Viability Of Mononuclear Cells Research Articles

Impacts of cryopreservation on phenotype and functionality of mononuclear cells in peripheral blood and ascites.

Mononuclear cells in peripheral blood and ascites are important clinical resources commonly used in translational and basic research. However, the impact of different cryopreservation durations and extra freeze-thaw cycles on the number and function of mononuclear cells is unknown. Peripheral blood samples (n = 21) and ascites samples (n = 8) were collected from healthy volunteers and ovarian cancer patients. Mononuclear cells were isolated, frozen, and thawed at 6 and 12 months. The impact of cryopreservation on cell viability, the phenotype, and the activation and proliferation of T cells were analyzed by flow cytometry. Single-cell sequencing was applied to investigate the underlying mechanism. The cell number and viability of mononuclear cells in peripheral blood and ascites were significantly decreased after cryopreservation. The T lymphocytes, especially CD4+ T cells, were affected the most significantly. By contrast, monocytes, natural killer (NK) cells, natural killer T (NKT) cells, and B cells were more tolerant. Meanwhile, T cell proliferation and IL-2 secretion are significantly affected after long-term cryopreservation. Mechanistically, the cell death induced by elevated reactive oxygen species (ROS) was involved in the reduction of CD4+ T cells after cryopreservation. Our data indicates that different subtypes of mononuclear cells exhibit different tolerance capacities upon cryopreservation. Thus, our research can provide evidence and support for individuals who are conducting experiments using frozen clinical patient-derived mononuclear cells, for basic research or clinical trials. In addition, extra caution is worthwhile when researchers compare immune cell functionality from peripheral blood or ascites across datasets obtained in different cryopreservation conditions.

Characterization of Nanoprecipitated PET Nanoplastics by 1H NMR and Impact of Residual Ionic Surfactant on Viability of Human Primary Mononuclear Cells and Hemolysis of Erythrocytes.

Manufactured nanoplastic particles (NPs) are indispensable for in vitro and in vivo testing and a health risk assessment of this emerging environmental contaminant is needed. The high surface area and inherent hydrophobicity of plastic materials makes the production of NPs devoid of any contaminants very challenging. In this study, we produced nanoprecipitated polyethylene terephthalate (PET) NPs (300 nm hydrodynamic diameter) with an overall yield of 0.76%. The presence of the ionic surfactant sodium dodecyl sulfate (SDS) was characterized by 1H NMR, where the relative ratio of NP/surfactant was monitored on the basis of the chemical shifts characteristic of PET and SDS. For a wide range of surfactant/NP ratios (17:100 to 1.2:100), the measured zeta potential changed from -42.10 to -34.93 mV, but with an NP concentration up to 100 μg/mL, no clear differences were observed in the cellular assays performed in protein-rich media on primary human cells. The remaining impurities contributed to the outcome of the biological assays applied in protein-free buffers, such as human red blood cell hemolysis. The presence of SDS increased the NP-induced hemolysis by 1.5% in protein-rich buffer and by 7.5% in protein-free buffer. As the size, shape, zeta potential, and contaminants of NPs may all be relevant parameters for the biological effects of NPs, the relative quantification of impurities exemplified in our work by the application of 1H NMR for PET NPs and the ionic surfactant SDS could be a valuable auxiliary method in the quality control of manufactured NPs.

Immune Profiling of Newly Diagnosed Multiple Myeloma (NDMM) Treated with Quadruplet Induction and Autologous Stem Cell Transplantation (ASCT) and Comparison with Achievement of Minimal Residual Disease (MRD) Negativity

Background Incorporation of anti-CD38 monoclonal antibodies (mAb) to the triplet backbone of proteasome inhibitor, immunomodulatory agent and corticosteroid has resulted in unprecedented depth of response including MRD negativity in transplant eligible NDMM. While numerous immunological mechanisms of modern quadruplet combinations are proposed, the modulation of the immunoprofiles bone marrow (BM) and peripheral blood (PB) with quadruplet therapy and ASCT are unknown. The baseline as well as longitudinal study of the composition and functionality of the immune microenvironment with treatment may have implications on disease response. Methods We prospectively collected clinically annotated patient (pt) samples from a phase II single institution clinical trial in transplant eligible NDMM (NCT04991103). Pts received quadruplet combination with daratumumab, bortezomib, lenalidomide and dexamethasone for six cycles followed by MRD adapted deferral of ASCT for pts who achieved MRD<10 -5. Pts deferring ASCT received three additional cycles of consolidation followed by maintenance. Pts who did not meet threshold for ASCT deferral received ASCT followed by maintenance therapy per protocol. We collected paired PB and BM samples at the time of screening (SCN), post-induction (I), and post-consolidation/ASCT (C). We purified and biobanked viable PB mononuclear cells (PBMCs). We thawed PBMCs, treated with FcR blocking Ab, stained with fluorochrome-labeled antibodies and a live/dead fixable stain, and then analyzed using the FACSymphony A5 SE flow cytometer. We identified immune cell subsets in PBMCs using FlowJo v10.2. We report data as percentage of each population within total live PBMCs. Results We collected 48 samples from 8 pts at three timepoints (SCN, I and C) and performed initial immunophenotyping analysis on the PBMCs. We observed no significant changes over time in the frequencies of peripheral CD45 +, CD3 +, NK T cells, monocytes or dendritic cells (not shown). We noted decreased frequencies of multiple B cell subsets, including naïve, transitional, memory and double negative cells (not shown), as well as CD56 +CD16 + NK cells (Fig. panel A) post-induction with no recovery following consolidation . While the frequencies of CD3 + T cells did not change with time, we observed a shift in the CD4:CD8 ratio, with an increase in the proportion of CD8 + T cells post-consolidation (Fig. panel B). Within the post-consolidation CD8 + T cell compartment, we noted increased frequencies of cells that presented with senescent (PD1 negCD57 +), exhausted (PD1 +CD57 neg) or anergic (PD1 +CD57 +) profiles. Consistent with this, the frequencies of terminally differentiated effector memory cells re-expressing CD45RA (TEMRA) CD8 + T cells increased at consolidation (Fig. panel C) and many of these cells exhibited an anergic profile (CD57 +PD1 +, Fig. panel D). Comparison of the baseline immune profiles and MRD levels post-induction revealed that the pt attaining MRD<10 -5 had the highest frequency of-NK cells as well as proportionally higher T CM/T EM and lower TEMRA/T Naive cells at diagnosis. This subject was able to defer ASCT per protocol and subsequently exhibited the lowest frequency of CD8 + TEMRA with an anergic phenotype (PD1 +CD57 +) post quadruplet consolidation. The subject with the least response to quadruplet induction and highest MRD post-induction had a high proportion of CD8 + T cells with an exhausted phenotype (PD1 +CD57 neg) at screening. Conclusion This is the first longitudinal analysis comparing MRD levels to the immune profiles of PB and BM cells derived from a cohort of NDMM patients prospectively treated with quadruplet induction and ASCT. We conclude that quadruplet induction negatively affects the NK cell and B cell compartment during induction and these compartments do not rapidly recover in the immediate post-transplant period. ASCT post quadruplet therapy is associated with preferential expansion of the effector CD8 + T cell compartments, including terminally differentiated TEMRA cells, which express markers associated with T cell exhaustion and anergy. Additional studies evaluating BM immune profile and T cell exhaustion are ongoing and shown at the meeting.

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.

Molecular hybrids of substituted phenylcarbamoylpiperidine and 1,2,4-triazole methylacetamide as potent 15-LOX inhibitors: Design, synthesis, DFT calculations and molecular docking studies

Inflammation is a multifaceted phenomenon triggered by potentially active mediators acutely released arachidonic acid metabolites partially in lipoxygenase (LOX) pathway which are primarily accountable for causing several diseases in humans. It is widely believed that an inhibitor of the LOX pathway represents a rational approach for designing more potent antiinflammatory leads with druggable super safety profiles. In our continual efforts in search for anti-LOX molecules, the present work was to design a new series of N-alkyl/aralkyl/aryl derivatives (7a-o) of 4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,2,4-triazole-3-thiol which was commenced in seriate formation of phenylcarbamoyl derivative (1), hydrazide (2), semicarbazide (3) and 4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,2,4-triazole-3-thiol (4). The aimed compounds were obtained by reacting 4-phenyl-5-(1-phenylcarbamoylpiperidine)-4H-1,2,4-triazole-3-thiol with assorted N-alkyl/aralkyl/aryl electrophiles. All compounds were characterized by FTIR, 1H-, 13C-NMR spectroscopy, EI-MS and HR-EI-MS spectrometry and screened against soybean 15-LOX for their inhibitory potential using chemiluminescence method. All the compounds except 7m and 7h inhibited the said enzyme remarkably. Compounds 7c,7l, 7j and 7a displayed potent inhibitions ranging from IC50 1.92 ± 0.13 µM to 7.65 ± 0.12 µM. Other analogues 7g, 7o, 7e, 7b, 7d, 7k and 7n revealed excellent inhibitory values ranging from IC50 12.45 ± 0.38 µM to 24.81 ± 0.47 µM. All these compounds did not reveal DPPH radical scavenging activity. Compounds 7i-o maintained > 90 % human blood mononuclear cells (MNCs) viability at 0.125 mM as assayed by MTT whilst others were found toxic. Pharmacokinetic profiles predicted good oral bioavailability and drug-likeness properties of the active scaffolds. SAR investigations showed that phenyl substituted analogue on amide side decreased inhibitory activity due to inductive and mesomeric effects while the mono-alkyl substituted analogues were more active than disubstituted ones and ortho substituted analogues were more potent than meta substituted ones. MD simulation predicted the stability of the 7c ligand and receptor complex as shown by their relative RMSD (root mean square deviation) values. Molecular docking studies displayed hydrogen bonding between the compounds and the enzyme with Arg378 which was common in 7n, 7g, 7h and baicalein. In 7a and quercetin, hydrogen bonding was established through Asn375. RMSD values exhibited good inhibitory profiles in the order quercetin (0.73 Å) < 7 g < baicalein < 7a < 7n < 7 h (1.81 Å) and the binding free energies followed similar pattern. Density functional theory (DFT) data established good correlation between the active compounds and significant activity was associated with more stabilized LUMO (lowest unoccupied molecular orbitals) orbitals. Nevertheless, the present studies declare active analogues like 7c, 7 l, 7a, 7j as leads. Work is ongoing in derivatizing active molecules to explore more effective leads as 15-LOX inhibitors as antiinflammatory agents.

Antibacterial and antibiofilm efficacy of colistin & meropenem conjugated silver nanoparticles against Escherichia coli and Klebsiella pneumoniae.

The progressive increase in infections caused by multidrug-resistant (MDR) Gram-negative bacteria and the emergence of resistance to last-resort antimicrobial drugs in recent years necessitate the development of new therapeutic strategies. This study was conducted to obtain nanostructured antimicrobials by conjugating colistin (COL) and meropenem (MEM) antibiotics with biosynthesized silver nanoparticles (bio-AgNPs) via the green synthesis method using Rosa damascena extract, and to investigate the antibacterial and antibiofilm activity of these nanostructures against Escherichia coli and Klebsiella pneumoniae strains. Ultraviolet-visible spectrophotometry, high-resolution-transmission electron microscopy, atomic force microscopy, X-ray diffraction, and Fourier transform-infrared spectroscopy analyses were performed to determine the physical and chemical properties of synthesized bio-AgNPs, COL@bio-AgNPs, MEM@bio-AgNPs, and COL&MEM@bio-AgNPs. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration of nanoparticles were determined on standard and MDR clinical strains. The antibiofilm efficacy and cytotoxic effect of nanoparticles were evaluated by the crystal violet dye method and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide dye method, respectively. The characterization analyses demonstrated that the synthesized nanoparticles had crystal structure and spherical morphology (5.6-30.2 nm in size). Antibiotic conjugated nanoparticles exhibited better antimicrobial activity and lower MIC values (0.125-4 µg/mL) on the tested strains compared to free antibiotics, and MIC values were decreased up to 1024-fold (p < 0.05). Antibiotic conjugated nanoparticles were found to be more effective in biofilm eradication than free antibiotics and bio-AgNPs and had a less inhibitory effect on peripheral blood mononuclear cell viability. The findings revealed that antibiotic-conjugated nanoparticles have the potential to be used as an effective antimicrobial drug against MDR E. coli and K. pneumoniae strains.

Downregulation of RCN1 promotes pyroptosis in acute myeloid leukemia cells.

Reticulocalbin-1 (RCN1) is expressed aberrantly and at a high level in various tumors, including acute myeloid leukemia (AML), yet its impact on AML remains unclear. In this study, we demonstrate that RCN1 knockdown significantly suppresses the viability of bone marrow mononuclear cells (BMMNCs) from AML patients but does not affect the viability of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood stem cells (PBSCs) from healthy donors in vitro. Downregulation of RCN1 also reduces the viability of AML cell lines. Further studies showed that the RCN1 knockdown upregulates type I interferon (IFN-1) expression and promotes AML cell pyroptosis through caspase-1 and gasdermin D (GSDMD) signaling. Deletion of the mouse Rcn1 gene inhibits the viability of mouse AML cell lines but not the hematopoiesis of mouse bone marrow. In addition, RCN1 downregulation in human AML cells significantly inhibited tumor growth in the NSG mouse xenograft model. Taken together, our results suggest that RCN1 may be a potential target for AML therapy.

Evaluation of the Impact of a New Cooling Cell Processor System on Islet Cell Isolation Facility.

Standard cell therapy equipment, including the gold standard cell processor to purify human islets for clinical transplantation, is rarely refrigerated, potentially exposing cells to elevated temperatures during the centrifugation step. Custom cooling systems have a direct benefit on human islet viability and function. The current study was designed to test the effectiveness of a newly developed, readily available cooled cell processor system requiring minimal modifications and to evaluate its impact on human cell viability and the GMP cleanroom environment.The cooler system, a mechanically refrigerated heat exchanger set at -30 °C was used to deliver cooled medical grade dry air to the cell processor bowl through a hole drilled in the centrifuge cover. With the limited availability of pancreas donors in Qatar, system validation was done with continuous density gradient purification of pooled human bone marrow buffy coat. Sterility, turbulence, and particle count were measured in class C and class B clean room environments. No turbulence developed around the cooled cell processor, and no excess 0.5 µm and 5 µm airborne particulates were generated as per cleanroom GMP standards. At the beginning and end of the collection steps, the temperature rose respectively to 21.50 °C ± 0.34 °C and 21.93 °C ± 0.20 °C in the non-cooled cell processor and to only 10.9 °C ± 0.17 °C and 11.16 °C ± 0.35 °C in the cooled- cell processor (p <0.05). The cooled cell processor led to both improved recovery (98%) of the mononuclear cell fraction and viability (100% ± 2%) post-processing. The new cooling system effectively reduces the heat produced by the cell processor while having no particulate impact on the GMP clean room environment. The cooled cell processor described here is an inexpensive ($16,000 without including taxes, customs clearance, and transportation) and minimally invasive method to provide robust cooling. Currently, this technology in the GMP cell therapy facilityis being applied to human islet cell isolation and transplantation for the clinical program.

Viability of mononuclear cells in leukocyte concentrates at the stages of their preparation, freezing, and thawing

Aim. To evaluate the viability of mononuclear cells (MNCs) in leukocyte concentrates (LCs) at the stages of their preparation, freezing, and thawing.Materials and methods. The study material included 44 LCs from donors of allogeneic hematopoietic stem cells (HSCs) and 189 autologous LCs from patients with oncohematological disorders. LCs were obtained from donors and patients by leukocytapheresis after mobilization of HSCs. LCs from patients were frozen with dimethyl sulfoxide (DMSO) used as a cryoprotectant at a final concentration of 5% and stored in liquid nitrogen. LCs were thawed before transplantation. A total of 161 LCs were immediately transfused to the recipient after thawing, and 28 LCs were washed from DMSO before transfusion. Flow cytofluorometry was used to determine the percentage of MNC populations that excluded 7-aminoactinomycin D (7-AAD).Results. The viability of peripheral blood MNCs in donors and patients was close to 100%. It was found that leukocytapheresis and cryopreservation with DMSO did not affect the viability of MNCs. The freezing of LCs with DMSO, storage in liquid nitrogen, and thawing resulted in a significant decrease in the content of viable MNCs (p = 0.0025), while no effect of LC storage duration on the viability of MNCs was revealed. Following DMSO removal from LCs, significantly more HSCs remained in a viable state than without washing (94.4 [94.5; 95.2] % vs. 86.7 [67.6; 92.9] %, (p = 0.0051); for other MNC populations, except monocytes, the differences in the viability index were also statistically significant.Conclusion. The viability of MNCs in LCs is recommended to be used as an independent characteristic of the transplant quality. In obtaining LCs and mixing them with the cryoprotectant DMSO, the viability of MNCs does not decrease, while in thawed LCs, it decreases significantly. Thawing of LCs with removal of DMSO allows to achieve the best viability of HSCs and most MNC populations.

Use of a commercial feed supplement based on yeast products and microalgae with or without nucleotide addition in calves

The use of feed additives with antioxidant and immune response modulatory activity could be a useful strategy in suckling calves to reduce morbidity and mortality. This strategy is based on several feed additives tested for these purposes. The aim of the paper is the examination of a commercial feed additive for adult cows for use in calves, with and without nucleotide supplementation. Seventy-five Holstein Friesian male calves were divided in 3 groups, with each calf randomly assigned to a group according to birth order. All calves received 2 L of pooled colostrum within 2 h of birth. The commercial feed supplement group was orally administered with 5 g/head of Decosel (dried brewer's yeast lysate (Saccharomyces cerevisiae), brewer's yeast walls (Saccharomyces cerevisiae), diatoms, spirulina, barley flour, calcium carbonate; Agroteam srl, Torrimpietra, Italy) and the nucleotides + commercial feed supplement group was orally administered with 5 g/head of an additive containing 2.5 g of Decosel and 2.5 g of nucleotides once daily from birth to 25 d. The control group was orally administered 20 mL of fresh water/head once daily. Calves that received the supplement and the nucleotides showed lower rates of protein and metabolizable energy conversion, with longer villi and greater crypt depth in duodenum. Moreover, the commercial feed supplement alone increased antioxidant capacity [2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) and ferric-reducing antioxidant power] in plasma some activity of antioxidant liver enzymes, and peripheral blood mononuclear cell viability after in vitro concanavalin A and H2O2 stimuli. Dietary supplementation with a commercial feed supplement containing yeast products (yeast cell walls and hydrolyzed yeast) and microalgae enhanced the redox balance and gut morphology in calves, allowing calves to improve their immune response, increasing resistance to stress. Moreover, these beneficial effects were strongly potentiated when dietary nucleotides were added to the supplement.

Verification of prognostic expression biomarkers is improved by examining enriched leukemic blasts rather than mononuclear cells from acute myeloid leukemia patients

BackgroundStudies have not systematically compared the ability to verify performance of prognostic transcripts in paired bulk mononuclear cells versus viable CD34-expressing leukemic blasts from patients with acute myeloid leukemia. We hypothesized that examining the homogenous leukemic blasts will yield different biological information and may improve prognostic performance of expression biomarkers.MethodsTo assess the impact of cellular heterogeneity on expression biomarkers in acute myeloid leukemia, we systematically examined paired mononuclear cells and viable CD34-expressing leukemic blasts from SWOG diagnostic specimens. After enrichment, patients were assigned into discovery and validation cohorts based on availability of extracted RNA. Analyses of RNA sequencing data examined how enrichment impacted differentially expressed genes associated with pre-analytic variables, patient characteristics, and clinical outcomes.ResultsBlast enrichment yielded significantly different expression profiles and biological pathways associated with clinical characteristics (e.g., cytogenetics). Although numerous differentially expressed genes were associated with clinical outcomes, most lost their prognostic significance in the mononuclear cells and blasts after adjusting for age and ELN risk, with only 11 genes remaining significant for overall survival in both cell populations (CEP70, COMMD7, DNMT3B, ECE1, LNX2, NEGR1, PIK3C2B, SEMA4D, SMAD2, TAF8, ZNF444). To examine the impact of enrichment on biomarker verification, these 11 candidate biomarkers were examined by quantitative RT/PCR in the validation cohort. After adjusting for ELN risk and age, expression of 4 genes (CEP70, DNMT3B, ECE1, and PIK3CB) remained significantly associated with overall survival in the blasts, while none met statistical significance in mononuclear cells.ConclusionsThis study provides insights into biological information gained/lost by examining viable CD34-expressing leukemic blasts versus mononuclear cells from the same patient and shows an improved verification rate for expression biomarkers in blasts.

Investigations of p-tolyloxy-1,3,4-oxadiazole propionamides as soybean 15-lipoxygenase inhibitors in comforting with in vitro and in silico studies

Inflammatory disorders are the prime contributor to public health issue and the development of more effective and safer anti-inflammatory drugs in addition to other therapeutic alternatives to treat inflammatory illnesses, particularly chronic inflammatory diseases, is one of the foremost current issues. In this regard, our present work is concerned with the synthesis of a new series of N-alkyl/aralkyl/aryl derivatives (7a–o) of 5-((p-tolyloxymethyl)-4H-1,3,4-oxadiazole-2-ylthio)propionamide which was instigated by the successive conversions of p-tolyloxyacetic acid into ester, hydrazide and 5-(p-tolyloxymethyl)-4H-1,3,4-oxadiazole-2-thiol. The planned compounds (7a–o) were attained by the reaction of 5-(p-tolyloxymethyl)-4H-1,3,4-oxadiazole-2-thiol with variety of N-alkyl/aralkyl/aryl electrophiles in potassium hydroxide and were characterized by FTIR, 1H-, 13C-NMR spectroscopy, EI-MS and HR-EI-MS spectrometry and probed for their inhibiting potential against soybean 15-lipoxygenase (15-LOX) enzyme. The compounds 7a, 7n, 7 g, 7e, 7h, 7i, 7j and 7b promulgated the potent inhibiting potential with IC50 values 9.43 ± 0.45, 16.75 ± 0.49, 19.45 ± 0.37, 21.32 ± 0.46, 22.64 ± 0.56, 23.53 ± 0.62, 24.32 ± 0.45 and 29.15 ± 0.57 µM, respectively, while excellent to good inhibitory activities were shown by 7o, 7 m, 7k, 7f, 7c, 7 l and 7d with IC50 values in the range 30.29 ± 0.56 to 52.54 ± 0.64 µM. Compounds 7i–o maintained 91.12 ± 1.5 to 98.23 ± 1.2% blood mononuclear cells (MNCs) viability at 0.25 mM by MTT assay whilst compounds 7d–h observed 46.51 ± 1.3 to 57.12 ± 1.4% viability where as the most toxic compounds were 7b (12.51 ± 1.4%), 7a (28.12 ± 1.5%) and 7c (38.23 ± 1.5%) as compared with controls. Pharmacokinetic profiles predicted good oral bioavailability and drug-likeness properties of molecules as per rule of five. Molecular docking studies displayed hydrogen bonding between the compounds and the enzyme with Arg378 which was common in 7n, 7 g, 7h and baicalein. In 7a and quercetin, hydrogen bonding was established through Asn375; Tyr512 and Val589 were also involved in bonding with other analogues. RMSD (root mean square deviation) values exhibited good inhibitory profiles in the order quercetin (0.73 Å)<7 g (0.98 Å)<baicalein (1.23 Å)<7a (1.27 Å)<7n (1.75 Å)<7h (1.81 Å) whereas the binding free energies (kJ/mol) increasing order: (−37.56 kJ/mol)< quercetin (−35.43 kJ/mol)<7n (−34.55 kJ/mol)<7 g (−33.38 kJ/mol)<7h (31.75 kJ/mol)<7a (−31.63 kJ/mol). Density functional theory (DFT) data established good correlation between the IC50 values of compounds and HOMO–LUMO (highest occupied and lowest unoccupied molecular orbitals) energy calculations. It was found that compounds with more stabilized LUMO orbitals had significant biological activity. The work collectively supports steps taken forward in search for potential ‘leads’ as anti-inflammatory agents wherein compounds 7a, 7n, 7i, 7j possessed ‘lead’ like properties for further investigations. Communicated by Ramaswamy H. Sarma

Effect of Culture Conditions of Lophocereus marginatus Endophytic Fungi on Yield and Anticancer and Antioxidant Activities

Culture conditions affect the production of secondary metabolites in endophytic fungi. Therefore, the aim of the present study was to evaluate the yield and anticancer and antioxidant activity of endophytic fungi extracts from the cactus Lophocereus marginatus, under different culture conditions. The strains Penicillium citrinum, Aspergillus versicolor, Metarhizium anisopliae, and Cladosporium sp. were fermented in different culture media (potato dextrose agar, Czapeck broth, and malt broth), types of inoculums (spore or mycelium), and shaking conditions (150 rpm or static) for one week. Methanol extracts were obtained from mycelia, which was followed by determining their yields and evaluating their effect on L5178Y-R murine lymphoma cells growth and human peripheral blood mononuclear cells (PBMCs) viability, using the 3-[4,5dimethylthiazol-2-yl]2,5-diphenyl tetrazolium bromide reduction colorimetric assay. In addition, antioxidant activity was determined by the 2,2-diphenyl-1-picrylhydrazyl test. We determined the half-maximal inhibitory concentration (IC50) values of tumor cell growth inhibition, the selectivity index (SI), and the antioxidant activity, as compared with the healthy cells control. The best yields were obtained with the Czapeck broth medium in all the evaluated strains, reaching values of 50.3%. Of the 48 extracts evaluated, only seven significantly (p < 0.01) inhibited tumor cell growth (IC50 < 250 µg/mL). A. versicolor extract showed the highest anticancer activity, after culturing spores (IC50 = 49.62 µg/mL; SI = 15.8) or mycelium (IC50 = 69.67 µg/mL; SI = 12.2) in malt broth, under static conditions. Extracts did not present significant antioxidant activity. In conclusion, we showed that culture conditions influenced the anticancer activity of L. marginatus endophytic fungi.

Manufacturing of CD34 + HPC-enriched, high-purity mononuclear cell products from umbilical cord blood.

The purpose of this study was to explore methods of selectively enriching CD34 + haematopoietic progenitor cells (HPC) in mononuclear cell (MNC) preparations, and to outline a procedure for cryopreservation and thawing of manufactured material. Density gradient centrifugation of umbilical cord blood was achieved using Ficoll-Paque™ media at 1.077g/mL and 1.065g/mL densities and Leucosep preparation tubes. Post-process samples were analysed for CD34 + and MNC content. Finally, MNCs were frozen down at a concentration of 8.5 × 106 cells/mL in CryoStor CS10 using an Asymptote VIAFreeze controlled rate freezer at a rate of - 2°C per minute, then thawed and analysed for viability and recovery. Processing with 1.065g/mL media selectively depleted non-HPC cell types, producing an approximately fourfold increase in CD34 + frequency (M ± 1SD = 1.4 ± 1.3%, P < 0.01) relative to the pre-process sample (M ± 1SD = 0.4 ± 0.3%), whereas 1.077g/mL media produced only a twofold enrichment (0.7 ± 0.6, P < 0.01). This was not accompanied by any significant forfeit of CD34 + recovery (79 ± 32% vs. 78 ± 32% respectively; P = 0.87). The MNCs generated by the 1.065g/mL procedure were of greater purity (96 ± 2%) than in the 1.077g/mL procedure (80 ± 7%, P < 0.01). Post-thaw, MNC viability was 95 ± 1% and CD34 + viability was 98 ± 1%. Ultra-pure MNCs rich in CD34 + HPCs can be generated with a simple, inexpensive modification to Ficoll-Paque™ media. These products can be easily cryopreserved using a simple controlled rate freezing procedure.

In Vivo Efficacy of Wound Healing under External (Bio)AgNCs Treatment: Localization Case Study in Liver and Blood Tissue

The present study reports on the in vivo application of (Bio)silver nanocomposite formulations (LBPC-AgNCs) on wound healing. Additionally, the present study emphasizes the limited uptake of silver by liver and blood tissues as well as the high viability of PBMCs following external LBPC-AgNCs treatment. The wound closure was monitored via stereoscopic microscope, a localization case study in liver and blood tissue was carried out by (Inductively Coupled Plasma-Mass Spectrometers (ICP/MS), and peripheral blood mononuclear cells (PMBC) viability was determined via flow cytometry technique. The silver formulation was applied externally on the site of the wound infection for a period of ten days. At the beginning of the experiment, a moderate decrease in body weight and atypical behavior was observed. However, during the last period of the experiment, no abnormal mouse behaviors were noticed. The wound-healing process took place in a gradual manner, presenting the regeneration effect at around 30% from the fourth day. From the seventh day, the wounds treated with the silver formulation showed 80% of the wound healing potential. The viability of PBMCs was found to be 97%, whereas the concentrations of silver in the liver and blood samples were determined to be 0.022 µg/g and 9.3 µg/g, respectively. Furthermore, the present report becomes a pilot study in transferring from in vitro to in vivo scale (e.g., medical field application) once LBPC-AgNCs have demonstrated a unique wound healing potential as well as a non-toxic effect on the liver and blood.

Mesenchymal Stem Cell Identification After Delayed Cord Clamping.

We sought to determine the feasibility of identifying and quantifying mesenchymal stem cells (MSCs) from umbilical cord blood (UCB) after delayed cord clamping in preterm and term births. We obtained 3mL of UCB at various gestational ages after delayed cord clamping. UCB separated by density gradient centrifugation within 4h of delivery was passed through magnetic bead micro-columns to exclude the CD34 + cell population. The samples were incubated with fluorescent-tagged mesenchymal cell marker antibodies CD 29, CD44, CD73, CD105, and hematopoietic cell marker CD45. The cell populations were analyzed by flow cytometry. Viable cells were assessed with 7-aminoactinomycin-D. The results were expressed in median (minimum to maximum) MSCs and compared between preterm and term samples. A total of 12 UCB samples (32-40weeks) were obtained, 10 of which demonstrated MSCs, accounting for 0.0174% (0-14.7%) of the viable UCB mononuclear cells. MSCs comprised 0.148% (0.0006-1.59%) and 0.116% (0-14.7%) of the viable UCB mononuclear cells in the term (n = 5), 38.4 ± 1.3weeks, and preterm (n = 7) samples, 34.6 ± 1.1, respectively, p = 0.17. There was an overall median of 96 (0-39,574) MSCs. There was no difference in the median numbers of MSCs identified between term and preterm UCB samples, 3384 (23-6042) and 36 (0-39,574), respectively, p = 0.12. Mesenchymal stem cells were identified and quantified in 5 of 7 preterm and all 5 term UCB 3-mL samples obtained after delayed cord clamping.

Parsing p-tolyloxy-1,3,4-oxadiazolepropanamides as 15-lipoxygenase inhibitors prop up by in vitro and in silico profiling including structure determination

In humans, tissue injury result in the production of an inflammatory response and arachidonic acid released from the damaged membrane phospholipids is oxidized in two separate pathways, that is, lipoxygenase (LOX) and cyclooxygenase (COX) pathways, resulting in the production of biologically active mediators. The present work deals with the synthesis of a series of new N-alkyl/N-aryl derivatives (6a-n) of 5-((p-tolyloxymethyl)-4H-1,3,4-oxadiazole-2-ylthio)propanamide and these analogues (6a-n) were characterized by modern spectroscopic and spectrometric techniques and scanned for their inhibition for soybean 15-LOX enzyme. The results showed the derivatives 6g, 6i and 6j as potent enzyme inhibitors with an IC50 values 8.5 ± 0.4 µM, 11.4 ± 0.6 µM and 19.7 ± 0.4 µM. Compounds 6g and 6i maintained blood mononuclear cells (MNCs) viability of 75.5 ± 1.4%, 95.6 ± 1.5%, but 6j was found toxic with viability of 32.6 ± 1.6%, respectively, as determined by MTT assay. Rest of the compounds displayed good to poor inhibitory profiles against the said enzyme with IC50 values from 24.6 ± 0.6 µM to 83.5 ± 0.5 µM which were also found nontoxic to MNCs under assay conditions except 6k which revealed only 23.6 ± 1.3% cellular viability. Calculated ADME properties predicted ‘drug-like properties’ of all compounds with good oral bioavailability profiles. Molecular docking studies showed that Lys526 and Trp772 in 6g established hydrogen bonding with the protein and π-alkyl (Val126, Val520, Pro530) and alkyl interaction (Arg533) further helped the ligand protein binding as depicted in quercetin where hydrogen bonding and π-alkyl interaction was recognized. In summary, the derivatives 6g and 6i demonstrated highly appreciable drug-like properties and warrant further investigations in search for potential leads as anti-LOX agents.

Abstract 22 CellStor Transfer Freezing Bag: Reliable Closed System for Complex Cord Blood Sample Processing and CD34+ Hematopoietic Stem Cell Cryopreservation

IntroductionHematopoietic stem cell (HSC) transplants are effective in the treatment of numerous disorders. Proper processing and storage of umbilical cord blood (UCB) and peripheral blood (PB) HSCs is invaluable for therapeutic applications. Although automated sample processing methods exist, they are unable to accommodate low volume, viscous, fibrin clotted, partially hemolyzed, or otherwise complex samples, resulting in the loss of precious HSCs. The CellBios single-use transfer freezing bag was designed to process peripheral and cord blood samples and freeze HSCs, including from those with the above-mentioned problems. Here we demonstrate the utility of these transfer freezing bags to salvage problematic UCB samples and recover precious HSC material suitable for banking and therapeutics.ObjectiveThe objective of this study was to develop a closed UCB sample processing and cryopreservation solution that is amenable to easy manual operation, can deliver excellent HSC viability and recovery, can eliminate contaminating red blood cells (RBCs), and is capable of processing complex/defective starting samples that are incompatible with conventional automated processing systems into therapeutic grade HSC banks.MethodsWe studied 100 randomly selected UCB samples, from various centers throughout India, that were processed and prepared for cryopreservation using our transfer freezing bag. We recorded the pre- and post-sample processing volumes, total nucleated cell counts (TNCC), hematocrit (HCT), CD34+ cell viability, and recovery.ResultsStudy results showed the following: 94% vs 98% CD34+ cell viability post processing, 77% vs 85% total mononuclear cell viability, 69% vs 85% TNCC recovery in problematic vs normal samples, respectively. It was possible to achieve low RBC carryover –91% HCT reduction post-processing in normal samples; more importantly, problematic samples also had RBC removal evidenced by HCT reduction of 45%.DiscussionWe have demonstrated efficient removal of RBCs to alleviate post-transplantation issues and maximized CD34+ HSC recovery in both normal and problematic UCB samples. Crucially, it was possible to salvage precious CB HSCs from defective samples that would be lost had they been processed in conventional automated workflows. The CellBios CellStor Transfer Freezing Bag Set offers best in class performance in preparing umbilical cord blood samples for cryostorage.

Promoting effect of Tmsb4x on the differentiation of peripheral blood mononuclear cells to dendritic cells during septicemia

BackgroundThymosin beta 4 × (Tmsb4x) has been highlighted as an important regulator in immune and inflammation responses. Promoted differentiation of mononuclear cells into dendritic cells (DCs) exert a beneficial effect on septicemia. Herein, we investigated the effects of Tmsb4x on the mononuclear cells to affect immune responses during septicemia. MethodsInitially, we isolated peripheral blood samples from healthy individuals and patients with septicemia for extraction of mononuclear cells, followed by Tmsb4x expression quantification. A cell model was constructed with mononuclear cells through lipopolysaccharide stimulation. The viability and apoptosis were evaluated in response to Tmsb4x silencing or re-expression. Additionally, the proportion of DCs was assessed by determining levels of inflammatory factors as well as by flow cytometric analysis. A mouse septicemia model was developed for in vivo validation. ResultsCell and animal models demonstrated decreased Tmsb4x expression in the setting of septicemia, which led to increased inflammatory response and reduced proportion of DCs, along with inhibited mononuclear cell viability and promoted apoptosis. However, restoration of Tmsb4x facilitated the differentiation of mononuclear cells into DCs. ConclusionTo conclude, upregulated Tmsb4x promoted the generation of DCs from mononuclear cells, which contributed to deep understanding of underpinning mechanisms in the development of septicemia.

Targeting new N-furfurylated 4-chlorophenyl-1,2,4-triazolepropionamide hybrids as potential 15-lipoxygenase inhibitors supported with in vitro and in silico studies

Lipoxygenases (LOXs) are a group of enzymes that catalyze the oxidation of polyunsaturated fatty acids and initiate the biosynthesis of secondary metabolites that are involved to control inflammation. In search of new and more potent LOX inhibitors, a series of new 3-(5-(4-chlorophenyl)-4-(2-furylmethyl)-1,2,4-triazole hybrids was prepared and screened for its LOX inhibitory potential. 4-Chlorobenzoic acid (a) was metamorphosed into N-furfuryl-5-(4-chlorophenyl)-4-(2-furylmethyl)-1,2,4-triazole (4) via intermediates like benzoate (1), hydrazide (2) and semicarbazide (3). Finally, triazole (4) was fused with propionamides (6a–o) and transformed it into the aimed derivatives (7a–o). The structural interpretations of the prepared derivatives (7a–o) were accomplished via FTIR, 1H-, 13C-NMR spectroscopy, EI-MS and HR-EI-MS spectrometry. The inhibitory potency of the compounds against soybean 15-LOX was determined by in vitro assay using chemiluminescence method. Compounds 7a and 7f exhibited potent LOX inhibitory profiles with IC50 21.83 ± 0.56 and 25.72 ± 0.51 µM, whereas 7d and 7e showed comparable inhibitory potential with IC50 values of 34.52 ± 0.39 and 39.12 ± 0.46 µM, respectively. Compounds 7a, 7f, 7d and 7e exhibited 65.58 ± 1.4%, 54.72 ± 1.3%, 58.52 ± 1.2% and 63.56 ± 1.4% blood mononuclear cells viability, respectively. Density functional theory and molecular docking studies further strengthened the studies of the synthesized compounds and these derivatives perceived to be potential ‘lead’ compounds in drug discovery as anti-LOX. Communicated by Ramaswamy H. Sarma