to there is an increase in the heavy metals anthropogenic in day to day life by various factors, which are causing serious issues or harmful health hazardous for all types of living organisms. Moreover, they are present everywhere in the universe it causes the contamination of the food, dietary, and processed materials. Accordingly, the present review article further summarizes the studies related to the determination of lead as a toxic impurity with a total of 134 references in the period 2000 to 2018. In this write-up, emphasize the one of the highly toxic heavy metal element Lead (Pb) and it's toxicity in the animals, humans, plants, and aquatic systems. In addition, the purpose of this article is to evaluate the effectiveness of established analytical techniques and trends in analytical methods like AAS; ICP-OES; ICP-MS; ASV; X-ray fluorescence spectrometry, these techniques significantly applicable for the quantitative analysis of Pb in various sources. The various regulatory authorities for Pb throughout the globe like IOSH, EPA, EMA, and CDCSO. This reveals the need and scope of further research in the field of heavy metal toxicity and development of new analytical techniques in meeting the needs of the life scientists. The present comprehensive review is an attempt to transform the state of knowledge into the findings that may act as a guideline for all the interested groups at different levels.

The orphan small nucleolar RNA (snoRNA) ACA11 is overexpressed as a result of the t(4;14) chromosomal translocation in multiple myeloma (MM), increases reactive oxygen species, and drives cell proliferation. Like other snoRNAs, ACA11 is predominantly localized to a sub‐nuclear organelle, the nucleolus. We hypothesized that increased ACA11 expression would increase ribosome biogenesis and protein synthesis. We found that ACA11 overexpression in MM cells increased nucleolar area and number as well as argyrophilic nuclear organizing regions (AgNORs). Supporting these data, samples from t(4;14)‐positive patients had higher AgNORs scores than t(4;14)‐negative samples. ACA11 also upregulated ribosome production, pre‐47S rRNA synthesis, and protein synthesis in a ROS‐dependent manner. Lastly, ACA11 overexpression enhanced the response to proteasome inhibitor in MM cells, while no effect was found in response to high doses of melphalan. Together, these data demonstrate that ACA11 stimulates ribosome biogenesis and influences responses to chemotherapy. ACA11 may be a useful target to individualize the treatment for t(4;14)‐positive myeloma patients.

Xenograft models are invaluable tools in establishing the current paradigms of hematopoiesis and leukemogenesis. The zebrafish has emerged as a robust alternative xenograft model but, like mice, lacks specific cytokines that mimic the microenvironment found in human patients. To address this critical gap, we generated the first “humanized” zebrafish that expresses human hematopoietic-specific cytokines (granulocyte-monocyte colony-stimulating factor, stem cell factor, and stromal cell-derived factor 1α). Termed GSS fish, these zebrafish promote survival, self-renewal and multilineage differentiation of human hematopoietic stem and progenitor cells and result in enhanced proliferation and hematopoietic niche-specific homing of primary human leukemia cells. Using error-corrected RNA sequencing, we determined that patient-derived leukemias transplanted into GSS zebrafish exhibit broader clonal representation compared to transplants into control hosts. GSS zebrafish incorporating error-corrected RNA sequencing establish a new standard for zebrafish xenotransplantation which more accurately recapitulates the human context, providing a more representative cost-effective preclinical model system for evaluating personalized response-based treatment in leukemia and therapies to expand human hematopoietic stem and progenitor cells in the transplant setting.

A central question in cell biology is how cells respond to stress signals and biochemically regulate apoptosis. One critical pathway involves the change of mitochondrial function and release of cytochrome c to initiate apoptosis. In response to apoptotic stimuli, we found that maspin—a noninhibitory member of the serine protease inhibitor superfamily—translocates from the cytosol to mitochondria and binds to cardiolipin in the inner mitochondrial membrane. Biolayer interferometry assay revealed that recombinant maspin binds cardiolipin with an apparent K(d),of ∼15.8 μM and competes with cytochrome c (apparent K(d) of ∼1.31 μM) for binding to cardiolipin-enriched membranes. A hydrophobic, lysine-rich domain in maspin consists of 27 aa, is located at position 268–294, and is responsible for the interaction of this protein with cardiolipin. Depletion of cardiolipin in cells significantly prevents maspin binding to the inner mitochondrial membrane and decreases cytochrome c release and apoptosis. Alteration to maspin’s cardiolipin binding domain changes its ability to bind cardiolipin, and tumor cells expressing this mutant have a low frequency of apoptosis. We propose a model of apoptosis in which maspin binds to cardiolipin, displaces cytochrome c from the membrane, and facilitates its release to the cytoplasm.—Mahajan, N., Hoover, B., Rajendram, M., Shi, H. Y., Kawasaki, K., Weibel, D. B., Zhang, M. Maspin binds to cardiolipin in mitochondria and triggers apoptosis.

Background Pediatric leukemias have a diverse genomic landscape associated with complex structural variants, including gene fusions, insertions and deletions, and single nucleotide variants. Routine karyotype and fluorescence in situ hybridization (FISH) techniques lack sensitivity for smaller genomic alternations. Next-generation sequencing (NGS) assays are being increasingly utilized for assessment of these various lesions. However, standard NGS lacks quantitative sensitivity for minimal residual disease (MRD) surveillance due to an inherently high error rate. Methods Primary bone marrow samples from pediatric leukemia (n = 32) and adult leukemia subjects (n = 5), cell line MV4–11, and an umbilical cord sample were utilized for this study. Samples were sequenced using molecular barcoding with targeted DNA and RNA library enrichment techniques based on anchored multiplexed PCR (AMP®) technology, amplicon based error-corrected sequencing (ECS) or a human cancer transcriptome assay. Computational analyses were performed to quantitatively assess limit of detection (LOD) for various DNA and RNA lesions, which could be systematically used for MRD assays. Results Matched leukemia patient samples were analyzed at three time points; diagnosis, end of induction (EOI), and relapse. Similar to flow cytometry for ALL MRD, the LOD for point mutations by these sequencing strategies was ≥0.001. For DNA structural variants, FLT3 internal tandem duplication (ITD) positive cell line and patient samples showed a LOD of ≥0.001 in addition to previously unknown copy number losses in leukemia genes. ECS in RNA identified multiple novel gene fusions, including a SPANT-ABL gene fusion in an ALL patient, which could have been used to alter therapy. Collectively, ECS for RNA demonstrated a quantitative and complex landscape of RNA molecules with 12% of the molecules representing gene fusions, 12% exon duplications, 8% exon deletions, and 68% with retained introns. Droplet digital PCR validation of ECS-RNA confirmed results to single mRNA molecule quantities. Conclusions Collectively, these assays enable a highly sensitive, comprehensive, and simultaneous analysis of various clonal leukemic mutations, which can be tracked across disease states (diagnosis, EOI, and relapse) with a high degree of sensitivity. The approaches and results presented here highlight the ability to use NGS for MRD tracking.

Acute leukemia represents the most common pediatric malignancy comprising diverse subtypes with varying prognosis and treatment outcomes. New and targeted treatment options are warranted for this disease. Patient-derived xenograft (PDX) models are increasingly being used for preclinical testing of novel treatment modalities. A novel approach involving targeted error-corrected RNA sequencing using ArcherDX HemeV2 kit was employed to compare 25 primary pediatric acute leukemia samples and their corresponding PDX samples. A comparison of the primary samples and PDX samples revealed a high concordance between single nucleotide variants and gene fusions whereas other complex structural variants were not as consistent. The presence of gene fusions representing the major driver mutations at similar allelic frequencies in PDX samples compared to primary samples and over multiple passages confirms the utility of PDX models for preclinical drug testing. Characterization and tracking of these novel cryptic fusions and exonal variants in PDX models is critical in assessing response to potential new therapies.

Background T-cell Acute Lymphoblastic Leukemia (T-ALL) / Lymphoblastic Lymphoma (LBL) represent a class of devastating hematologic cancers with high rates of relapse and mortality in both children and adults. Development of CAR-T cell therapies for T-cell cancers has been complicated by induction of fratricide and the high risk of malignant cell contamination of the drug product in the autologous setting. Previously, Cooper et. al., demonstrated that CRISPR/Cas9 gene-editing to delete CD7 prevented self-killing, and deletion of the T-cell receptor alpha constant (TRAC) enabled the use of healthy donor-derived T-cells to manufacture CD7-targeted CAR-T cells without risk of malignancy and mitigating the risk of GvHD. Here we present preclinical data supporting the safety and efficacy of WU-CART-007, an IND ready, off-the-shelf, and fratricide resistant CD7-targeted CAR-T cell for the treatment of CD7+ T-cell malignancies. Methods WU-CART-007 was manufactured using T cells isolated from healthy donors by deletion of CD7 and TRAC, followed by CAR transduction, cell expansion, depletion of residual TCRa/b+ cells and cryopreservation. Donors were confirmed negative for a panel of adventitious viruses. CD7/TRAC deletion and CAR transduction were confirmed by flow cytometry. Off-target editing profile was assessed by GUIDE-Seq. The binding kinetics to human CD7 were conducted by bio-layer interferometry and CD7 selectivity was confirmed by cell microarray with a library of HEK-293 cells expressing approximately 6000 human proteins. The in vitro activity of WU-CART-007 was interrogated by co-culture with human CD7+ CCRF-CEM T-ALL cells and the potential on-target, off-tumor activity was assessed by co-culture with a panel of immune and non-immune primary human cells. In vivo anti-tumor functionality was confirmed in immunocompromised NSG mice after the establishment of low or high tumor burden CCRF-CEM xenografts engineered to express green fluorescent protein (GFP) and click beetle red (CBR) luciferase. The impact of WU-CART-007 on normal hematopoiesis was assessed using CD34+ humanized NCG mice. Results Several successful full-scale manufacturing runs were completed with consistently high dual CD7/TRAC deletion, transduction efficiency, and cell viability. Drug product was primarily composed of a T cell memory phenotype. Off- target nuclease analysis by GUIDE-seq and targeted NGS confirmed no evidence of off-target editing events. The WU-CART-007 scFv exhibited high affinity and exquisite specificity for human CD7. In vitro co-incubation experiments confirmed strong cytotoxicity against CD7-expressing cells including CCRF-CEM T-ALL cells, primary T and NK cells, but not CD7- cells such as myeloid cells, B cells, hepatocytes, astrocytes, cardiomyocytes, epithelial cells, and endothelial cells. Importantly, no cytotoxicity was observed against hematopoietic progenitor cells in human bone marrow or cord blood following co-incubation with WU-CART-007. Similarly, WU-CART-007 treatment of a non-tumor bearing humanized mouse model resulted in transient reductions in CD7+ cells (T-cells and NK cells) but not CD7- cells (myeloid and B cells), and the impacted cells recovered after circulating WU-CART-007 cells were no longer detectable. Assessment of in vivo anti-tumor efficacy revealed that WU-CART-007 effectively inhibited tumor progression (>99% TGI) in both low and high burden CCRF-CEM tumor models and improved survival in a dose-dependent manner, while CAR- cells were inactive, confirming CD7-dependent activity. Conclusions These preclinical studies support the use of WU-CART-007 in clinical trials and highlight the potential of WU-CART-007 to be a well-tolerated and active therapy for patients with CD7+ T-cell malignancies. A first in human Phase 1/2 trial in patients with R/R T-ALL/LBL is currently open for enrollment (NCT# 04984356). Disclosures No relevant conflicts of interest to declare.

Data from high-income countries suggest that women receive less intensive diagnostic and therapeutic management than men for acute coronary syndrome (ACS). There is a paucity of such data in the Indian population, which is 69% rural and prior studies focused mostly on urban populations. The objective of the present study was to identify the gender based differences in ACS management, if any, in a predominantly rural population.Data from 35 hospitals across Himachal Pradesh covering90% of state population were collected for one year (July 2015-June 2016). A total of 2118 ACS subjects met inclusion criteria and baseline characteristics, in-hospital treatments and mortality rates were analyzed.Women constituted less than one-third of ACS population. Women were older compared to men and were more likely to present with NSTEMI/UA. Misinterpretation of initial symptoms and late presentation were also common in women. Fewer women received optimal guideline based treatment and PCI (0.9% vs 4.2%, p0.01). Compare to men, women more often had Killip class1 (27.3% vs 20.4%, p0.01) and higher in-hospital mortality (8.5% vs 5.6%, p=0.009). On multivariate analysis the association between female gender and mortality was attenuated (adjusted odds ratio [OR]=1.36 [0.77-2.38]).The present study from India, is the first of its kind to evaluate the gender based differences among ACS patients, in a predominantly rural population. Our analysis demonstrates a significant gender based difference between symptom awareness and delay in presentation, management and in-hospital outcome. Further studies are warranted across other parts of country to investigate this gender disparity.

Existing metrics for grading mitral regurgitation (MR) are limited and fraught with high interobserver variability. We developed and evaluated a Doppler-based, semiquantitative novel index (Mitral Regurgitation Severity Index [MRSI]) of MR severity. In a total of 125 patients (70 in the derivation cohort and 55 in the validation cohort), MRSI was calculated as a ratio of time velocity integral of mitral inflow (continuous-wave Doppler-TVI MV) to the time velocity integral of the left ventricle outflow (pulse-wave Doppler-TVI LVOT). Inter-rater agreement for MRSI and predictive ability of the MRSI were then assessed. In the derivation cohort, MRSI differed significantly between patients with severe MR (2.6 ± 0.51) and mild-moderate (nonsevere) MR (1.4 ± 0.18) and a cutoff of ≥1.8 was associated with optimal diagnostic accuracy. In the validation cohort, MRSI exhibited excellent agreement between a level II and a level III reader with a mean difference of -0.14 (95% confidence limit of agreement: -0.80 to 0.53), correlation coefficient of 0.88 (p

Overexpression of the multiple myeloma set domain (MMSET) Wolf-Hirschhorn syndrome candidate 1 gene, which contains an orphan box H/ACA class small nucleolar RNA, ACA11, in an intron, is associated with several cancer types, including multiple myeloma (MM). ACA11 and MMSET are overexpressed cotranscriptionally as a result of the t(4;14) chromosomal translocation in a subset of patients with MM. RNA sequencing of CD138+ tumor cells from t(4;14)-positive and -negative MM patient bone marrow samples revealed an enhanced oxidative phosphorylation mRNA signature. Supporting these data, ACA11 overexpression in a t(4;14)-negative MM cell line, MM1.S, demonstrated enhanced reactive oxygen species (ROS) levels. In addition, an enhancement of cell proliferation, increased soft agar colony size, and elevated ERK1/2 phosphorylation were observed. This ACA11-driven hyperproliferative phenotype depended on increased ROS levels as exogenously added antioxidants attenuate the increased proliferation. A major transcriptional regulator of the cellular antioxidant response, nuclear factor (erythroid-derived 2)-like 2 (NRF2), shuttled to the nucleus, as expected, in response to ACA11-driven increases in ROS; however, transcriptional up-regulation of some of NRF2’s antioxidant target genes was abrogated in the presence of ACA11 overexpression. These data show for the first time that ACA11 promotes proliferation through inhibition of NRF2 function resulting in sustained ROS levels driving cancer cell proliferation.—Mahajan, N., Wu, H.-J., Bennett, R. L., Troche, C., Licht, J. D., Weber, J. D., Maggi, L. B., Jr., Tomasson, M. H. Sabotaging of the oxidative stress response by an oncogenic noncoding RNA.

Dis3 encodes a conserved RNase that degrades or processes all RNA species via an N-terminal PilT N terminus (PIN) domain and C-terminal RNB domain that harbor, respectively, endonuclease activity and 3′–5′ exonuclease activity. In Schizosaccharomyces pombe, dis3 mutations cause chromosome missegregation and failure in mitosis, suggesting dis3 promotes cell division. In humans, apparently hypomorphic dis3 mutations are found recurrently in multiple myeloma, suggesting dis3 opposes cell division. Except for the observation that RNAi-mediated depletion of dis3 function drives larval arrest and reduces tissue growth in Drosophila, the role of dis3 has not been rigorously explored in higher eukaryotic systems. Using the Drosophila system and newly generated dis3 null alleles, we find that absence of dis3 activity inhibits cell division. We uncover a conserved CDK1 phosphorylation site that when phosphorylated inhibits Dis3’s exonuclease, but not endonuclease, activity. Leveraging this information, we show that Dis3’s exonuclease function is required for mitotic cell division: in its absence, cells are delayed in mitosis and exhibit aneuploidy and overcondensed chromosomes. In contrast, we find that modest reduction of dis3 function enhances cell proliferation in the presence of elevated Ras activity, apparently by accelerating cells through G2/M even though each insult by itself delays G2/M. Additionally, we find that dis3 and ras genetically interact in worms and that dis3 can enhance cell proliferation under growth stimulatory conditions in murine B cells. Thus, reduction, but not absence, of dis3 activity can enhance cell proliferation in higher organisms.

For decades, cytogenetic markers have played a major role in the diagnosis and classification of pediatric and adult cancers. However, many chromosomal lesions less than 50,000 base pairs (bp) are not typically detected by traditional cytogenetic studies, and these alterations are currently an area of active focus to determine the relationship with disease severity and outcome. Recent advancements in DNA sequencing technology have aided in our ability to detect numerous genomic alterations, ranging from a single-nucleotide to chromosomal-scale lesions, from a single genome. However, the routine implementation of sequencing-based strategies for the detection of minimal residual disease (MRD) has been limited because of the inherent error rate of the NGS platforms. With error rates of 0.5–2.0%, the ability to detect clinical significant remaining disease at lesser frequencies is limited. However, a variety of strategies and platforms are rapidly advancing, which will enable sequencing with the sensitivity and specificity to not only detect very rare levels of refractory or residual disease but also guide oncologists in precision therapeutic selection. This chapter details key considerations, limitations, and advancements in next-generation sequencing technologies, with a focus on error-corrected sequencing using unique molecular indexing, in the application of accurately detecting minimal residual disease.

Hypertrophic cardiomyopathy (HCM) affects the right ventricle (RV) because of the anatomically hypertrophied septum and plausibly by extension of the myopathic process to the RV. We sought to investigate RV strain in patients with left ventricular hypertrophy secondary to either HCM or hypertension (H-LVH). Our cross-sectional study included 32 patients with HCM, 21 patients with H-LVH, and 11 healthy subjects, who were evaluated with transthoracic echocardiography. Using a dedicated software package, bi-dimensional acquisitions were analyzed to measure segmental longitudinal strain in apical views. Right ventricular global longitudinal strain (GLS) was calculated by averaging septal and right free wall strains. The HCM and H-LVH groups were comparable for age and demographic characteristics. Right ventricular tricuspid annular plane systolic excursion was not significantly different between HCM and H-LVH subjects. Moreover, RV GLS, septal and lateral RV myocardial strain were significantly impaired in patients with HCM (all p0.001). Regional and global RV strain parameters were not significantly impaired in H-LVH compared to healthy controls An RV GLS cut-off value of14.9% differentiated HCM and H-LVH with a 90% sensitivity and a 95% specificity (p0.001). RV strain parameters are impaired in patients with HCM. Assessment of two-dimensional RV strain parameters could help differentiate between HCM and H-LVH.

Despite the high prevalence of rheumatic heart disease (RHD) in developing countries such as India, data on characteristics, complications, and treatment practices are lacking. The HP-RHD (Himachal Pradesh Rheumatic Heart Disease) registry aimed at reporting these parameters in patients with RHD from a northern state of India.A total of 2,005 consecutive patients of RHD were enrolled over a period of 6 years (2011 to 2016) in the present study. The clinical characteristics, complications, and treatment practices were systematically recorded.The mean age for patients with RHD was 40.3 ± 14.3 (range 5 to 83 years). RHD predominantly affected females (72.3%) and population from rural background (92%). Multivalvular involvement was frequent (43.2%), mitral valve was the commonest affected valve (83.3%). The majority of the patients had moderate-to-severe valvular dysfunction (69.3%). Mitral and tricuspid valve involvement was more frequent in female subjects compared with more frequent aortic valve involvement in male subjects (p 0.001). The major adverse cardiovascular events were recorded in 23.4% patients at the time of registry and comprised mainly advanced heart failure (15.6%), peripheral embolism (4.1%), and stroke (3.9%). The independent risk determinants of major adverse cardiovascular events (were advanced age (odds ratio [OR]: 1.01; 95% confidence interval [CI]: 1.00-1.02), severe mitral stenosis (OR: 1.73; 95% CI: 1.34-2.20), severe tricuspid regurgitation (OR: 2.11; 95% CI: 1.48-3.02), presence of pulmonary artery hypertension (OR: 1.33; 95% CI: 1.04-1.69), and atrial fibrillation (OR: 1.64; 95% CI: 1.28-2.11). Evidence-based use of oral anticoagulant therapy was documented in 77.7% of high-risk patients. Only 28.5% of study population was receiving secondary prophylaxis.Complications in patients with RHD increase with age and worsening valvular dysfunction. Programs focused on early detection and evidence-based management will assist in improving outcomes.

The current definition of peripartum cardiomyopathy (PC) is restricted to patients with left ventricular systolic dysfunction (ejection fraction [EF]45%). Data on peripartum heart failure (HF) with normal EF are sparse. We describe clinical characteristics of patients with normal (≥55%) and patients with low (45%) left ventricular ejection fractions (LVEFs). Electronic medical records (2006 to 2013) of our tertiary care center were retrospectively screened to identify peripartum HF with normal EF, defined as an entity meeting Framingham criteria for HF with symptom onset during the last month of pregnancy or up to 5 months after delivery and with an EF of ≥55%. Clinical characteristics, echocardiographic parameters, and outcomes of these patients were compared with age-matched control patients with traditionally defined PC (EF45%). A total of 25 patients with PC and EF≥55% were identified. Exclusion of hypertension (n=9), preeclampsia (n=1), and diabetes mellitus (n=2) yielded 13 patients with PC and EF≥55%. Age-matched patients with traditional PC (EF45%) constituted controls (n=16). Compared with patients with PC and low LVEF, patients with PC and normal LVEF had lower B-type natriuretic peptide levels, systolic and diastolic left ventricular dimensions, left atrial size, and incidence of decompensated HF during delivery (p0.05). Compared with historical age-matched controls, patients with normal LVEF exhibited attenuated E' mitral annular velocities. On follow-up, these patients were associated with a lower New York Heart Association functional class. In conclusion, peripartum HF with normal LVEF appears to be a distinct entity.

Precursor T-cell acute lymphoblastic leukemia (T-ALL) is a genetically heterogenous hematologic malignancy resulting from accumulation of molecular lesions in a multistep process. Although survival rates have improved considerably, event free survival for patients with T-ALL are generally inferior compared to B-cell ALL. Genetic alterations are important determinants of responsiveness to therapy and serve as targets for molecularly tailored therapies. More than 75 chimeric fusion genes have been reported in T-ALL, the majority of which encode factors involved in transcriptional regulation, while only a smaller percentage codes for tyrosine kinases. We report a case of relapsed T-ALL, despite low risk stratification at the time of diagnosis, harboring a novel fusion protein SPTAN1-ABL1. Primary bone marrow specimen collected at diagnosis was transplanted in NSG-B2m mice and propagated as a patient-derived xenograft (PDX) line. For transcriptomic characterization, RNA isolated from primary and PDX samples was subjected to error-corrected targeted next-generation sequencing using ArcherDX FusionPlex HemeV2 kit. Bioinformatics analysis identified the novel SPTAN1-ABL1 gene fusion in which exon 2 of SPTAN1 was fused with exon 4 of ABL1. This fusion was confirmed by Sanger sequencing. Translation of the fusion product sequence showed in-frame fusion leading to the generation of a chimeric protein containing N-terminal SPTAN1 and C-terminal ABL1 with intact kinase domain. SPTAN1 encodes non-erythryocytic-1-spectrin-alpha protein, an actin-binding protein, with N-terminal domain possessing oligomerization activity. Because oligomerization of ABL1 promotes its kinase activity, it is possible that SPTAN1-ABL1 possesses constitutive kinase activity. The full-length SPTAN1-ABL1 fusion protein was cloned in a mammalian expression vector and expressed in BaF3 cells. SPTAN1-ABL1 fusion was detected at similar allelic frequencies in primary and PDX samples indicating the concordance between the two. Furthermore, treatment of engrafted mice with dasatinib (Qd10, 5 mg/Kg, p.o.) significantly prolonged survival compared to untreated mice (n=5 each, P Citation Format: Anilkumar Gopalakrisnapillai, Erin Crowgey, Demetria Ruhl, Darcy Hamill, Nitin Mahajan, Todd Druley, E. Anders Kolb, Sonali P. Barwe. Identification of a novel fusion protein SPTAN1-ABL1 in a child with T-cell acute lymphoblastic leukemia: Functional characterization and therapeutic implications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-322.

Background Peripartum cardiomyopathy (PPCM) is a rare life-threatening condition of unclear etiology. Data on the use of cardiac magnetic resonance (CMR) imaging to characterize PPCM are limited. This study was done to assess the role of CMR imaging for the diagnosis and prognostication of patients with PPCM. Methods Medical records of a tertiary medical center were screened for PPCM patients with CMR imaging done within the past 5 years (2006-2011). Images were reviewed by 2 expert radiologists (blinded to clinical data) using cine sequences for chamber function and size, T2-weighted images for the determination of edema (T2-ratio), and late gadolinium enhancement (LGE) sequences for myocardial tissue characterization. Results Ten PPCM patients (aged 28 ± 6 years, 90% African American) had a total of 15 CMR examinations: 4 in the acute phase (within 7 days of diagnosis) and 11 during follow-up (median, 12 months; range, 1-72 months). Left ventricular ejection fraction was decreased in all 4 initial scans. Elevated T2 ratio (> 2) seen in 1 patient decreased on follow-up imaging. LGE was seen in 1 of the 4 acute-phase scans and in 4 of the 11 follow-up phase scans. These 4 patients had multiple readmissions because of heart failure exacerbations and persistently low left ventricular ejection fraction on subsequent echo- cardiograms. Conclusions LGE seems to be associated with a poor prognosis in the setting of PPCM. CMR imaging seems to have promising practical implications in the diagnosis and prognostication of PPCM patients.

Henoch-Schönlein purpura (HSP) is an acute systemic vasculitis with unknown etiology, although several studies have found HSP to be related to cytokines such as tumor necrosis factor α, interleukin (IL)-1, and adhesion molecules. In the present study we determined the levels of cytokines such as IL-18 and endothelin-1 (ET-1) in children with HSP. Subjects were divided into three groups (group 1, 20 subjects with HSP; group 2, 10 subjects belonging to group 1 during their follow-up 4 to 6 months later; and group 3, 16 controls who were healthy siblings of the subjects). IL-18 and ET-1 levels were determined using enzyme immunoassay and expressed as mean ± standard deviation. We observed higher IL-18 levels in children with HSP (767.6 ± 145.1 pg/mL) than in controls (614.6 ± 66.54 pg/mL, p 0.05), but IL-18 levels were found to be significantly lower in subjects with HSP in remission (502.7 ± 60.81 pg/mL) than in those who were in an active phase (1,050 ± 244.5 pg/mL, p 0.05, n = 10). ET-1 levels were found to be significantly higher in subjects with HSP (1.93 ± 0.19 pg/mL) than in controls (1.10 ± 0.13 pg/mL, p 0.05), although no significant difference was observed in ET-1 levels between subjects in group 1 (1.88 ± 0.30 pg/mL) and group 2 (1.91 ± 0.120, p 0.05, n = 10). A positive correlation was observed between IL-18 and ET-1 levels in subjects with HSP (correlation coefficient [r] = 0.5254, p 0.01). These results suggest that levels of IL-18 and ET-1 are worth monitoring during the clinical course of the disease, but caution must be exercised in extrapolating data based on small study samples.

Maspin is a member of the serine protease inhibitor (serpin) superfamily and displays tumor-suppressing activity by controlling cell migration, proliferation, apoptosis, and adhesion. Here, we provide evidence that maspin acts as a reactive oxygen species (ROS) scavenger through oxidation of three structurally exposed cysteine thiols to sulfenic acid. Ablation of these cysteine residues in maspin resulted in a significant increase in total ROS production in mouse mammary TM40D cells. Also, cells containing a triple-cysteine mutant of maspin showed elevated ERK1/2 activity, a downstream target of ROS, and enhanced proliferation and colony formation. These findings establish a novel mechanism by which maspin utilizes its cysteine thiols to inhibit oxidative stress and cell growth.

Aim: The aim is to review the English literature for post-traumatic superolateral dislocation of mandibular condyle (SDMC),discuss their dynamics and clinical management and to propose to modify the existing classification of SDMC. Patients and Methods: A literature search was carried at Pubmed, Sciencedirect, Google and references from reported articles were crosschecked to look for the cases of SDMC from 1969 to 2015 in English language. Also, we have reviewed 11 of our patients with total of 18 superolateral dislocated intact or sagittal split condyles ,who visited our unit in the previous two years. Results: In our retrospective analysis 58 cases of SDMC were found in the literature, of which 38 had intact mandibular condyles and 20 had sagittal split. Early and intact SDMC were successfully managed conservatively with closed reduction, whereas old cases and largely fractured condyles necessitated open reduction. Additionally, we observed an unusual dislocation associated with fracture of contralateral posterior mandible(angle) in our series which did not gratify the existing classification. Conclusion: Alteration of the existing classification was required to accommodate the unusual type of dislocation.

The Ets proteins are a family of transcription factors characterized by an evolutionarily conserved DNA-binding domain and have diverse biological functions including tumor suppressor as well as tumor promoter functions. They are regulated via a complex and diverse number of mechanisms and control key cellular processes. Prostate-derived Ets transcription factor (PDEF), a unique member of the ETS family, is present in tissues with high epithelial content are hormone-regulated, such as prostate, breast, salivary glands, ovaries, colon, airways, and stomach tissues. PDEF (prostate-derived Ets factor) is also referred to as SPDEF (SAM pointed domain containing Ets transcription factor), PSE (mouse homolog), or hPSE (human PSE) in the literature and is the sole member of the PDEF ETS sub-family. The role of PDEF in cancer development is still not fully elucidated though. The present article focuses on the key findings about the PDEF's biological functions, interacting proteins, and its target genes. There is a strong urge to focus on the clinical studies in larger cohort, which elucidate the regulation of PDEF and its target genes, to determine the potential of PDEF as biomarker. Based on the studies discussed in the present article, one can anticipate that PDEF offers a great potential for developing therapeutics against cancer.

Objectives The purpose of this study was to estimate the effect of long-term exposure to lower plasma low-density lipoprotein cholesterol (LDL-C) on the risk of coronary heart disease (CHD). Background LDL-C is causally related to the risk of CHD. However, the association between long-term exposure to lower LDL-C beginning early in life and the risk of CHD has not been reliably quantified. Methods We conducted a series of meta-analyses to estimate the effect of long-term exposure to lower LDL-C on the risk of CHD mediated by 9 polymorphisms in 6 different genes. We then combined these Mendelian randomization studies in a meta-analysis to obtain a more precise estimate of the effect of long-term exposure to lower LDL-C and compared it with the clinical benefit associated with the same magnitude of LDL-C reduction during treatment with a statin. Results All 9 polymorphisms were associated with a highly consistent reduction in the risk of CHD per unit lower LDL-C, with no evidence of heterogeneity of effect (I 2 = 0.0%). In a meta-analysis combining nonoverlapping data from 312,321 participants, naturally random allocation to long-term exposure to lower LDL-C was associated with a 54.5% (95% confidence interval: 48.8% to 59.5%) reduction in the risk of CHD for each mmol/l (38.7 mg/dl) lower LDL-C. This represents a 3-fold greater reduction in the risk of CHD per unit lower LDL-C than that observed during treatment with a statin started later in life (p = 8.43 × 10 −19 ). Conclusions Prolonged exposure to lower LDL-C beginning early in life is associated with a substantially greater reduction in the risk of CHD than the current practice of lowering LDL-C beginning later in life.

Takayasu's arteritis (TA) is an inflammatory fibrosing arteritis affecting predominately the aorta and its main branches. Pathogenesis of this disease remains enigmatic. Despite the numerous studies, the role of adventitia in vascular lesion formation in the setting of TA has been ignored. Virtually nothing is known about the mechanism regulating inflammation in the adventitia in the setting of TA. The present study included subjects with Takayasu's arteritis and normal healthy control subjects. Isolated T cells from peripheral blood mononuclear cells (PBMCs) using nylon wool and HUT-78 (human cutaneous T lymphoma cell line) were stimulated with PHA for 24 h. Stimulated cell were fixed with paraformaldehyde and fractionated into membrane, cytosolic and nuclear fractions. These cellular fractions were co-cultured with human fibrosarcoma cell line (HT-1080) and transcriptional expression of matrix metalloproteinases (MMP-1, 3, 9 and TIMP-1) was determined using semiquantitative RT-PCR. Stimulation of MMPs-TIMP synthesis by HT-1080 cells was mimicked by a membranous fraction derived from activated T-cell isolated from TA subjects and activated HUT-78 cells, whereas cytosolic and nuclear fractions were ineffective. In conclusion, for the first time we provide evidence for the presence of a cell surface-specific antigenic moiety on T-cells of TA subjects, which is responsible for activation of fibroblasts (cells predominantly present in adventitia) to enhance MMP production and, therefore, may lead to extracellular matrix degradation.

BACKGROUND Despite the high prevalence of rheumatic heart disease (RHD) in developing countries such as India, data on characteristics, complications, and treatment practices are lacking. The HP-RHD (Himachal Pradesh Rheumatic Heart Disease) registry aimed at reporting these parameters in patients with RHD from a northern state of India. METHODS A total of 2,005 consecutive patients of RHD were enrolled over a period of 6 years (2011 to 2016) in the present study. The clinical characteristics, complications, and treatment practices were systematically recorded. RESULTS The mean age for patients with RHD was 40.3 ± 14.3 (range 5 to 83 years). RHD predominantly affected females (72.3%) and population from rural background (92%). Multivalvular involvement was frequent (43.2%), mitral valve was the commonest affected valve (83.3%). The majority of the patients had moderate-to-severe valvular dysfunction (69.3%). Mitral and tricuspid valve involvement was more frequent in female subjects compared with more frequent aortic valve involvement in male subjects (p

Introduction Chromosomal rearrangements generating gene fusions are more common in pediatric malignancies compared to adults, and possess diagnostic, prognostic and therapeutic value. Traditionally chromosomal rearrangements including structural variants (SVs) are identified using karyotyping and fluorescence in situ hybridization (FISH); however, these methods are not sensitive for small rearrangements and single nucleotide variants (SNVs). The need to detect these cryptic SV and SNVs in pediatric cancers demands the development of assays to analyze complex RNA molecules. Methods Primary bone marrow samples obtained from Nemours Biobank were used for RNA isolation. Targeted error-corrected RNA sequencing using ArcherDX HemeV2 kit was conducted on 30 primary pediatric leukemia samples and their corresponding mouse passaged xenograft samples. RNA data (fastq) was analyzed via a custom cloud environment leveraging ArcherDx Version 5.1.3 software. The gene fusion data produced by the Archer panel was initially correlated with diagnostic FISH data available for each primary sample. Results Ten out of 30 primary bone marrow samples possessed gene fusions detected by routinely tested FISH probes for diagnostic purposes, and including ETV6-RUNX1, BCR-ABL1, TCF3-PBX1, and KMT2A. In addition, this approach detected cryptic gene fusions in 10 samples that were negative for chromosomal rearrangements via FISH, including SPTAN1-ABL1, RUNX1-MKL1, NUP98-NSD1, P2RY8-CRLF2 and TCF3-HLF. The remaining 10 samples, which did not possess detectable gene fusions, showed abnormal exon usage and domain duplications for several, key oncogenes along with novel mutations. A comparison of the primary sample and mouse passaged xenograft sample revealed that majority of gene fusions representing the abundant clone remained consistent between the primary and xenograft sample in secondary and tertiary passages. Certain gene fusions representing minor clones appeared and disappeared in xenograft samples and subsequent passages in mice in comparison to the primary patient sample, highlighting the heterogeneity of the disease. Thus, the presence of major driver mutations at similar allelic frequencies in xenografts compared to primary samples and over multiple passages confirms the utility of xenograft models for preclinical drug testing. Discussion Using a novel approach that utilizes targeted error-corrected sequencing, all the aberrations detected by clinical diagnostic testing were verified, plus several novel fusion events were identified with high confidence. This method also validated the concordance between primary and xenograft samples. Characterization of these novel cryptic fusions and exonal variants in leukemogenesis will enable identification of new drug targets and prognostic factors for pediatric leukemia. Citation Format: Sonali P. Barwe, Anilkumar Gopalakrishnapillai, Nitin Mahajan, Todd Druley, E. Anders Kolb, Erin L. Crowgey. Identification of novel fusion genes and expression variants in primary and patient-derived xenograft samples of pediatric leukemia using error-corrected RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2076.

Background Receptor for advanced glycation end products (RAGE) may play an important role in inflammatory processes and endothelial activation. Extracellular newly identified RAGE binding protein (EN-RAGE), natural pro-inflammatory ligand for RAGE. The role of C-reactive protein (CRP) as a mediator in inflammation and atherosclerosis is the subject of recent investigations worldwide. In the present study, we investigated the effect of CRP on RAGE and EN-RAGE gene expression in THP-1 monocytic cell line. MAP kinases (ERK, p38 and JNK) were exploited as possible signaling pathways involved in the signal transduction by CRP. Further, atorvastatin was used as a therapeutic modality for modulation of these genes in the presence of CRP. Materials and methods Time and dose-dependent experiments were carried out in the presence of CRP. Specific MAPK pathways inhibitors were used to elucidate the signaling pathways involved. Effect of atorvastatin was also determined in the presence of CRP on the expression of these genes. Results Time and dose-dependent experiments revealed that, treatment of THP-1 cells with 100 μg of CRP/ml/10 6 cells for 24 h, augmented the expression of RAGE and EN-RAGE genes by 2.5–3.5 folds and 3.5–4.5 folds respectively. CRP acted via FcγRII and utilized ERK, p38 and JNK pathways to transduce signals. Atorvastatin in a dose of 20 μM, was able to attenuate up-regulation of CRP-induced genes ( p Conclusion Our data strongly suggests that blockade of RAGE–EN-RAGE by statins at an early stage may prevent inflammation in atherosclerosis and counteract the harmful effects mediated by CRP.

Aim: Accelerated extracellular matrix breakdown caused by the increased activity of matrix metalloproteinases (MMPs) has been implicated in several rheumatological disorders and systemic vasculitides, especially Takayasu’s arteritis and Kawasaki disease. Therefore, the aim of the present study was to investigate the potential role of MMPs in Henoch–Schonlein purpura (HSP), an acute type of systemic vasculitis in children. Methods: We studied the activity of MMP-2 and MMP-9 in the sera using gelatin zymography and the transcriptional expression in peripheral blood mononuclear cells using semi-quantitative RT-PCR in 20 patients with HSP in acute and convalescent phase and in 20 healthy children, who were siblings of the subjects with same age group. Results: All 20 children with HSP showed increased levels of serum activity of MMP-2 and MMP9 in acute phase as compared with their convalescent phase [MMP-2 (p > 0.05); MMP-9 (p > 0.05)] and their control counterparts [MMP-2 (p < 0.001); MMP-9 (p < 0.001)]. Similarly, transcriptional expression of MMPs was found to be higher in the acute phase of HSP than in convalescent phase [MMP-2 (p < 0.05); MMP-9 (p < 0.001)] and in their healthy controls [MMP-2 (p < 0.001); MMP-9 (p < 0.01)]. Conclusion: The presence of excessive transcriptional expression and gelatinolytic activity of MMPs may be downstream to the actual aetiopathogenetic factors.