PB Samples Research Articles

Identification of pancreatin inhibitors from Thai medicinal Piper plants for antidiabetic and anti-obesity activities using high-performance thin-layer chromatography-bioautographic assay

Exploring the potential of natural products against diabetes and obesity is in demand nowadays. Pancreatic α-amylase and pancreatic lipase are the drug targets to minimize the absorption of glucose from starch and fatty acids from lipids, respectively. In this study, five Piper species, namely P. sarmentosum (Ps), P. wallichii (Pw), P. retrofractum (Pr), P. nigrum (Pn), and P. betle (Pb), which are commonly used as food ingredients and traditional medicines, were evaluated for their inhibitory activities against pancreatin using the microtiter plate method. Additionally, pancreatin inhibitors were identified through a cost-effective high-performance thin-layer chromatography (HPTLC)-bioautography developed using red starch and p-nitrophenyl palmitate, corresponding to anti-amylase and -lipase activities, respectively. Of the 15 samples tested, leaf samples from Pb, which had the highest total phenolic and total flavonoid contents, exhibited remarkable inhibitory activity against pancreatin, with a relative amylase inhibitory capacity (RAIC) ranging between 4.260 × 10−5 and 4.861 × 10−5 and a reciprocal half-maximal inhibitory concentration (1/IC50, PTL) of 0.390–0.510 (mg/mL)−1. Additionally, Ps samples demonstrated the second-ranked anti-pancreatin activity. Principal component analysis indicated that total phenolic content contributed to the anti-pancreatin activities of Pb samples. The anti-pancreatin bands were isolated and identified as caffeic acid, myricetin, genistein, piperine, and eugenol. Myricetin, in the roots of Ps samples, showed notable anti-pancreatin activity, which was consistent with results from the in silico prediction toward pancreatic α-amylase and pancreatic lipase. Caffeic acid and eugenol were present in Pb samples. In conclusion, the developed cost-effective pancreatin HPTLC-bioautography efficiently identified amylase and lipase inhibitors from Piper herbs, which supported the use of these plants for antidiabetes and anti-obesity.

Enhancing mechanical and microstructural properties of Pb composite anode by the addition of W-Co3O4 ceramic particles fabricated by Accumulative Roll Bonding

In this research, the accumulative roll bonding (ARB) technique was employed as a severe plastic deformation method to manufacture a unique bi-metal Pb/W composite reinforced by Co3O4 ceramic oxide. For this purpose, composites with different amounts of reinforcement particles (0.25, 0.5, 1, 1.5, and 2 wt%) were successfully produced using the ARB process (up to 10 cycles). Also, the mechanical properties were evaluated using microhardness, tensile, and shear punch tests. For microstructural investigations, the X-ray diffraction method (XRD), transmission electron microscopy (TEM), and fractography analysis by scanning electron microscopy (SEM) were employed. The Pb-0.5%W/Co3O4 following 10 ARB cycles exhibited optimal mechanical and microstructural properties. For this composite, the tensile strength showed a markable increase of 2.17 times, yield strength by 4.7 times, shear strength by 3.6 times, and hardness by 4.2 times compared to the pure Pb sample subjected to the same ARB cycles. However, the strain was reduced by 3.85 times. These enhanced properties are also followed by increased stiffness and improved anode dimensional stability, creep resistance, and operational lifespan.

Effect of metal chalcogenides on modifying the structural and optical properties of ZnWO4 nanostructure

Hydrothermal and thermolysis techniques were used to create ZnWO4 and 0.9ZnWO4-0.1MS (M = Cd, Zn, Pb) samples. The formed phases in each sample and their structure and microstructure were examined using Raman spectroscopy, Fourier-transform infrared spectroscopy and Rietveld refinement of Synchrotron x-ray diffraction data. The composition and morphology of the formed samples were investigated via scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDS). The average crystallite size remains relatively constant regardless of the different metal chalcogenides. MS/ZnWO4 samples have two distinct morphologies. The UV diffuse reflectance spectroscopy (UV-DRS) technique is used to investigate the linear and nonlinear optical parameters for different samples. Alloying ZnWO4 with CdS or PbS affects the photo response region of the sample to include visible light. MS/ZnWO4 (MS=CdS, ZnS, PbS) samples have optical band gap values of 2.56, 2.43, 2.99 and 3.1 eV, respectively. The CdS/ZnWO4 and ZnS/ZnWO4 samples exhibit the highest refractive index and optical dielectric constant values within the visible range. CdS or PbS/ZnWO4 samples show the highest nonlinear optical parameters values within the visible range. The impact of MS samples on the photoluminescence emission of ZnWO4 was explored. The CIE chromaticity diagram reveals that MS/ZnWO4 (M = Zn and Pb) samples display a different degree of blue color while CdS/ZnWO4 sample reveals green-yellow colors.

Kinetics and Biology of Circulating Tumor Cells (CTCs) and Measurable Residual Disease (MRD): Two Dynamic High-Risk Clones in Multiple Myeloma (MM)

BACKGROUND CTCs and MRD are two small clones associated with a high risk of progression in smoldering MM (SMM) and post treatment relapse in active MM. Despite growing knowledge about their clinical significance and biological features, these questions remain unanswered: 1) What is the prognostic value of CTC and MRD kinetics? 2) Is MM dissemination and on-treatment resistance driven by specific genomic alterations? 3) Are CTCs and MRD genetically related? AIM Analyze the kinetics and molecular features of CTCs and MRD in SMM and MM patients. METHODS CTC kinetics were investigated using NGF in 164 SMM patients with ≥3 assessments collected every 6 months in the iMMunocell study. MRD kinetics were investigated using NGF in 277 MM patients with ≥4 assessments in BM aspirates after induction, transplant, consolidation, and yearly during maintenance (GEM2012MENOS65-GEM2014MAIN trials). Unsupervised clustering based on CTC and MRD kinetics was performed using CONNECTOR. Molecular features of BM tumor cells at diagnosis were compared to paired CTCs in 94 SMM and MM patients and to paired MRD in 96 MM patients. In 10 cases, genomic data from tripartite BM tumor cells, CTCs, and MRD was available. Tumor samples were isolated based on patient-specific aberrant phenotypes using FACS. Whole exome and RNA-seq were performed in 343 and 217 samples, respectively. RESULTS Based on the assessment of CTCs in 754 PB samples, SMM patients clustered into 3 groups: sustained undetectable (n = 20), stable (n = 71) and evolving (n = 73) CTC levels. With a median follow-up of 2 years, the respective 2y progression-free rates were 100%, 94% and 81% ( P =.003). Risk stratification based on CTC kinetics (C-index 0.68) outperformed a single baseline assessment (C-index 0.61) and, along with the IMWG 20/2/20 model, had independent prognostic value (HR 3.4; P = .01). Patients with high risk cytogenetics showed more frequently evolving CTC levels. Studying genetic drivers of tumor egress, we found CTCs to closely resemble BM tumor cells (88% concordance at copy number [CNA] and somatic mutational [SNV] level). No recurrent cell-specific SNV or CNA was identified. CTC's transcriptional profile revealed 268 DEGs enriched in interferon alpha, gamma response and cell-adhesion pathways. Next, the prognostic value of MRD dynamics was analyzed according to 1,759 assessments, identifying 3 patient clusters: sustained undetectable (n = 100), stable (n = 104), and evolving (n = 73) MRD levels. Respective 6-year rates of PFS after consolidation were 90%, 75% and 5% ( P <.001), and of OS were 94%, 95% and 66% ( P<.001). Risk stratification based on MRD kinetics (C-index 0.82) outperformed a single MRD assessment before maintenance (C-index 0.62), the R-ISS (C-index 0.57) and R2-ISS (C-index 0.61) at diagnosis. MRD cells showed heterogeneous genomic evolution (60% median concordance) diverging from BM tumor cells at diagnosis due to de novo CNA and SNV that emerged at a subclonal level, particularly after transplant. After induction, MM driver mutations were detected or remained clonal in 13/19 (68%) MRD, whereas after transplant, this was true for only 5/13 (38%) MRD. No recurrent cell-specific SNV or CNA was identified. The transcriptional signature of MRD was treatment dependent, with common molecular features of on-treatment resistance found in all 96 patient-paired samples analyzed after various treatment stages. The 334 DEGs were commonly enriched in KRAS signaling hallmarks. Genomic profiles of tripartite BM tumor cells, CTCs and MRD from 10 MM patients confirmed a patient-specific mutational landscape with genomic heterogeneity in MRD. For instance, one patient exhibited BRAF & DIS3 mutations and del(13q) detected at baseline in BM tumor cells and CTCs, which persisted in MRD. After induction, de novo clonal SNVs, including a NEK11 mutation, emerged, followed by the appearance of 3 subclonal CNA after transplant, possibly contributing to resistance. CONCLUSIONS This is the largest dataset analyzing the kinetics and molecular features of CTCs and MRD in SMM and active MM. We showed superior prognostic value of CTC and MRD kinetics over single assessments. Tumor dissemination seems to be driven by transcriptional priming rather than acquired secondary genetic events, while on-treatment resistance is linked to genomic and transcriptional evolution, without recurrent genetic alterations and with common molecular signatures of resistance.

Bone Marrow Cytokines Concentrations in Myelodysplastic Neoplasms: Its Correlation with the Immune Populations and Clonal Hematopoiesis

Background: Myelodysplastic neoplasms (MDS) are a clonal, heterogenic disease of the hematopoietic stem cell. Pathophysiology is mainly related to intrinsic myeloid abnormalities, epigenetic changes and immune system deregulation that leads to changes in the bone marrow microenvironment. MDS patients have shown broad and heterogenic changes in cytokine concentrations compared to controls. An elevation of pro-apoptotic and inflammatory cytokines and chemokines has been described. Nevertheless, the role of inflammatory soluble factors is still poorly understood. We aim to describe cytokine concentrations in the bone marrow of MDS patients and how it correlates with the immune population and clonal hematopoiesis. Methods: We prospectively analyzed 60 bone marrow plasma samples. Forty-four MDS patients without RS or 5q deletion, 8 healthy donors (HD) and 9 cytopenia of unknown significance (CUS). Cytokine/chemokine concentration were analyzed with ThermoFisher commercial kit ProcartaPlexTM Multiplex immunoassay of 32-PLEX (EGF/eotaxin/FGF-2/G-CSF/GM-CSF/HGF/INFalpha/ IFNgamma/IL-1alpha,/IL1-b/IL-10/IL-12/IL-13/IL-15/IL-17a/IL-1RA/IL-2/IL-22 /IL3 /IL-4/IL-5/IL-6/IL-7/IL-8/IL-9), carried out by Luminex® platform. Furthermore, we analyzed NK subpopulations differentiating an immature population (CD56 brigthCD16 −) from a mature one (CD56 dimCD16+). Lymphocyte T sub-populations were studied in PB samples determined as CD45+CD3+ and the different expression of the following monoclonal antibodies CD4/CD8/CCR7/CD45RA/CD27/CD28/CD25/CD127/CXCR3/CXCR6. The analysis was performed, by flow cytometry, on fresh samples, following a membrane staining protocol with 10-colors/3 lasers. Finally, molecular analysis was performed by NGS using the Oncomine Myeloid Research Assay included 40 genes. Results: A total of 60 subjects were included (median age was 71 years-old and 35% were female). Auto-immune disease was present in 6.6% (Figure 1). Regarding chemokines, CCL11 a pro-inflammatory chemokine was increased in MDS (59.73pg/ml) compared to CUS (41.24pg/ml) and HD (22.63pg/ml), also CXCL9, with a complex role in tumor progression, being 29.08pg/ml in MDS, 17.14pg/ml in CUS and 1.13pg/ml in HD. On the other hand, CCL2 and CCL4 showed lower concentrations in MDS vs HD but higher than CUS (Figure 2). Interleukins analysis showed, IL-1RA higher concentrations in MDS (139.9pg/ml), than HD (36.4pg/mL) and no detection in CUS. IL- 2 was detected only in HD (7.61pg/mL) and IL-3 showed greater concentrations in HD and CUS (20.75pg/ml) compared to MDS (13.84pg/ml). IL-6 did not showed differences among the three groups; in 18.3% subjects (11/60) concentrations were ≥10pg/ml and 8 were MDS. IL-8, a pro-inflammatory/proangiogenic cytokine, showed increased concentrations in only 23% subjects (14/60); 13 were MDS. Regarding growth factors, we observed EGF only in HD (28.16%), whilst HGF and VEGF-A showed higher concentrations in MDS. Mature NK sub-populations were increased in CUS (76.16%) vs MDS (11.58%), (p=0.006) and HD (10.66%), (p=0.01). Moreover, CD8+ T lymphocytes between CUS 49.79% vs MDS 14.29% and HD 1.68 (p=0.05) and Th2 in MDS of 22.71% vs CUS 17.5 (p=0.031) without differences to HD 22.54%. Forty-six MDS subjects presented ≥3 somatic mutations, those presented higher concentrations of CCL11 (73.56pg/mL), HGF (497.7pg/mL), CCL2 (95.3pg/ml) and VEGF-A (506pg/mL). Interestingly, we found that among CUS subjects 66.6% (6/9) presented mutations, 2 DMT3A (VAF < 5%), 1 IDH1 (VAF 3.7%), 1 SRSF2 (VAF 38.9%), 1 TET2 (VAF 39.5%), 1 ZRSR2 (VAF 35%), 1 PHF6 (VAF 39.8%). Finally, regarding IPSS-R risk, we observed higher concentrations of inhibitory proteins like IL-10 and cytokines involved in the regulation of tumor microenvironment like CXCL10 or CCL2 in HR-MDS, while in LR-MDS we observed a more cytotoxic and inflammatory profile (Figure 2). Conclusions: Our study describes a heterogenic background of MDS. Suggesting an increase in some cytokines related to cell recruitment, migration, and proliferation in the bone marrow of MDS and an exhaustive and inhibitory environment in those patients with high-risk characteristics, pointing out the important role of immune subpopulations and environment in the clonal progression.

A Randomized Double-Blind Placebo-Controlled Phase II Multi-Center Study of Inflammation Modification of Canakinumab to Prevent Leukemic Progression of Clonal Cytopenias of Unknown Significance (CCUS): Impact Study

Background and Significance: High-risk Clonal Cytopenia of Undetermined Significance (HR-CCUS) is defined by prolonged cytopenias (> 4 months) in 1 or more cell lineages plus myeloid-disorder-associated mutations. CCUS patients with spliceosome mutations, DNMT3A, TET2, or ASXL1 mutations in combination with at least 2 other somatic mutations appear to have an extremely high rate of progression to myeloid neoplasms (MN) with a 95% 5-year cumulative probability of developing a MNsuch asMDS, MPN, or AML (13-fold increased risk compared to patients with cytopenia classified). Several studies implicate the inflammasome in CH progression to MDS/AML. Interleukin (IL)-1β is a key upstream cytokine in the inflammasome that strongly induces IL-6. Mutations in another key protein in the inflammasome, cryopyrin, result in high systemic levels of IL-1β and are associated with auto-inflammatory diseases. We hypothesize that the preclinical and clinical data showing a benefit of IL-1β inhibition in patients with CH, is due to reduced downstream activations of inflammasome proteins. Canakinumab is a high-affinity human monoclonal anti-human IL-1β antibody- FDA approved for the treatment of IL-1β driven inflammatory and oncologic diseases. By binding specifically to human IL-1β, canakinumab blocks the interaction of IL-1β with the IL-1 receptor, leading to inhibition of its downstream targets, thereby preventing IL-1β-induced gene activation and the production of inflammatory mediators. A phase II clinical trial investigating the use of canakinumab in combination with azacitidine is currently studying the impact canakinumab may have on disease progression in relapsed/refractory low or intermediate risk MDS patients (NCT04239157). This has now been extended to newly diagnosed MDS as new emerging data looks promising [unpublished data]. No significant toxicities have been reported. Thus, we hypothesize that the treatment of HR-CCUS patients, for whom there is no current disease modifying therapy, with an inflammasome targeting antibody, would prevent or significantly delay development of a MN, improve cytopenias and decrease the clone size as well as prevent further clonal evolution. Study Design and Methods: This study (NCT05641831) is a Phase II multicenter, randomized, two-arm, double-blind placebo-controlled study in patients with HR-CCUS (Figure 1). Patients with HR-CCUS, no known hematological malignancy, and any mutational combination as deemed appropriate by the inclusion criteria - are randomized to the study Arm or control Arm. Patients assigned to the study arm will be eligible to receive canakinumab, 300 mg subcutaneously every 3 months for 2 years. Patients assigned to the control arm will receive placebo every 3 months for 2 years. Bone marrow biopsies will be done for both arms every 6 months to monitor for disease progression (WHO 2016). PB samples will be collected every 3 months to monitor for disease progression. Patient-reported outcomes will be collected at the time of disease assessment. Statistical plan: The null hypothesis assumes the time to overt MN (MDS/MPN/CMML/AML) is equal in the control and study arms (H 0: HR=1) versus the alternative hypothesis that the risk of an event is lower in the study arm (H 1: HR<1). Assuming exponential survival, 71 events (overt MN) are required for the final analysis to detect a hazard ratio of 0.55 (median 2 years for the control arm versus 3.636 years for the study arm) using a log-rank test with 80% power and one-sided significance level of 5%. This includes one interim futility analysis when 50% of the targeted number of events (36) have been observed. At the time of the interim analysis, futility will be declared if the hazard ratio is greater than 1 and in favor of the control arm. The futility boundary is statistically non-binding in that there is not a final significance level adjustment. If the futility boundary is crossed, then the study will terminate early; patients randomized to the control arm will continue standard of care off study and patients randomized to the study arm will discontinue treatment with Canakinumab. Accounting for 5% drop-out, this calculation assumes accrual of 89 patients over 3 years and a minimum follow-up of 5 years (or until withdrawal, death, or study closure; earliest event). To date, 5 patients have been randomized and enrolled on study (Table 1).

G-CSF Induces MAIT Cells to Exert Anti-Intestinal Gvhd Effects Dependent on CXCR6 Mediated Chemotaxis

Background: Graft-versus-host disease (GVHD) is a major complication leading to transplantation failure and affecting overall survival after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Granulocyte colony-stimulating factor (G-CSF) can induce T-cell tolerance and altering graft cell components to enhance GVHD prevention in HSCT. MAIT cells are a group of innate immune T cells. It has unique biological properties, including mucosal tissue enrichment, activation of microbial riboflavin derivatives and tissue repair properties, which determine MAIT cells play an important role in mucosal immunity. Previous studies found that a high MAIT cell counts in G-CSF-induced grafts (G-grafts) is associated with a low incidence of gut acute GVHD (aGVHD). However, the effect of G-CSF mobilization on the phenotype, function and its regulatory mechanism in GVHD of MAIT cells has not been elucidated. Methods: MAIT cells in PB samples from four healthy donors before and after G-CSF treatment were isolated for Single-cell RNA sequencing (Figure A). Flow cytometry, In vitro experiment and NOD-SCID-IL-2Rg−/− mice (NSG mice) were performed to observe the phenotypic, functional changes and the dynamic distribution of G-CSF induced donor-derived MAIT cells in recipients. Results: A total of 80709 MAIT cells were divided into 14 clusters. Based on key type-associated marker genes and gene sets, MAIT0 (clusters 5 and 9), MAIT1 (cluster 7), MAIT2 (clusters 4 , 11), Cycling MAIT (clusters 12, 13), Activation (clusters 0, 8), ISG (cluster 10) and MAIT17 (clusters 1,2,3,6) cells were identified (Figure B,C). The subset frequencies and expression signatures of immunosuppression (MAIT2 and Cycling cells from cluster 12) and tissue repair (Activation and MAIT17) were both increased at both transcriptome (Figure D) and protein levels, suggesting that G-CSF enhanced the immune tolerance and tissue repair of MAIT cells. In vitro further confirmed that G-CSF-induced MAIT cells showed a stronger inhibition for T cell proliferation (Figure E). Besides, overexpressed genes related to immune regulation in G-CSF induced MAIT cells were strongly enriched in cluster 12 with high CSF3R (G-CSF receptor) expression (Figure F). The cluster 12 also showed the enrichment of IL-17 and TNF signaling pathway favoring tissue repair function, while downregulation of Graft-versus-host disease pathway upon G-CSF mobilization, indicating that G-CSF-CSF3R interaction is the potential mechanism by which G-CSF mediates the immune regulation of MAIT cells. Based on whether recipients develop gut aGVHD or not after allo-HSCT, the matched donor grafts after G-CSF mobilization were divided into two groups termed as post-G-CSF-no gut aGVHD (n=3) and post-G-CSF-gut aGVHD group (n=1), respectively. MAIT cells in post-G-CSF-no gut aGVHD group overexpressed CSF3R and its related target genes, immunosuppression and tissue repair signatures compared with post-G-CSF-gut aGVHD. Besides, MAIT cells from G-PB of recipients without gut aGVHD had a higher CD114 expression (encoded by CSF3R) compared with gut aGVHD (Figure G). These results suggest that the enhanced immunosuppression and tissue repair function of MAIT cells induced by G-CSF may exert anti-gut aGVHD effects. The NSG mice confirmed MAIT cells in G-PB to tissue chemotaxis especially gut tissues in the recipient. A higher frequency of MAIT cells was detected in gut tissues in the low GVHD score group (GVHD Score＜3) compared with the high GVHD score group on post-transplant day 14 (Figure H), suggesting recruited MAIT cells in gut tissues favor anti-gut aGVHD effect. Moreover, CXCR6 was the most differentially expressed chemotactic receptor gene between post-G-CSF-no gut aGVHD and post-G-CSF-gut aGVHD group (Figure I). At the protein level, the expression of CXCR6 were significantly upregulated upon G-CSF administration (Figure I), while that of CCR6 and CXCR6 was non-significant. These results suggest that CXCR6 is necessary for G-CSF induced MAIT cell recruitment to the gut tissue. Conclusions G-CSF-CSF3R interactions enhance immunosuppression and tissue repair functions of MAIT cells, while CXCR6 recruits these functional MAIT cells from PB to gut tissues to exert anti-gut aGVHD effect. Hence, CSF3R and CXCR6 expression in G-CSF mobilized MAIT cells is crucial to exert anti-gut aGVHD effect, and has the potential to become predictive markers of gut aGVHD.

Minimal Residual Disease Detected by the 7NB-mRNAs ddPCR Assay Is Associated with Disease Progression in High-Risk Neuroblastoma Patients: A Prospective Multicenter Observational Study in Japan.

High-risk neuroblastoma (HR-NB) patients remain far from obtaining optimal outcomes, with more than 50% relapse/regrowth rate despite current intensive multimodal therapy. This originated from the activation/proliferation of chemoresistant minimal residual disease (MRD). MRD with a significant prognostic was reported by several quantitative PCR (qPCR) or droplet digital PCR (ddPCR) assays quantitating different sets of NB-associated mRNAs (NB-mRNAs). The 7NB-mRNAs ddPCR assay quantitating CRMP1, DBH, DDC, GAP43, ISL1, PHOX2B, and TH mRNAs was reported to outperform other qPCR assays by a retrospective in-house observational study. In the present study, the Japan Children's Cancer Group (JCCG) Neuroblastoma Committee conducted a prospective multicenter observational study aimed at evaluating a prognostic value of MRD in bone marrow (BM-MRD) and peripheral blood (PB-MRD) detected by 7NB-mRNAs ddPCR assay. Between August 2018 and August 2022, 7 HR-NB patients who registered for JCCG clinical trials (JN-H-11 and JN-H-15) were enrolled. A total of 19 BM and 19 PB samples were collected, and 4/15 BM and 4/15 PB samples were classified as progressive disease (PD)/non-PD samples. BM-MRD and PB-MRD estimated area under curve (AUC) of 0.767 and 0.800 with a significant accuracy (AUC > 0.7). The present study validated a prognostic value of BM-MRD obtained by a previous study (AUC 0.723) and revealed the significant accuracy of PB-MRD as well as BM-MRD.

B-355 Measurable Residual Disease Detection in Mantle Cell Lymphoma (MCL) Subjects Using NGS B-Cell MRD Assay

Abstract Background Mantle Cell Lymphoma (MCL) is an aggressive, rare form of non-Hodgkin B-cell lymphoma. Measurable Residual Disease (MRD) assessment can provide useful information when assessing and tracking response to therapy, refining treatment and predicting clinical outcome for subjects with B-cell lymphoproliferative diseases. One method of MRD detection is to identify and track specific clonal immunoglobulin heavy chain (IGH) gene rearrangement sequences on a next-generation sequencing (NGS) platform. Here, we present the NGS-based B-cell MRD Assay with 10−6 sensitivity for detecting MRD in malignancies in mantle cell lymphoma (MCL). Methods Residual DNA samples from different specimen types (44 BM and 77 PB) at different post-treatment time points were obtained from anonymized 47 MCL subjects that had been enrolled in a study approved by the Spanish Group of Lymphoma and Autologous Stem Cell Transplantation (clinical trial: NCT02682641, publication reference: 10.1200/JCO.21.02321). 121 of these follow up samples were tested by the B-cell MRD Assay by tracking the clonal sequences detected in corresponding baseline samples. Among them, 90 follow up samples were paired PB and BM samples. The B-cell MRD Assay workflow consists of DNA extraction and PCR based library preparation with proprietary multiplex master mixes (MMx) targeting either IGH Framework 1 (FR1) or 3 (FR3) regions, which both track the patient- and tumor-specific CDR3 sequence. Purified libraries are then equimolar pooled and sequenced on Illumina’s NextSeq Dx platform. The FASTQ output files from sequencing are analyzed using Invivoscribe’s B-Cell MRD Software-NextSeqDx. Results from Invivoscribe were compared to the results from Salamanca lab that were generated by qPCR. Results Based on the results from B-cell MRD Assay, 115/121 (95%) samples were valid, 42/115 (37%) valid samples had, “MRD Detected”, and 73/115 (63%) samples had, “MRD Not Detected”, with an “MRD Detection” rate of 28% in PB and 50% in BM specimen types. When comparing MRD detectable status between paired PB and BM samples from 36 MCL subjects, 30/44 (68%) paired samples had same MRD call and were concordant, whereas 14/44 (32%) calls were discordant. More specifically, 12/14 (86%) discordant samples had PB specimen type with a “MRD Not detected” call, whereas the paired BM specimen showed a “MRD Detected” call. Overall, in comparison with the MRD results (n = 84) provided by Salamanca lab using qPCR method, the B-cell MRD Assay demonstrated concordance of 80%, PPA of 79%, and NPA of 80% for MRD detection in MCL samples. Conclusion Good concordance was observed between the qPCR used at Salamanca and Invivoscribe’s B-cell MRD assay. It was important to note that the BM specimen type was determined to be more sensitive in detecting MRD when compared to PB in assessment of paired PB and BM samples. These results demonstrate that the B-cell MRD Assay can be utilized for MRD detection in follow-up sample types for MCL subjects.

Peripheral Blood DNA Methylation Signatures and Response to Tofacitinib in Moderate-to-severe Ulcerative Colitis.

Predictive biomarkers for treatment efficacy of ulcerative colitis [UC] treatments are lacking. Here, we performed a longitudinal study investigating the association and potential predictive power of genome-wide peripheral blood [PB] DNA methylation signatures and response to tofacitinib treatment in UC. We recruited moderate-to-severe UC patients starting tofacitinib treatment, and measured PB DNA methylation profiles at baseline [T1], after 8 weeks [T2], and in a subset [n = 8] after a median of 20 weeks [T3] using the Illumina Infinium HumanMethylation EPIC BeadChip. After 8 weeks, we distinguished responders [R] from non-responders [NR] based on a centrally read endoscopic response [decrease in endoscopic Mayo score ≥1 or Ulcerative Colitis Endoscopic Index of Severity ≥2] combined with corticosteroid-free clinical and/or biochemical response. T1 PB samples were used for biomarker identification, and T2 and publicly available intraclass correlation [ICC] data were used for stability analyses. RNA-sequencing was performed to understand the downstream effects of the predictor CpG loci. In total, 16 R and 15 NR patients, with a median disease duration of 7 [4-12] years and overall comparable patient characteristics at baseline, were analysed. We identified a panel of 53 differentially methylated positions [DMPs] associated with response to tofacitinib [AUROC 0.74]. Most DMPs [77%] demonstrated both short- and long-term hyperstability [ICC ≥0.90], irrespective of inflammatory status. Gene expression analysis showed lower FGFR2 [pBH = 0.011] and LRPAP1 [pBH = 0.020], and higher OR2L13 [pBH = 0.016] expression at T1 in R compared with NR. Our observations demonstrate the utility of genome-wide PB DNA methylation signatures to predict response to tofacitinib.

A Study of the Properties of UV-Aged and Low Formaldehyde Emissions Particleboards Manufactured with Bio-Based Wood Protein Adhesives

The environmental crisis and the safeguarding of the population's health has led to research into different ways of mitigating harmful gases. Among the emissions that the wood industry has sought to reduce are those of formaldehyde, which is why new green adhesive methods for wood panels have been investigated in recent years. In this research, particleboard with two bio-based wood adhesive (PB-bbwa) formulations. The first PB-bbwa formulation, based on proteins obtained from compounds from the alcoholic beverage industry, and the second PB-bbwa formulation, based on proteins from a mixture of compounds from the alcoholic beverage and food industries, were manufactured and tested to evaluate the physical–mechanical, thermal and formaldehyde emission properties of untreated and UV-treated formulations at a laboratory scale. The results of the physical properties obtained in the PB-bbwa were similar or even better than those of the control PB. Additionally, PB-bbwas improve on the control PB sample’s Janka hardness by least 28%, and a decrease in thermal conductivity in the edgewise position and formaldehyde emissions by 12% and 88%, respectively, in comparison to the control PB. The tests performed evidenced that PB-bbwas showed comparable performance against the control PB made with urea-formaldehyde and satisfied international standard requirements.

ENGINEERING CHARACTERIZATION OF GUM ARABIC BONDED PARTICLE BOARD MADE FROM RICE HUSK AND SAWDUST

The Particle board (PB) was developed using Rice Husk (RH) and Sawdust (SD) with Gum Arabic (GA) resin as binder. The aim of the research work was to explore the engineering viability of GA bonded particle board made from agro-industrial wastes for sustainable environment. It was also to investigatealternative to the high-in-demand conventional particle board made from wood and urea melamine or amino-formaldehyde which is known to be carcinogenic. The RH/GA/SD materials used to produce samples were cheap and locally sourced. For the experiment, the rice husk (RH) content was kept constant although at 65%but the sawdust (SD) and gum arabic (GA) were varied at percentage levels of 5/30%, 10/25%, 15/20%, 20/15%, and 25/10%. After characterization the density of the Pb samples ranged from 447.71 Kg/m3 to 698.84Kg/m3. The compressive, split tensile and flexural strengths ranged from 2.24 to 23.31N/mm2, 1.52 to 3.04 N/mm2 and 1.776 to 4.752N/mm2 respectively. Modulus of elasticity ranged from 33.20 to 158.4N/mm2. The results showed that all the mechanical parameters tested met the requirement of the standards, except for moisture intrusion. The maximum value of water absorption was 7.62% for 2 minutes’ immersion in water as against 0.66% and 3.45% in 12 minutes recommended by European standards. The produced particle board is considered structurally viable in that at its ultimate strength it can serve as load-bearing in spite of relatively low density. To mitigate against water absorption, use of wax, veneer and paints can be introduced.

Geochemical evaluation and environmental risk assessment of heavy metals: A case study from Ireland using Tellus stream sediment data (2011–2017)

A comprehensive assessment of the distribution, spatial mapping and ecological risk of heavy metals in stream sediments of 17 counties of Ireland based on the Tellus geochemical survey programme in Ireland was carried out. Average trace metal concentrations of chromium (Cr), nickel (Ni), copper (Cu), lead (Pb), zinc (Zn), vanadium (V), cobalt (Co), arsenic (As), and major and minor oxides iron oxide (Fe2O3), manganese oxide (MnO) in the stream sediments were above the background values, but lower than Irish sediment quality guidelines for livestock and Canadian sediment quality guidelines (ISQG); threshold effect level (TEL) and probable effect level (PEL). 69.65% of Ni, 22.13% of Zn, and 96.7% of Pb samples exceeded the low-effect-range (ERL) values, whereas only 21%, 1.80% and 0.69% of the same elements exceeded the median effect range (ERM) values. Hotspots of Zn (>12000 mg kg−1), Pb (>10,000 mg kg−1), Fe2O3 (61.38%), MnO (26%), Cr (>2300 mg kg−1) and Ni (874.2 mg kg−1) were observed at Wicklow, Dublin, Mayo, Galway and Cavan counties. Catchment geochemistry was identified for the presence of heavy metals in the stream sediments. The geo-accumulation index (Igeo) indicated Kildare to be the least contaminated county with Igeo < 0 for Cr, Ni, Cu, Zn, Pb and As for 50% of the samples. The enrichment factor (EF) indicated that some sites located in Mayo and Donegal were highly contaminated. PInemerow followed the trend, Tipperary > Dublin > Waterford > Monaghan > Donegal > Wexford > Galway > Wicklow > Cavan > Meath > Kilkenny > Leitrim > Mayo > Louth > Sligo > Kildare > Carlow. Based on potential ecological risk index (PERI), 83.1% of the sites posed low ecological risk (PERI <150), 11.9% sites posed moderate risk (150 < RI < 300) and high ecological risk was posed by 0.3% (PERI >1200) of the sites. Lithology, topography and anthropogenic factors influenced the distribution and ecological risks of heavy metals. Non-carcinogenic and carcinogenic risk for adults and children were above the recommended value.

Sediment provenance and dispersal in the early Eocene Dongying Depression, Bohai Bay Basin, Eastern China: Evidence from detrital zircon geochronology, geochemistry and petrology

Comprehensive provenance studies of syn-rift basin fills are required to better understand possible sources of clastic detritus and sediment routing systems. The Eocene fill of the Bohai Bay Basin in eastern China represents a syn-rift succession, where subsurface datasets permit investigation of sediment sources and sinks. New detrital zircon U–Pb samples (441 detrital zircon grains) from six wells were combined with elemental geochemical analysis of siliciclastic sediment, sandstone petrography and palynology to investigate depositional ages and provenance. This study demonstrates the importance of integrating geochronometry, geochemistry, petrology and palynology datasets to fully unravel syn-rift sediment provenance and routing. Zircons of the Dongying Depression were derived principally from an active continental margin island-arc setting characterized by felsic acid magmas, and subordinately from a recycled orogenic belt. The Shicun and Binnan faults controlled the relationship between sediment routing systems and source areas, explaining spatial differences of provenance signals in the depression. The routing system around the Shicun fault is characterized by a dominance of late Paleozoic and Paleoproterozoic zircons and subordinate Mesozoic zircons. In contrast, increased Mesozoic zircons in samples from south of the Binnan fault provide evidence of Mesozoic magmatism in this area. The early Eocene sediments record the signal of early Cretaceous magmatism in the North China Craton, but lack a record of substantial syn-depositional magmatic activity since the Paleogene in the Dongying Depression. Zircons from early Cretaceous strata and pre-existing zircons from the Xing-Meng Orogenic Belt and Inner Mongolia paleo-uplift were transported to the Yanshan and Luxi areas together. These sediments entered the Dongying Depression in the early Eocene, following a period of recycling. In addition, a Precambrian basement signal indicates possible denudation of Neoarchean to Proterozoic rocks in the Luxi uplift and from the Binnan fault footwall. Overall, the provenance signals of the basin reflect zircon recycling from an Early Cretaceous succession, denudation of ancient magmatic rock masses and the absence of syn-sedimentary magmatism.

The beneficial effects of bare and CMC-supported α-FeOOH, Fe3O4, and α-Fe2O3 nanoparticles on growth, nutrient content, and essential oil of summer savory (Satureja hortensis L.) under Cd, Pb and Zn stresses.

This research studies the impacts of iron oxide nanoparticles (FeONPs) on alleviating the toxic effects of cadmium (Cd), lead (Pb), and zinc (Zn) on summer savory (Satureja hortensis L.). Different types of soil additives, including bare and carboxymethylcellulose (CMC)-supported hematite (α-Fe2O3), goethite (α-FeOOH), and magnetite (Fe3O4), were applied at three rates (0, 0.25, and 0.5% w/w) to a Cd, Pb, and Zn-contaminated soil sample. The experimental results showed that the application of FeONPs increased plant height, dry weights of shoot and root, and yield and content of essential oil. Bare and CMC-supported FeONPs increased the content of K, P, and Fe in the aerial parts of summer savory. However, these soil additives reduced the contents of Cd, Pb, and Zn in plant tissues. CMC-supported FeONPs proved to be more efficient additives in diminishing the toxic effects of Cd, Pb, and Zn in summer savory compared to their bare forms. Bare and CMC-supported goethite NPs were able to restrict the uptake of Cd, Pb, and Zn by summer savory roots in the metal-contaminated soil. The application of CMC-supported goethite at an application dose of 0.5% (w/w) increased shoot dry weight, shoot concentrations of K, P, and Fe, and yield of essential oil by about 62.6, 76.6, 77.1, 210, and 230%, respectively. Conversely, they reduced shoot concentrations of Cd, Pb, and Zn by about 64.6, 68.7, and 40.6%, respectively, compared to the control. These are significant results and indicate that CMC-supported goethite is likely to be the most effective soil additive in diminishing the toxicity of Cd, Pb, and Zn to metal-stressed summer savory.

17th International Conference on Malignant Lymphoma, Palazzo dei Congressi, Lugano, Switzerland, 13 - 17 June, 2023.

Introduction: FL is characterized by a heterogeneous clinical course. The biologic importance of the tumour microenvironment in FL pathogenesis is well established. However, detailed information on the immune profile in PB has not been previously investigated, which becomes very relevant in the current era of the immunotherapy. The aim of this study was to characterise the PB immune profile of FL patients at diagnosis, first relapse (R1) and subsequent relapses (>R2) and compare them with healthy controls. Moreover, R1 was also analysed according to histological transformation (HT) and POD24 status. Methods: We collected PB samples of FL patients at diagnosis (n = 42), R1 (n = 22) and >R2 (n = 16) from April 2019 to August 2022 (median age at diagnosis: 65 years [30–82]; 22M/20F). 15 PB samples from healthy donors were also obtained (median age: 31 years [28–65]; 5M/10F). All individuals signed informed consent. The identification of major subsets of T-cells, B-cells, NK cells, monocytes, neutrophils, eosinophils, basophils, dendritic cells (DC) and myeloid suppressor cells (MSC) was performed by multiparameter flow cytometry. Euroflow methodology was used to set up the Omnicyt™ flow cytometer and acquired at least 150.000 events. All data were analysed using Infinicyt™ software version 2.0 (Cytognos, Salamanca, Spain). Results: Compared with healthy controls, FL patients showed a lower CD4+/CD8+ ratio due to an increase in CD8+ lymphocytes and a decrease in total CD4+ lymphocytes. In addition, FL patients had a significantly lower percentage of naïve T lymphocytes, a higher percentage of effector memory and effector lymphocytes, both CD4+ and CD8+. Of note, regulatory T lymphocytes (Treg) and Th1 cells were increased in FL patients, while NK-cells were diminished. Interestingly, differences in DC and MSC were also found between healthy controls and FL patients (Figure). These differences remained when comparing the immune populations between diagnosis and relapse and deepened with each relapse. No differences were observed between groups in the percentage of neutrophils, monocytes, eosinophils, or total B lymphocytes. A subanalysis performed in R1 samples according to HT and POD24 status revealed that HT-R1 presented significantly a smaller CD8+ central memory population compared with non HT-R1. POD24 patients displayed a lower CD4+/CD8+ ratio and a higher proportion of Th1 cells (p < 0.05). In both adverse prognostic groups, the percentage of myeloid DC and neutrophils was significantly lower. The research was funded by: This study has been funded by Instituto de Salud Carlos III (ISCIII) through the projects PI19/00887 to A.L.-G. and E.G., and PI19/00925 to L.M. Andrea Rivero was supported by a grant from Hospital Clinic de Barcelona during the conduct of the study. Keywords: indolent non-Hodgkin lymphoma, microenvironment No conflicts of interests pertinent to the abstract.

Health Risk Assessment Via the Consumption of Clam (Corbicula largillierti) Accumulated Heavy Metals (Pb) and Coliform Bacteria at Kapuas River, West Borneo

As population activity is increasing and the Kapuas river is still a means of transportation, causing Corbicula largillierti to accumulate heavy metals (Pb) and Coliform bacteria in their bodies. This makes it dangerous to be consumed by humans. Thus, this research has two main aims. First, to find out the concentration of Pb and Coliform inside Corbicula largillierti that lives at Kapuas river, Pontianak and the river’s water. Second, to analyze the health risk of consuming the clams. Furthermore, the Atomic Absorption Spectrophotometer (AAS) is used to analyze the Pb sample and the Most Probable Number (MPN) method is used to analyze the Coliform. The result of this research shows that concentration Pb and Coliform from the Kapuas river have exceeded the limits designated for aquatic biota based on the Decree of the State Minister for the Environment No. 51 of 2004. In addition, the health risk is measured using Estimated Daily Intake (EDI), Target Hazard Quotient (THQ) and Target Carcinogenic Risk (TR) with scores for each are &gt;10 times the RfD, &gt;1 and 10−3. This result shows that based on the clam Corbicula largillierti consumption for 70 years life span of 60 kg body weight, had a high and negative effect on health risk problems and had a cancer risk for consumers. However, the health risk problem might increase much worse due to the long and continual period of consumption exceeding estimated daily intake.

High-temperature creep behaviors of polybutene-1 with different chain microstructure and molecular weight

The creep resistance of polymer determines the dimensional stability of product under stress. The isotactic polybutene-1 (iPB) with outstanding high-temperature creep resistance and stress-crack resistance is applied widely in the field of hot-water pipes. Although the chain information including weight-average molecular weight (Mw) and aggregate structures of isotactic polybutene-1 (iPB) influence the creep resistance, the crucial factor affecting the creep behavior greatly is unclear. In this work, a series PB samples with varied Mw, isotacticity and configurational sequence were synthesized and characterized based on Gel Permeation Chromatography (GPC), solvent fractionation and Nuclear Magnetic Resonance Spectroscopy (NMR). The Differential Scanning Calorimetry (DSC), Wide-angle X-ray diffraction (WAXD) and Small angle X-ray scattering (SAXS) were used to characterize the aggregation structures of PB samples. The stress-strain behaviors and 95 °C creep deformation of these PB samples were testes by tensile test and Dynamic Mechanical Analysis (DMA), respectively. It was found that these PB samples showed different chain microstructures like 88–99 wt% high isotactic PB (HiPB) fractions, 90–97.6 mol% tetra-meso placements (mmmm) in HiPB fraction, and 50 × 104-160 × 104Mw. Increasing the molecular weight, isotacticity or configurational sequence mmmm enhanced the creep resistance of PB, while mmmm configurational sequence played more important role in creep resistance of PB through adjusting the aggregation structure (crystalline domains) of the final product greatly. Exploring the influencing factors of creep resistance provided guidance for the synthesis of high-performance PB for high-temperature pipe application.

Abstract A05: Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies

Abstract Targeted therapies(TT) combining FLT3 inhibitors with hypomethylating agents (HMA) and Venetoclax (Ven) are highly effective against FLT3-ITD acute myeloid leukemia (AML) with high response rates. Signaling mutations along with emergence of rarer mutations are the key contributors to primary and secondary resistance to TT. We hypothesize that resistant leukemia clones have unique proteomic profiles that facilitate survival under therapy pressure(TP) and deciphering proteomic profiles at the single-cell level will delineate resistance mechanisms and adaptive responses driven by TP. We performed multiplexed single-cell proteomic analysis of serial PB and BM samples (n:162) from patients treated with FLT3i+HMA+/-VEN using CyTOF. Unsupervised analysis identified leukemia cells and non-malignant cellular elements of the leukemia compartment in an unbiased manner. Next, we interrogated the leukemia proteomic landscape to identify leukemia associated proteomic features. Since Ven targets BCL2 and FLT3i modulates the expression of anti-apoptotic molecules we assessed the expression of apoptosis regulators and found that leukemia cells with immature phenotype, including those with leukemia stem cell(LSC) phenotype, almost always expressed moderate-high levels of BCL2. Monocytic cells(MC) lacked BCL2 expression and had the highest levels of MCL1. Despite expressing moderate levels of MCL1 and BCL-XL, TT was effective in substantially eliminating CD34+ immature leukemia cells. We observed a relative enrichment of MCs, either benign or malignant, after TT. Since cells with MC phenotype were inherently resistant to TT we hypothesized that leukemia cell subsets having similar proteomic profiles to MCs will persist after TT. Phenotypic interrogation of leukemia cells (UMAP) revealed that LSCs generally clustered on the opposite pole distant from MCs and leukemia cells on poles facing MCs were more differentiated. Remarkably, FLT3-ITD mutation partners differentially altered the proteomic landscape. NPM1 mutant FLT3-ITD AML cells displayed a less diverse leukemia architectural organization with differentiation block. Contrarily, signaling mutations diversified the leukemia landscape into a diverse continuum of differentiation states. We also observed that RAS/MAPK mutations could overcome differentiation block and gave rise to a differentiation continuum encompassing less-differentiated, transitional and differentiated leukemia cells. The transitional CD34+ leukemia cells, mapped in close vicinity to MCs, and differentiated MCs that preferentially persisted at D28, had active signaling pathways and expressed CD36. The presence of CD34+ leukemia cells with persistent signaling at D28 was indicative of poor clinical outcomes. Conclusion: Multiplexed single-cell proteomic analysis identified a unique mode of resistance and proteomic features of surviving cells in with FLT3-ITD AML patients treated with TT, and elucidated how different mutation partners in FLT3-ITD AML differentially altered proteomic landscape to drive resistance to TT. Citation Format: Muharrem Muftuoglu, Musa Yilmaz, Mahesh Basyal, Li Li, Naval Daver, Michael Andreeff. Multiplexed CyTOF analysis of FLT3-ITD AML landscape identifies proteomic profiles associated with resistance to targeted therapies [abstract]. In: Proceedings of the AACR Special Conference: Acute Myeloid Leukemia and Myelodysplastic Syndrome; 2023 Jan 23-25; Austin, TX. Philadelphia (PA): AACR; Blood Cancer Discov 2023;4(3_Suppl):Abstract nr A05.

Artificial thymic organoids generated with peripheral blood CD34 +cells represent a quick and powerful tool to determine the nature of severe T cell lymphopenia

Abstract The study of early T-cell development in humans is challenging because of the scarce availability of thymic samples and limitations of in vitro T-cell differentiation assays. We have previously shown that artificial thymic organoids (ATOs), generated by aggregating DLL4-expressing stromal cells with CD34 +cells isolated from bone marrow (BM) or mobilized peripheral blood (MPB), are a reliable tool to determine the hematopoietic-intrinsic or extrinsic nature of severe T-cell lymphopenia. However, BM or MPB samples are not often available and delays in obtaining these samples might dangerously postpone the patient’s clinical management decision. We thus set out to evaluate whether we could obtain reliable results from ATOs generated with CD34 +cells isolated from PB. The main challenge of using CD34 +cells from un-mobilized PB is that the frequency of these cells is much lower than that found in BM or MPB. For the generation of ATOs with BM or MPB CD34 +cells we used a minimum of 5000 cells/ATO, but these numbers would not be feasible for PB samples. We thus generated ATOs with decreased numbers of CD34 +cells, and we determined that 1000 cells/ATO was the minimum necessary to achieve efficient T-cell differentiation. Additionally, we slightly modified the experimental protocol and added SCF for the first 3 weeks of culture, in addition to IL7 and FLT3L, which are present throughout the T-cell differentiation assay. We also established that we could efficiently generate ATOs when starting from fresh blood (up to 3–4 days) and a minimum amount of 5–7 ml, for infants and early pediatric samples. Reduced efficiency in ATO generation was however obtained when isolating CD34 +cells from PB drawn more that 5–6 days earlier or from frozen samples. This work was supported by the Division of Intramural Research, NIAID, NIH.