HD Samples Research Articles

A-031 Development of novel ELISAs for total GDF-15 and H-specific GDF-15

Abstract Background To develop sensitive and specific ELISAs for the quantitative measurement of total human GDF-15 and H-specific (H202D mutant) GDF-15 in human serum, and other biological fluids. Growth/differentiation Factor 15 (GDF-15) is a divergent member of the TGF-β superfamily of growth factors. It is encoded in humans by a gene in chromosome 19. The human GDF-15 gene encodes for a protein of 308 amino acid residues which consists of a signal sequence (residues 1-29), pro-domain (30-194), and mature growth factor domain (195-308). The molecular weight of a mature GDF-15 dimer is 25 kDa. Approximately 25% of humans have a missense polymorphism in the GDF-15 gene resulting in mutation of histidine 202 to aspartate (histidine 6 in the mature domain), close to the N-terminus of the mature growth factor. This variant is associated with phenotypes in prostate cancer, hyperemesis gravidarum, (severe morning sickness in pregnancy), and rheumatoid arthritis. Methods Highly specific and reproducible total GDF-15 (AL-1014-r) and H-specific GDF-15 (AL-1018-r) ELISAs have been developed using specific monoclonal antibodies to help estimate the total and mutant (DD) concentration in serum in the respective assays. The ELISAs use 10 uL sample volume and are calibrated to recombinant human GDF-15 from R & D Systems (Biotechne, USA). These ELISAs were validated for their specificity using HH, DD, HD recombinant preparations and their circulating levels in male, female, 1st, and 2nd-trimester serum samples. Results The Total GDF-15 ELISA assay detects total GDF-15 including histidine 202 to aspartate mutation (HH, HD & DD variant) in the mature domain in equimolar proportions. The H-specific GDF-15 assay is specific to histidine at 202aa in the GDF-15 sequence and does not detect histidine 202 to aspartate mutation (DD variant). Median Levels of total GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 847.9 pg/mL, 1182.4 pg/mL, 12759.7 pg/mL, and 12951.2 pg/mL. Median Levels of H-specific GDF-15 in healthy males (n=18), females (n=17), 1st trimester (n=20), and 2nd trimester (n=20) were 723.2 pg/mL, 641.3 pg/mL, 8381.4 pg/mL and 4652.1 pg/mL. Method comparison between total GDF-15 and commercial GDF-15 assay using serum samples (HH, wild type) in the range of 400-2500 pg/mL yielded a slope of 0.98 (r=0.97). The HD and DD mutant samples in the total assay recovered at twice the concentrations of the commercial assay. The total and intact assays are highly reproducible with the total coefficient of variation less than 7%. Conclusions Use of total GDF-15 assay in combination with the GDF-15 H-specific assay (DD nondetectable) ELISA can accurately estimate the mutant (DD) concentration in serum. This will enhance the knowledge of the GDF-15 measurements in the literature as the commercial assays were compromised for its immunoreactivity to the HD DD genotypes which is highly prevalent.

The study of environmental impact from fabric dyeing process in Lahanam area, Songkhone district, Lao PDR

The aim of this research was to examine water contamination resulting from the fabric dyeing process in the Lahanam area, located in Songkhone district, Lao PDR. Lahanam is a famous village known for its fabric dyeing and weaving activities, utilizing both natural and chemical dyes which result in the discharge of colors into local water resources. Water samples were collected from three different sources: household dyeing water (HD), community wastewater (CW) and the nearby river, Xebanghieng (XR). Qualitative and quantitative analyses were performed on the water samples. Atomic Absorption Spectroscopy (AAS) was employed to measure the presence of heavy metals such as lead (Pb), arsenic (As), cadmium (Cd) and mercury (Hg). Additionally, tests were conducted to determine the biological oxygen demand (BOD), dissolved oxygen (DO), ammonia contents and pH in the water of Xebanghieng. The results revealed that HD samples contained significantly higher amounts of all four heavy metals (lead 0.217 mg L–1, cadmium 0.105 mg L–1, arsenic 0.029 mg L–1 and mercury 0.0014 mg L–1), with lead, cadmium and arsenic exceeding standard limits. These data indicate potential environmental impact of chemical dyeing in the village in the future. Therefore, it is recommended to replace the current dyeing process with more sustainable alternatives, such as natural dyes. GRAPHICAL ABSTRACT HIGHLIGHTS The water quality of the river in the dyeing village in Lao PDR Heavy metals in wastewater after dyeing process. The impact of chemical dyeing.

Children with Hirschsprung disease exhibited alterations in host-microbial co-metabolism after pull-through operation.

This study aims to compare the fecal metabolome in post pull-through HD with and without HAEC patients and healthy young children using nuclear magnetic resonance (NMR) spectroscopy. Fresh fecal samples were collected from children under 5years of age in both post-pull-through HD patients and healthy Thai children. A total of 20 fecal samples were then analyzed using NMR spectroscopy. Thirty-four metabolites identified among HD and healthy children younger than 5years were compared. HD samples demonstrated a significant decrease in acetoin, phenylacetylglutamine, and N-acetylornithine (corrected p value = 0.01, 0.04, and 0.004, respectively). Succinate and xylose significantly decreased in HD with HAEC group compared to HD without HAEC group (corrected p value = 0.04 and 0.02, respectively). Moreover, glutamine and glutamate metabolism, and alanine, aspartate, and glutamate metabolism were the significant pathways involved, with pathway impact 0.42 and 0.50, respectively (corrected p value = 0.02 and 0.04, respectively). Differences in class, quantity, and metabolism of protein and other metabolites in young children with HD after pull-through operation were identified. Most of the associated metabolic pathways were correlated with the amino acids metabolism, which is required to maintain intestinal integrity and function.

Single Nuclei Multiomics Analysis of Transcriptional and Chromatin Accessibility of Tumor Cells Uncovers Molecular Signatures and Regulatory Elements of Malignant Clonal Evolution in Multiple Myeloma

Category Multiple Myeloma and Plasma Cell Dyscrasias: Basic and Translational Introduction Multiple myeloma (MM) is a hematological cancer resulting from abnormal proliferation of plasma cells (PC) in bone marrow. The progression of the disease from Monoclonal Gammopathy of Undetermined Significance (MGUS), Smoldering Multiple Myeloma (SMM), to MM involves multiple drivers and dynamic interactions of premalignant/malignant cells. In the advancement of single-cell technologies, the same-cell readouts of nucleus RNA sequencing (snRNA-seq) and Assay for Transposase-Accessible Chromatin sequencing (snATAC-seq) provide a less biased measurement of gene expressions and chromatin accessible regions. Profiling and analyzing both omics data at different stages of MM may reveal essential molecular signatures and regulatory elements for microenvironmental modification and evolution. These insights could benefit MM patients as novel targets for early therapeutic intervention at different stages of progression. Methods Bone marrow aspirates were collected from 33 individuals: Healthy donors (n=3), patients with MGUS (n=2), SMM (n=5), MM (n=21), and Plasma Cell Leukemia (PCL; n=2). Viable mononuclear cells underwent CD138+ sorting and nuclei isolation prior to library preparation. Libraries were prepared with the Chromium Multiome kit (10X Genomics) and sequenced on NovaSeq S4 flow cells at UAMS. The clonal V(D)J arrangements of each sample and expression level from HD samples were used to distinguish malignant (MM) from normal plasma cells (PC). The MM data from snATAC-seq and snRNA-seq were integrated using Signac with the weighted nearest neighbor methods from Seurat. All inter- and intra-tumor clusters were evaluated for markers of high-risk disease, differential gene expression, differentially accessible regions, and transcription factor activities. Results We performed snRNA-seq and snATAC-seq sequencing to generate multi-stage, molecular classified-specific chromatin accessibility and transcriptional profiles of plasma cell neoplasms comprising 439,134 cells across 33 samples. The malignant plasma cells for each sample were extracted by the unique clonotypic signature of the immunoglobulin gene rearrangement. Of these, we detected the highest clonality in MGUS samples, suggesting clonal competition at the premalignant stage. Based on our primary microarray data, we detected the overexpression of molecular signatures for most cells in each sample, confirming the intratumor cell heterogeneity. Furthermore, we used the ratio of 70-gene signature of aggressive disease and proliferation signatures to investigate the clonal composition of MM cells corresponding to the risk of relapse. Surprisingly, the proportion of high-risk cells increased respectively from low to high-risk samples. The expansion of high-risk clonality might exhibit competition among malignant cell subpopulations regarding microenvironmental adaptation. The multiomics integration generated less distinct clusters between MGUS and SMM, indicating the gradual complex alterations of subclonal gene expression and chromatin accessibility in malignant transformation. Interestingly, most accessible regions were found in intronic/distal intergenic regions implying the long-range gene regulation from cis-regulatory elements. Based on the positive/negative correlation of binding motifs within differentially accessible regions, we identified unique enriched transcription factors for each disease stage, suggesting the mechanism patterns in the evolutionary process of malignant cells. Conclusion In the largest multiomics study to date of CD138+ cells, we demonstrate the malignant clonal evolution using gene expression and chromatin accessibility profiling from patients with early-stage disease to highly aggressive form. Throughout the time of disease progression, we found an increase in the proportion of malignant cells with high-risk signatures. Also, we uncover unique signatures of chromatin accessibility for each molecular classification that can further refine our understanding of functional heterogeneity in multiple myeloma. Collectively, our joint single nuclei profiling reveals additional insights into the malignant clonal evolution and investigates novel regulatory elements involved in disease progression.

Early Subclones Showing Aberrant Co-Expression of Stem Cell, T Cell and Myeloid Genes Can be Detected By Flow Cytometry in MYD88 Mutated Waldenström's Macroglobulinemia Patients

Background: Our RNA-Seq analysis of 249 untreated MYD88 mutated WM patients showed aberrant expression of stem cell, pre-B-cell, T-cell and myeloid genes in early WM B clones. This expression pattern was associated with inferior progression free survival with BTK inhibitor therapy and diminished with advancing disease. We therefore sought to validate this early signature at the protein level using flow cytometry and evaluate its potential as a clinical assay to monitor WM disease evolution. Methods: RNA-Seq predictions were validated on an independent cohort of treatment naïve MYD88 mutated WM patients and healthy (HD) samples. We designed a 13-color panel for flow cytometry detection of surface markers CD19, CD138, k and l immunoglobulin (Ig) light chains, CD34, CD117 (KIT), CD4, CD8A, CD33, FCGR3A (CD16), CEACAM8 (CD66b), pre-BCR (using an antibody that recognizes a unique conformational epitope of the BCR complex formed by the association of IGLL1 and VPREB1); Zombie Yellow was used to label dead cells. Peripheral B (PBMC) and/or BM mononuclear cells (BMMC) were isolated from WM and HD samples by density gradient centrifugation using Ficoll-Paque medium. Data acquisition was performed for at least 10^6 nucleated cells per tube in BDLSR Fortessa flow cytometer using the FACSDiva software. Results: RNA-Seq analysis showed high expression of genes atypical of mature B-cells that significantly diminished with increasing disease burden. For validation purposes, we selected genes reaching at least a 10-fold drop in their expression levels between the earliest and the most advanced disease cohort. In particular, the T-cell and myeloid genes including CD4, CD8A, FCGR3A, CD33, CEACAM8 as well as the prePCR surrogate light chain genes IGLL1 and VREPB1 respectively decreased from 67.6, 7.1, 80.5, 23.2, 8.3, 114, 38.3 to 2.4, 0.9, 1.7, 0.7, 0.4, 12.2 and 4.3 transcripts per million (TpM). A subclonal stem cell signature was detected, including CD34 and KIT, which dropped from 1.67 and 0.64 to 0.15 and 0.06 TpM, respectively. It is well established that WM represents a mature, post germinal center clone. Consistent with this notion, expression of memory B-cell and plasma blast genes in our RNASeq data set, including PRDM1, XBP1 and SPIB, was high in all patients regardless of disease progression and expression of the studied atypical genes. Our independent patient validation cohort had a median age of 68 (range 50-88 years), BM involvement by immunohistochemistry of 40% (range 10-95%), hemoglobin of 13.2 (range 8.1-13.4 g/dL) and serum IgM of 686 (range 536-1,151 mg/dL). An IgMk monoclonal spike was detected in all cases. All patients were mutated for MYD88. Within the CD19+k+-selected cells, we detected a subpopulation of cells co-expressing CD34, KIT, CD4, CD8A, FCGR3A, CD33, CEACAM8 and preBCR in all the patients, including in PBMC. As predicted, these markers were relatively enriched in samples with lower BM disease burden ( Figure 1, 2). Even in those with the highest BM involvement, the subclone was still detectable in both cellular compartments. Conversely, HD controls showed normal B cell populations and no evidence of the aberrant subclone. Conclusions: In WM primary cells, we observed the existence of a clonal B cell population that appears to re-activate stem cell programming early on and progressively is replaced by clones bearing more typical B cell markers with disease expansion. This study lays the foundation to develop a widely accessible methodology to accurately diagnose IgM MGUS/early-stage WM and monitor disease evolution. Flow-based sorting of these subclones will allow targeted multi-omic studies with insights into disease pathogenesis and targeted therapeutic applications.

Influence of the Combined Magnetic Field and High Dilution Technology on the Intrinsic Emission of Aqueous Solutions

Liquids prepared by sequential multiple dilutions with mechanical action (highly diluted or HD solutions) are able to influence certain properties of adjacent solutions without direct contact, which is mediated by their emission in the infrared (IR) frequency range. These properties do not manifest when HD solutions are prepared in a geomagnetic field-free chamber. Here we studied the influence of a magnetic field and the intensity of mechanical treatment on the intrinsic emission of HD solutions of antibodies (Ab) to IFNγ and their effect on the adjacent water. IR-emission spectra were recorded using a Fourier-transform IR spectrometer. Magnetic field treatment reduced the intrinsic emission intensity of all HD samples; non-contact incubation with HD Ab prepared with intense (iHD Ab) shaking or gentle (gHD Ab) mixing reduced the emission intensity of HD water as well. The emission intensity of intact water was affected only by iHD Ab. Pre-treatment of HD Ab with a magnetic field did not modify their non-contact effect on intact water. We confirmed the presence of a non-contact effect and determined what factors it depends on (treatment with a magnetic field and the intensity of shaking when preparing HD solutions). The intensity of water emission both in the presence of HD Ab and in the presence of a magnetic field changes in a similar way.

Cerrado and Pantanal fruit flours affect gut microbiota composition in healthy and post-COVID-19 individuals: an invitro pilot fermentation study.

Cerrado and Pantanal plants can provide fruits with high nutritional value and antioxidants. This study aims to evaluate four fruit flours (from jatobá pulp, cumbaru almond, bocaiuva pulp and bocaiuva almond) and their effects on the gut microbiota in healthy (HD) and post-COVID-19 individuals (PC). An invitro batch system was carried out, the microbiota was analysed by 16S rRNA amplicon sequencing and the short-chain fatty acids ratio was determined. Furthermore, the effect of jatobá pulp flour oil (JAO) on cell viability, oxidative stress and DNA damage was investigated in a myelo-monocytic cell line. Beyond confirming a microbiota imbalance in PC, we identified flour-specific effects: (i) reduction of Veillonellaceae with jatobá extract in PC samples; (ii) decrease in Akkermansia with jatoba and cumbaru flours; (iii) decreasing trend of Faecalibacterium and Ruminococcus with all flours tested, with the exception of the bocaiuva almond in HD samples for Ruminococcus and (iv) increase in Lactobacillus and Bifidobacterium in PC samples with bocaiuva almond flour. JAO displayed antioxidant properties protecting cells from daunorubicin-induced cytotoxicity, oxidative stress and DNA damage. The promising microbiota-modulating abilities of some flours and the chemopreventive effects of JAO deserve to be further explored in human intervention studies.

DNA methylation of mitochondrial DNA shows variation in human brain.

AbstractBackgroundThere is growing evidence for the role of DNA methylation in regulating the transcription of mitochondrial genes, particularly in neurodegenerative disorders characterized by mitochondrial dysfunction including Alzheimer’s disease (AD). However, to date, a cross‐comparative analysis of the mitochondrial DNA methylome in neurodegenerative disorders has yet to be undertaken.MethodHere, we present an interrogation of the mitochondrial DNA methylome at single base resolution, using pyrosequencing, across different types of neurodegenerative disorders. We performed a targeted study design to investigate the D‐Loop methylation of the mtDNA in the entorhinal cortex (EC) for a pilot cohort of 26 AD, 22 Dementia with Lewy bodies (DLB) and 26 control samples, matched as closely as possible for age and sex. This research forms the basis of a larger study which will compare D‐Loop methylation in several brain regions including the EC, superior temporal gyrus and cerebellum in AD, DLB, Vascular dementia, Huntington’s (HD) and Parkinson’s disease (PD) samples. The striatum and substantia nigra, will also be analyzed in the HD and PD samples respectively.ResultWe have identified DNA methylation differences at the D‐Loop in different neurodegenerative diseases. In particular, we have found two statistically significant sites that show a decrease in percentage methylation of approximately 4% and 3% in the EC of the DLB brain samples compared to controls.ConclusionWe have discovered differences in DNA methylation across the mitochondrial genome between different types of neurodegenerative disorders in human brain samples using pyrosequencing. Moving forward we will take this approach and expand into the larger cohort to further investigate the role of mitochondrial epigenetic mechanisms in neurodegenerative disorders.

DNA methylation profiling across brain regions in Huntington’s disease.

AbstractBackgroundHuntington’s disease (HD) is an autosomal dominant condition that occurs due to the expansion of a CAG trinucleotide repeat in the Huntingtin (HTT) gene. The translated HTT protein has an expanded polyglutamine sequence thought to cause deleterious effects. These effects cause severe neurodegeneration in the striatum. In HD there is also clear atrophy in the cerebellum and a reduction in cortical thickness, including in the entorhinal cortex (EC). Epigenome wide association studies (EWAS) of DNA methylation differences in HD have thus far been limited to the cerebral cortex and use older technologies such as the Illumina 450K array. In this study, we performed an EWAS of HD in the striatum, EC and cerebellum, using the Illumina EPIC methylation array to profile 850, 000 sites across the genome.Method120 striatum, EC and cerebellum DNA samples from 22 control and 20 HD subjects were selected and matched for sex, age and post‐mortem interval. The samples were chosen to be as free from co‐existing pathologies as possible. The majority of subjects were represented in each brain region. The DNA was bisulfite converted, randomized and profiled using the EPIC array. The methylation intensities for each sample were subjected to quality control (QC). Following QC, the samples were analyzed in R for differences in methylation between HD and control samples within each brain region, before being subjected to cross‐regional analysis.ResultWe have used linear regression models to identify differentially methylated positions (DMPs) in specific brain regions in HD. We have used regional analyses to identify differentially methylated regions (DMRs) consisting of multiple adjacent DMPs. Finally, downstream analyses have highlighted epigenetically altered pathways in disease.ConclusionThis study builds a clearer picture of DNA methylation profiles in HD in disease relevant brain regions. Future studies should integrate this with other levels of genomic regulation.

Pretreatment Method for Chloramine-T Decon Sample Before GC Analysis of HD and VX.

Chloramine-T, especially its solution in weak acidity, is one of the decontaminants for chemical warfare agents (CWAs), HD, and VX. A high CWAs recovery from decontamination (decon) sample via pretreatment was essential for evaluating decontamination effects. This paper performed experiments to optimize pretreatment methods to extract residual CWAs from chloramine-T decon samples before GC analysis. Effects of two neutralization methods, destroying decon activity by 15% Na2SO3 or decreasing decon activity by 3% NH3·H2O or 4% NaOH, were studied. Results showed they were all suitable for the HD decon sample, but only 4% NaOH was ideal for the VX decon sample. As for extractant, compared with dichloromethane, petroleum ether was more suitable for recovering CWAs from decon samples. A high recovery above 80% could be obtained for HD and VX samples ranging from 10mg/L to 10,000mg/L when optimized neutralization and extraction methods were simultaneously carried out.

Effect of Sodium Hydroxide, Succinic Acid and Their Combination on Densified Wood Properties

The effect of NaOH, succinic acid and their combination used as pretreatments on selected properties of densified wood samples of pisang putih (Mezzettia spp.), nyatoh (Palaquium spp.) and sepetir (Sindora spp.) has been studied. The properties of these samples were then compared with those of untreated wood (control) and densified wood samples without any pretreatment (HD wood). For alkali-pretreatment, after immersion in 10% NaOH (w/w) for 1, 2 and 3 h, the samples were immediately compressed; for acid-pretreatment, the sample was impregnated with 10% succinic acid (w/w) using a vacuum of −50 kPa for 30 min and a pressure of 7 bar for 2 h before pressing; while for the combination-pretreatment, the sample was initially immersed in 10% NaOH then impregnated with 10% succinic acid (w/w) and finally densified/compressed. Compression was applied in a radial direction and carried out in an open system using a hot press machine at 180 °C and 7 MPa specific pressure for 30 min with a 40% compression ratio. The results showed that densified wood with or without pretreatment had a higher specific gravity (SG) than the control, whereas sepetir wood showed the greatest increase in SG. The alkali- and combination-pretreatments resulted in a higher C-set value than in the control, HD and acid-pretreated samples. Acid-pretreatment resulted in the best improvement in dimensional stability compared to the other pretreatments. Increasing the immersion time in NaOH solution was proven to be able to increase dimensional stability both in alkali- and combination-pretreatments, although water absorption values varied.

Single-Cell RNA Sequencing Analysis Reveals Mechanisms of Initiation and Progression in Chronic Myelomonocytic Leukemia

Despite advances in the genetic characterization of chronic myelomonocytic leukemia (CMML), the molecular mechanisms that drive the disease during its distinct phases remain unclear. To uncover vulnerabilities in CMML that could be therapeutically targeted to halt its evolution, we sought to dissect at the single-cell level the cellular and transcriptomic changes that occur in the hematopoietic system at the time of CMML's initiation and its progression after hypomethylating agent (HMA) therapy.To evaluate the molecular mechanisms underlying CMML maintenance, we performed single-cell RNA sequencing (scRNA-seq) analysis of lineage-negative (Lin -)CD34 + hematopoietic stem and progenitor cells (HSPCs) and bone marrow (BM) mononuclear cells (MNCs) isolated from untreated CMML patients (n=5 and 6, respectively) and age-matched healthy donors (HDs; n=2 and 3, respectively). Our integrated analysis revealed that CMML Lin -CD34 + HSPCs had a predominant granulomonocytic differentiation route with an increased frequency of early and committed myeloid-monocytic progenitors at the expense of HSCs and megakaryocyte/erythroid progenitors (Fig. 1a). Differential expression analysis among the clusters revealed that most transcriptomic differences occurred in CMML HSCs, which were characterized by the upregulation of genes involved in oxidative phosphorylation, type I interferon (IFN) and IFNγ response, myeloid development, and inflammatory signaling and had downregulated expression of genes involved in TNFα-mediated NF-κB activation (Fig. 1b). These data suggest that CMML HSCs undergo metabolic reprogramming and demand a higher level of mitochondrial activity to maintain activated monocytic differentiation in response to inflammatory signaling.Consistent with these results, scRNA-seq analysis of MNCs isolated from the same HD and CMML BM samples showed that monocytes were significantly increased at the expense of erythroid precursors and B cells in CMML (Fig. 1c). CMML monocytes had upregulated genes involved in IFNγ response, oxidative phosphorylation, MYC targets, NF-κB activation, and inflammation (e.g., S100A9, CCL3, IL1B). Interestingly, among the anti-apoptotic BCL2 family members, only the NF-κB transcriptional target BCL2A1 was significantly overexpressed.To investigate the mechanisms of resistance to HMA therapy, we performed integrated scRNA-seq analysis of sequential Lin -CD34 + cells and BM MNCs isolated from CMML patients at the time of disease initiation and progression after HMA therapy failure. CMML progression was driven by a significant expansion of lympho-myeloid progenitors (LMPPs) at the expense of earlier HSCs , which exacerbated myelomonocytic differentiation in the HSPC compartment (Fig. 1d). Expanded LMPPs were characterized by higher levels of IFNγ response, NF-κB survival signaling, and cell cycle regulators. Accordingly, scRNA-seq analysis of MNCs cells from the same patients showed significantly increased frequencies of monocytes and a reduction of naïve CD4 +/CD8 + T cells and effector memory CD8 + T cells. Differential expression analysis of the 2 sample groups in the monocyte population identified five different cellular clusters, one of which emerged only at progression (Fig. 1e). This population was characterized by high expression levels of inflammatory cytokines and the anti-apoptotic modulators MCL1 and BCL2A1. Together, these data suggest that CMML progression arises from immature myeloid progenitors at the stem cell level and that downstream monocytes undergo transcriptomic rewiring and acquire survival mechanisms that induce therapy resistance and further accelerate disease progression.In conclusion, our results elucidate the differentiation hierarchies and transcriptional programs associated with CMML's initiation and its progression after HMA therapy. Our data suggest that therapies targeting downstream effectors of NF-kB-mediated survival signaling could overcome treatment failure. [Display omitted] DisclosuresWei: Daiichi Sanko: Research Funding. Kantarjian: AbbVie: Honoraria, Research Funding; Immunogen: Research Funding; KAHR Medical Ltd: Honoraria; Jazz: Research Funding; Ipsen Pharmaceuticals: Honoraria; Astellas Health: Honoraria; NOVA Research: Honoraria; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Astra Zeneca: Honoraria; Ascentage: Research Funding; Aptitude Health: Honoraria; Daiichi-Sankyo: Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Precision Biosciences: Honoraria; Taiho Pharmaceutical Canada: Honoraria.

Effects of Stocking Density on Stress Response and Susceptibility to Infectious Hematopoietic Necrosis Virus in Rainbow Trout.

The goals of this study were to examine the effect of stocking density on the stress response and disease susceptibility in juvenile rainbow trout (Oncorhynchus mykiss). Fish were sorted into one of 2 stocking densities (high density "HD", 20-40 kg/m³) or (low density, "LD", 4-8 kg/m³) and 3 stress indices (cortisol levels in serum and water, and neutrophil: lymphocyte (N:L) ratios from blood smears) were measured at multiple time points over 21 d. Serum cortisol was significantly increased at 1 h in LD samples and at 14 d in HD samples. Water cortisol concentrations were significantly higher in LD tanks as compared with HD tanks on day 14. N:L ratios were significantly higher in HD tanks on day 14 as compared with LD tanks and with baseline. The effect of stocking density on mortality after exposure to infectious hematopoietic necrosis virus (IHNV) was compared between fish held in HD or LD conditions, with or without prior acclimation to the different density conditions. No significant differences in survival were found between HD and LD treatments or between acclimated and nonacclimated treatments. Cumulative results indicate that 1) 1 to 4 gram rainbow trout did not generally demonstrate significant differences in stress indices at the density conditions tested over a 21-d period, 2) independent differences were found in 3 stress indices at day 14 after sorting into LD and HD holding conditions; and 3) LD and HD stocking densities did not have a significant effect on mortality due to IHNV.

Effect of Co and Zr content on the magnetic properties of SmFe11Ti-based alloys produced by hydrogenation-disproportionation-desorption-recombination

The recent increasing demand for magnetic materials for applications such as electric motors in electric vehicles, as well as issues with restrictions to the supply of certain rare-earth elements, has led to renewed interest in SmFe-based alloys. The SmFe12-type crystal structure is especially desirable due to its high saturation magnetisation, but it is difficult to obtain this metastable phase on its own. Co, Zr, and Ti substitutions have been found to improve the magnetic properties and stabilise the hard magnetic Sm1Fe11Ti1 phase, while melt spinning and the hydrogenation disproportionation desorption recombination (HDDR) process have been found to produce fine grain size, and thus high coercivity. Therefore, in this work the effect of Co and Zr content on the magnetic properties of melt-spun and HDDR processed (Sm1-xZrx)(Fe1-yCoy)11.2Ti0.8 samples has been examined, where 0 ⩽ x ⩽ 0.3 and 0 ⩽ y ⩽ 0.25. Melt-spun, HD, and DR samples were analysed to determine the effect of varying the Co or Zr content. Amorphous Sm1Fe11Ti1 phase was found in all melt-spun samples. Co was found to exist in equal amounts in the α-(Fe, Co) and (Fe, Co)2(Ti, Zr) phases in the HD treated samples, while Zr existed mostly within the (Fe, Co)2(Ti, Zr) phase. The crystal size of the α-Fe or α-(Fe, Co) phase was shown to increase with increasing Co content but decreased with increasing Zr content. Increasing Co content generally improved Mr and Ms in the DR treated samples, whereas increasing Zr content was generally detrimental to Mr, Ms, and Hcj. However, each sample had a different fraction of the Sm1Fe11Ti1 phase, and higher Sm1Fe11Ti1 phase fractions were found to increase the Mr, Ms, and Hcj. Values of Mr, Ms, and Hcj corrected for the fraction of Sm1Fe11Ti1 phase were highest in the (Sm0.8Zr0.2)(Fe0.8Co0.2)11.2Ti0.8 sample. This composition should be investigated further to determine if superior magnetic properties can be produced, by optimising the DR treatment conditions to obtain 100 % Sm1Fe11Ti1 phase.

Simultaneous measurement of 16 bisphenol A analogues in house dust and evaluation of two sampling techniques

Bisphenol A (BPA) is a high production volume (HPV) chemical used to make polycarbonate (PC) plastics and epoxy resins. Due to its potential adverse health effects, BPA has been restricted or banned in some products and thus many alternatives to BPA have been introduced into the market. Most of these alternatives such as bisphenol S (BPS), bisphenol F (BPF), and bisphenol M (BPM), have similar chemical structures to BPA and termed as BPA analogues (BPAAs). In this study, a method was developed for the simultaneous determination of 16 BPAAs in indoor dust by gas chromatography coupled with tandem mass spectrometry (GC-MS/MS). The method was sensitive, with method detection limits (MDLs) ranging from 0.001 μg/g to 0.017 μg/g. The method demonstrated good recoveries (>80%) for the majority of the target compounds and was applied to the analysis of dust samples collected from 2007 to 2010 in 13 cities across Canada under the Canadian House Dust Study (CHDS). Samples were collected using two different techniques: household vacuum dust samples collected from household vacuum bags (HD); fresh vacuum dust (FD) samples collected by a trained technician from dry floor surfaces. In addition to BPA with 100% detection in 119 HD samples, BPS, BPF, and BPM were detected with high detection frequencies (>75%) with median (range) concentrations of 0.242 (<0.017–35.1) μg/g, 0.123 (<0.007–2.67) μg/g, and 0.028 (<0.005–0.908) μg/g, respectively, suggesting their wide use in various applications. Other BPAAs were not detected or detected with very low frequencies and at low levels. This study shows that BPAA concentrations in dust sampled from household vacuum bags compare well with concentrations observed in fresh dust collected from the same homes, suggesting that HD sampling could be a cost-effective alternative to FD sampling. The method was also applied to the analysis of a standard reference material – NIST SRM 2585 (organic contaminants in house dust). So far there are no certified values for BPAAs in SRM 2585. In this study only three BPAAs were detected in SRM 2585, with mean values of 34.2 (±2.3) μg/g for BPA, 1.88 (±0.22) μg/g for BPS, and 0.297 (±0.052) μg/g for BPF.

Short-chain and medium-chain chlorinated paraffins in Canadian house dust and NIST SRM 2585.

A method for the analysis of short-chain and medium-chain chlorinated paraffins (SCCPs and MCCPs) in house dust was developed. The method is based on sonication extraction, sample cleanup by solid phase extraction (SPE), and separation and detection by gas chromatography coupled with mass spectrometry (GC/MS) operated in electron capture negative ion (ECNI) chemical ionization mode. The method is sensitive, with method detection limits (MDLs) down to 0.22μg/g for SCCPs and 0.55μg/g for MCCPs. The overall recoveries of the method were 104 (± 11)% and 108 (± 16)% for SCCPs and MCCPs, respectively. The method was successfully applied to the analysis of SCCPs and MCCPs in NIST standard reference material (SRM 2585, organic contaminants in house dust) and a subset of house dust samples collected under the Canadian House Dust Study (CHDS). Average concentrations of SCCPs and MCCPs in SRM 2585 (n = 12 replicates) were 7.58 (± 0.43) μg/g for SCCPs and 16.4 (± 2.1) μg/g for MCCPs, respectively. A comparison was made between CP concentrations in paired dust samples collected using two different methods from the same homes: fresh or "active" dust (FD) collected by technicians and a sample taken from the household vacuum cleaner (HD). Spearman rank analysis showed a significant positive correlation (p < 0.01) between FD and HD samples for both MCCPs and SCCPs. CPs were detected in every house dust sample (n = 48 HD samples), with median (range) concentrations of 6.2 (4.0 - 57) μg/g and 19 (5.9-901) μg/g for SCCPs and MCCPs, respectively. Widely scattered CP levels and 100% detection frequency in this preliminary set of 48 HD samples suggest a wide variability in Canadian household exposures to CPs.

Application of MootralTM Reduces Methane Production by Altering the Archaea Community in the Rumen Simulation Technique.

The reduction of methane emissions by ruminants is a highly desirable goal to mitigate greenhouse gas emissions. Various feed additives have already been tested for their ability to decrease methane production; however, practical use is often limited due to negative effects on rumen fermentation or high costs. Organosulphur compounds from garlic (Allium sativum) and flavonoids have been identified as promising plant-derived compounds which are able to reduce methane production. Here, we evaluated the effects of a combination of garlic powder and bitter orange (Citrus aurantium) extracts, Mootral, on ruminal methane production, ruminal fermentation and the community of methanogenic Archaea by using the rumen simulation technique as ex vivo model. The experiment consisted of an equilibration period of 7 days, an experimental period of 8 days and a withdrawal period of 4 days. During the experimental period three fermenters each were either treated as controls (CON), received a low dose of Mootral (LD), a high dose of Mootral (HD), or monensin (MON) as positive control. Application of Mootral strongly reduced the proportion of methane in the fermentation gas and the production rate of methane. Moreover, the experimental mixture induced a dose-dependent increase in the production rate of short chain fatty acids and in the molar proportion of butyrate. Some effects persisted during the withdrawal period. Both, single strand conformation polymorphism and Illumina MiSeq 16S rRNA amplicon sequencing indicated an archaeal community distinct from CON and MON samples in the LD and HD samples. Among archaeal families the percentage of Methanobacteriaceae was reduced during application of both doses of Mootral. Moreover, several significant differences were observed on OTU level among treatment groups and after withdrawal of the additives for LD and HD group. At day 14, 4 OTUs were positively correlated with methane production. In conclusion this mixture of garlic and citrus compounds appears to effectively reduce methane production by alteration of the archaeal community without exhibiting negative side effects on rumen fermentation.

Expanding the number of phthalates monitored in house dust

ABSTRACTPhthalates have been used as plasticisers for several decades in various industry and consumer products. A method was developed for the determination of 13 not commonly monitored phthalates in household dust. The method was based on solvent extraction using sonication, sample clean-up by solid phase extraction (SPE), and analysis using isotope dilution gas chromatography-tandem mass spectrometry (GC/MS/MS). The method was applied to the analysis of dust samples collected using two vacuum sampling techniques from 38 urban Canadian homes: a sample of fresh or ‘active’ dust (FD) collected by technicians and a composite sample taken from the household vacuum cleaner (HD). Spearman rank correlations between HD and FD samples were significant for six phthalates with median concentrations above their method detection limits (MDLs), suggesting that the HD samples provide comparable results with FD samples. Seven phthalates were detected and quantified in a Canada-wide set of 126 household dust samples, among which six phthalates were detected at frequencies higher than 87%, with median (range) concentrations of 1.9 (<0.42–240) (μg/g) for diisohexyl phthalate (DIHxP), 3.8 (<0.16–260) (μg/g) for di-n-heptyl phthalate (DHepP), 6.6 (<1.1–1170) (μg/g) for diisooctyl phthalate (DIOP), 1.1 (<0.12–390) (μg/g) for di-n-octyl phthalate (DOP), 6.3 (<0.16–430) (μg/g) for dinonyl phthalate (DNP), and 1.8 (<0.18–850) (μg/g) for di-n-decyl phthalate (DDP). High detection frequencies and widely scattered concentration levels of these phthalates in this preliminary set of 126 samples suggested a high variability in potential exposure to phthalates in Canadian homes. NIST SRM 2585 (organic contaminants in house dust) was also analysed; eight phthalates were detected, with concentrations ranging from 6.0 μg/g for DOP to 79 μg/g for DIHxP. The results from SRM 2585 may contribute to the certification of phthalate concentration values in this SRM.

An intelligentized strategy for endogenous small molecules characterization and quality evaluation of earthworm from two geographic origins by ultra-high performance HILIC/QTOF MS(E) and Progenesis QI.

Animal-derived medicines have been a vital component for traditional Chinese medicine. However, their quality control remains challenging due to the large polarity of the contained endogenous small molecules (ESMs) that are difficult to separate by reversed-phase HPLC. Herein, an intelligentized strategy by ultra-high performance hydrophilic interaction chromatography/quadrupole time-of-flight mass spectrometry (HILIC/QTOF-MS(E)) is presented, and used for the ESMs characterization and differentiation of two geographic origins of earthworm (Guang Di-long, GD; Hu Di-long, HD) as a case study. Chromatographic separation was performed on a BEH Amide column (2.1 × 100mm, 1.7μm). The MS(E) data in both negative and positive ion modes were acquired to record the high-accuracy MS and MS/MS data of all precursor ions. Automatic data processing was enabled by use of Progenesis QI software. As a consequence, 926 metabolites among 4705 features and 761 among 3418 features were characterized in the negative and positive modes, respectively, by searching the human metabolome database (HMDB). To reduce the false positive identifications, structural confirmation was conducted by comparison with the reference standards (tR and MS, MS/MS data) or matching with theoretical data or commercial library. Principal component analysis (PCA) of the GD and HD samples showed distinct classifications. Further orthogonal partial least squares discriminant analysis (OPLS-DA) and variable importance in projection (VIP) plot revealed the potential discriminatory markers between GD and HD. The present study provides a powerful and practical strategy that facilitates the primary metabolites characterization and quality evaluation of animal-derived medicines more efficiently. Graphical Abstract A general flowchart illustrating the application of ultra-high performance HILIC/QTOFMS(E) coupled with data processing by Progenesis QI to characterization of the endogenous small molecules and discriminatory analysis of animal-derived traditional medicine: earthworm as a case study.

Coral transcriptome and bacterial community profiles reveal distinct Yellow Band Disease states in Orbicella faveolata.

Coral diseases impact reefs globally. Although we continue to describe diseases, little is known about the etiology or progression of even the most common cases. To examine a spectrum of coral health and determine factors of disease progression we examined Orbicella faveolata exhibiting signs of Yellow Band Disease (YBD), a widespread condition in the Caribbean. We used a novel combined approach to assess three members of the coral holobiont: the coral-host, associated Symbiodinium algae, and bacteria. We profiled three conditions: (1) healthy-appearing colonies (HH), (2) healthy-appearing tissue on diseased colonies (HD), and (3) diseased lesion (DD). Restriction fragment length polymorphism analysis revealed health state-specific diversity in Symbiodinium clade associations. 16S ribosomal RNA gene microarrays (PhyloChips) and O. faveolata complimentary DNA microarrays revealed the bacterial community structure and host transcriptional response, respectively. A distinct bacterial community structure marked each health state. Diseased samples were associated with two to three times more bacterial diversity. HD samples had the highest bacterial richness, which included components associated with HH and DD, as well as additional unique families. The host transcriptome under YBD revealed a reduced cellular expression of defense- and metabolism-related processes, while the neighboring HD condition exhibited an intermediate expression profile. Although HD tissue appeared visibly healthy, the microbial communities and gene expression profiles were distinct. HD should be regarded as an additional (intermediate) state of disease, which is important for understanding the progression of YBD.