Cell Colony-forming Units Research Articles

Lignin-derived compounds assisted with Kocuria marina H-2 and Pseudomonas putida B6-2 co-culture enhanced naphthalene biodegradation

The implementation of environmentally friendly and sustainable remediation strategies positively impacts solid waste management. In this study, the Kocuria marina H-2 and Pseudomonas putida B6-2 co-culture system demonstrated enhanced naphthalene biodegradation efficiency compared to single-strain cultures. Under optimal conditions of 35 °C, 200 rpm/min, and a 1:1 ratio of the co-culture system, the naphthalene biodegradation potential was further increased. Notably, the addition of both ethylenediamine-pretreated lignin and p-hydroxybenzoic acid significantly elevated naphthalene degradation rates to 68.5 %. In addition, the oil-liquid surface tension decreased, while cell surface hydrophobicity and colony-forming units increased with the addition of lignin-derived compounds. The modification of naphthalene bioavailability by ethylenediamine-pretreated lignin would accelerate the uptake and transport of hydrocarbons via ABC transporters and flagellar assembly. Importantly, genes related to bacterial chemotaxis and fatty acid biosynthesis were upregulated during the co-metabolism of naphthalene and p-hydroxybenzoic acid, further enhancing naphthalene bioconversion.

Cannabidiol Demonstrates Remarkable Efficacy in Treating Multidrug-Resistant Enterococcus Faecalis Infections In Vitro and In Vivo

The growing prevalence of multidrug-resistant Enterococcus faecalis infection has become a global concern. There is a demand for alternative antibacterial agents, such as herbal alternatives, such as Cannabidiol, that are cost-effective, non-toxic and efficient. This study investigates the Minimum Inhibitory Concentration (MIC) for planktonic cells and the Minimum Biofilm Eradication Concentration (MBEC) for biofilm formation in multidrug- resistance isolates of E. faecalis isolates, particularly focusing cannabidiol activity. E. faecalis isolates with strong biofilm presence, vancomycin, levofloxacin, and daptomycin displayed high MIC and MBEC values. Cannabidiol exhibited a significantly lower MIC (1 μg/mL) for planktonic cells and a low MBEC (2 μg/mL). Moreover, at low concentrations (2 µg/mL), cannabidiol demonstrated notable reductions in biofilm biovolume, bacterial cell viability and colony-forming unit compared to vancomycin, levofloxacin, and daptomycin. The mice treated with cannabidiol (100 mg/kg) exhibited a significant reduction in E. faecalis bacterial load in internal organs and increased the survival. In conclusion, the findings underscore the promising antibiofilm properties of cannabidiol against E. faecalis, indicating its potential as a novel therapeutic agent.

CARACTERIZACIÓN DE SISTEMAS DE PRODUCCIÓN LECHERA Y SALUD DE LA UBRE EN EL NORTE DE ANTIOQUIA, COLOMBIA

<p><strong>Background: </strong>The sociodemographic characterization of milk producers allows knowing their particular conditions in terms of social, political and economic structure through indicators that describe them as social actors, interaction networks, conflict situations and events related to milk production. <strong>Objective:</strong> To characterize the diversity of dairy production systems in Northern Antioquia with a focus on the producer, the farms, access to services and their impact on the biological indicators of udder health. <strong>Methodology:</strong> A cross-sectional study was designed <span style="text-decoration: line-through;">that</span> including 216 milk producers within distributed in 9 dairy municipalities in northern Antioquia. Surveys to characterize the producer, the farm and access to services for health and milk production were carried out. As biological indicators of udder health, the annual average<span style="text-decoration: line-through;">s</span> of bulk tank somatic cell counts (BTSCC) and colony-forming units (CFU) of each farm were analyzed. Statistical analyzes were performed with SPSS version 25.0 and a p < 0.05. <strong>Results: </strong>The biological indicators of udder health in the studied farms presented poor sanitary quality according to the BTSCC for 67 % of the producers and poor hygienic quality with CFU in for 22 % of the participating farms. Briceño producers showed the lowest production in contrast to Santa Rosa de Osos milk producers, who have the highest production. In the linear regression models, 18.2 % of the BTSCC was explained by the location in relation to the municipality, the size of the production system and the type of milking. The CFU values were explained in 29.8 % by the municipality, the cooperative association or livestock association, the milk storage system, the offer of laboratory services for milk and cows, the area of the farm dedicated to grazing, the number of animals on the farm and the average daily production per cow. <strong>Implications:</strong> This study shows the importance of understanding udder health issues not only at the cow level from a biomedical approach. The understanding of udder health from the farm in the sociodemographic context shows that its location, access to services, technology, education and associativity establish opportunities to improve production and competitiveness of milk production. <strong>Conclusions:</strong> udder health can be positively or negatively affected by the location of the production system, the size of the production system, the type of milking, the association to a cooperative or livestock association, the milk storage system, the supply of laboratory services for milk and cows, the area of the farm dedicated to grazing, the number of animals on the farm and the average daily production per cow. To consider these variables is fundamental in decision-making in mastitis control and preventive care for udder health.</p>

Quality Control in Human Adipose-Derived Stromal/Stem Cells and Tissue Engineering Fat Models for Aging Studies.

Adipose tissue is recognized not only as an endocrine organ but also as a reservoir for adipose-derived stromal/stem cells (ASCs). ASCs have stimulated the interest of both the scientific and medical communities due to their therapeutic potential and applications in tissue engineering and regenerative medicine. ASCs are leveraged for their multipotency and their paracrine function. ASC behavior is highly variable and donor dependent. Donor age, body mass index, disease status, sex, and ethnicity can lead to differential overall function and quality. The impact of donor age and passage on ASC behavior has been well documented, impacting cell proliferation and differentiation potential and thus must be taken into careful consideration when conducting in vitro studies. Pooling of ASCs from different donors reduces heterogeneity among individual donors and produces ASCs with a consistent differentiation and paracrine profiles, an advantage for studies in biological aging. This chapter provides a detailed overview for studies related to quality control for ASC pools considering biological and chronological aging in ASCs. There are hallmarks of biological aging and specific assays associated with the evaluation of each hallmark. Nevertheless, here we present the assays that provide a standardized characterization and qualification of donor pools for their regenerative potential, considering chronological and biological age of the pool. The assays included in this chapter are considered quality control standards to evaluate cell proliferation, differentiation, colony-forming units, and cellular senescence from different donor age and cell passage cohorts.

Abstract 3179: Spindle assembly checkpoint as a therapeutic target for TP53-mutated myeloid neoplasms

Abstract Introduction TP53-mutated (TP53mut) myeloid neoplasms (MN) are a distinct entity of myeloid neoplasms with no effective therapies and poor survival (Grob et al, Blood 2022). The TP53-deficient state facilitates the downstream dysfunction of the spindle assembly checkpoint (SAC), predisposing to genomic instability. Recently, a SAC protein, threonine tyrosine kinase (TTK), has emerged as an attractive therapeutic target. Upregulation of TTK has been described in various solid tumors. It is stipulated that targeting SAC proteins might result in extreme level of genomic instability that even cancer cells could not tolerate. We hypothesized that inhibition of TTK with a small molecular inhibitor—TTKi may offer a promising therapeutic strategy for TP53mut MN. Method We evaluated relative TTK mRNA expression and its association with TP53mut and complex karyotypes (CK) in the BEAT AML cohort (Tyner et al., 2018). We used myeloid cell lines, OCI-AML-3, THP-1, and U937 cell lines that harbor-near diploid, moderately aneuploid, and highly aneuploid karyotype, respectively. The efficacy of TTKi CFI-402257 (AbMole) was tested using cell viability, apoptosis, and colony-forming unit (CFU) assays using the standard protocol. Finally, we tested the efficacy of CFI-402257 using the diagnostic bone marrow mononuclear cell (BMMC) cells from TP53mut MN patients. Results Among 591 AML patients in the BEAT AML cohort (41.6% diploid, 35.9% non-CK, 22.7% CK), TTK mRNA expression was progressively higher with increasing chromosomal abnormality burden. TTK expression was 1.96-fold higher in TP53mut AML compared to TP53wt AML (P=0.0008). Further stratified by TP53mut VAF, there was a trend towards higher expression of TTK in TP53mut VAF≥50 compared to the cases with VAF<50 (P=0.06). Next, the treatment with TTKi (CFI-402257) led to a dose-dependent reduction in cell viability across all cell lines, with TP53mut cells (U937 and THP-1) being more sensitive than TP53-wildtype (TP53wt) OCI_AML-3 cells. Similarly, TTKi inhibited the CFU potential of THP-1, U937, and OCI-AML-3 cells. Finally, TTKi induced dose-dependent apoptosis in TP53mut MN patient BMMC but not in healthy donors, resulting in a significant reduction in colony-forming potential. Conclusion Overexpression of TTK was strongly associated with TP53mut and CK in AML. Inhibition of TTK-induced dose-dependent apoptosis in all myeloid cell lines tested with TP53mut highly aneuploid cell line (U937)—traditionally the most chemorefractory phenotype—being the most sensitive. TTKi induced preferential and dose-dependent inhibition of viability and cell proliferation potential in TP53mut MN compared to healthy donor cells, implying a therapeutic window. Further pre-clinical and clinical studies are indicated to evaluate SAC inhibition as a therapeutic strategy for TP53mut MN. Citation Format: Arini Arsana, Huixing Huang, Arief Al-Kali, Mithun Shah. Spindle assembly checkpoint as a therapeutic target for TP53-mutated myeloid neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3179.

Effect of supplementary sodium hypochlorite agitation techniques on an exvivo oral multispecies biofilm during passive disinfection of simulated immature roots.

To compare the effect of different sodium hypochlorite (NaOCl) agitation techniques on an exvivo oral multispecies biofilm during passive disinfection of simulated immature roots. Extracted human teeth were prepared to simulate immature roots. They were infected with a dental plaque-derived multispecies biofilm and cultured for 14 days. The roots were randomly designated into four groups: (1) negative control (PBS), (2) 1.5% NaOCl (CNI), (3) CNI + Ultrasonic activation (UA), (4) CNI + EasyClean agitation (ECA), (5) CNI + XP-endo finisher agitation (XPF), and (6) positive control (6% NaOCl). Biofilm samples were collected from the root canals and used to determine the number of viable cells (colony-forming units), scanning electron microscopy, and 16S rRNA gene sequencing. The mean colony-forming units per mL (CFU/mL) were analysed using One-way anova. 16S rRNA sequencing data were analysed for alpha (observed OTUs, Shannon index, and Chao1) and beta diversity (Bray-Curtis dissimilarities). The LEfSe analysis was used to determine the effect of treatment procedures on the abundance of root canal microbiota. The significance was set at .05. PBS and CNI samples had significantly higher CFU/mL counts than UA, ECA, XPF, and 6% NaOCl samples (p < .05). The pre-treatment, PBS, and CNI groups had significantly greater alpha diversity than the UA, ECA, XPF, and 6% NaOCl groups (p < .05). NaOCl agitation groups and the 6% NaOCl group achieved a more pronounced reduction in bacteria from the genera Fusobacterium, Actinomyces, Porphyromonas, and Capnocytophaga. The effectiveness of passive disinfection protocols was enhanced by NaOCl agitation techniques, suggesting that this supplementary method can improve the outcome of revitalization procedures.

In Vitro Characterization of Fat Grafts Processed Using the REVOLVE ENVI System versus Decantation

Background: This preclinical study evaluated benchtop/in vitro properties and fat viability and activity of grafts processed using the REVOLVE ENVI 600 system compared with decantation and evaluated properties of REVOLVE ENVI waste. Methods: Lipoaspirate from six donors was processed using REVOLVE ENVI or decantation. The composition of each graft, hematocrit/red blood cell content, fat particle size/macrostructure, viable adipocyte count, and adipocyte activity were analyzed. Stromal vascular fraction was analyzed for viable progenitor cell count and colony-forming units. Results: REVOLVE ENVI grafts had a higher mean (±SD) fat content at 85.6% ± 6.1% than decanted grafts at 72.1% ± 4.0% (P &lt; 0.001), with negligible free oil (0.4% ± 1.1%) and cellular debris (&lt;0.1%), whereas REVOLVE ENVI waste contained primarily aqueous fluid (91.0% ± 2.2%) with negligible viable fat. REVOLVE ENVI grafts had significantly lower hematocrit levels (P &lt; 0.001) and contained significantly more large fat globules (P &lt; 0.001) than decanted grafts or REVOLVE ENVI waste. The percentage of tissue particles of more than 1000 µm was highest for REVOLVE ENVI grafts at 61.6% ± 9.2% (decantation: 52.5% ± 13.4%; REVOLVE ENVI waste: 0.49% ± 1.50%), and the percentage of particles less than 200 µm was lowest for REVOLVE ENVI grafts at 15.7% ± 2.6% (decantation: 32.2% ± 8.9%; REVOLVE ENVI waste: 97.9% ± 4.5%). REVOLVE ENVI grafts contained 145.2% ± 36.0% more viable adipocytes, 145.7% ± 46.2% greater activity, 195.5% ± 104.2% more progenitors in SVF, and 363.5% ± 161.2% more SVF colony-forming units than decanted grafts. Conclusion: Fat grafts processed using REVOLVE ENVI demonstrated greater viability and activity than decanted grafts in vitro.

Unraveling Hematotoxicity of α-Amanitin in Cultured Hematopoietic Cells.

Amanita phalloides poisonings account for the majority of fatal mushroom poisonings. Recently, we identified hematotoxicity as a relevant aspect of Amanita poisonings. In this study, we investigated the effects of the main toxins of Amanita phalloides, α- and β-amanitin, on hematopoietic cell viability in vitro. Hematopoietic cell lines were exposed to α-amanitin or β-amanitin for up to 72 h with or without the pan-caspase inhibitor Z-VAD(OH)-FMK, antidotes N-acetylcysteine, silibinin, and benzylpenicillin, and organic anion-transporting polypeptide 1B3 (OATP1B3) inhibitors rifampicin and cyclosporin. Cell viability was established by trypan blue exclusion, annexin V staining, and a MTS assay. Caspase-3/7 activity was determined with Caspase-Glo assay, and cleaved caspase-3 was quantified by Western analysis. Cell number and colony-forming units were quantified after exposure to α-amanitin in primary CD34+ hematopoietic stem cells. In all cell lines, α-amanitin concentration-dependently decreased viability and mitochondrial activity. β-Amanitin was less toxic, but still significantly reduced viability. α-Amanitin increased caspase-3/7 activity by 2.8-fold and cleaved caspase-3 by 2.3-fold. Z-VAD(OH)-FMK significantly reduced α-amanitin-induced toxicity. In CD34+ stem cells, α-amanitin decreased the number of colonies and cells. The antidotes and OATP1B3 inhibitors did not reverse α-amanitin-induced toxicity. In conclusion, α-amanitin induces apoptosis in hematopoietic cells via a caspase-dependent mechanism.

Periodontal ligament stem cell colony-forming units of fluorosed and nonfluorosed teeth: An in vitro study

Abstract Introduction: The continued quest for extracting possible differences in the colony forming units (CFUs) of periodontal ligament stem cells (PDLSCs) between fluorosed and nonfluorosed, led to the current original project to evaluate if any such difference exists. Materials and Methods: For this study, the soft tissues adhering to eight healthy fluorosed and seven nonfluorosed extracted teeth were scraped using a curette. Results: The results of this study demonstrate similar CFUs by both fluorosed and nonfluorosed PDLSCs (P = 0.97), but nonfluorosed PDLSCs showed higher CFUs. Moreover, this is the first study where CFUs on PDSLCs were compared between fluorosed and nonfluorosed groups. Conclusion: This would influence the regeneration or renewal capacity of PDLSCs following treatment or on the physiologic basis, respectively. This is the first study of its kind; therefore, further studies are in progress on age-related changes in PDLSCs to evaluate the potential markers and various tissue-forming abilities.

CYP26 Activity Maintains the Bone Marrow Niche and Stem Cell Homeostasis

Vitamin A and its active compound, retinoic acid (RA), manifest pleomorphic activities in hematopoiesis. Vitamin A homeostasis is maintained systemically through hepatic CYP26. Emerging data suggests that CYP26 mediated RA metabolism also plays a role in controlling hematopoietic stem and progenitor cells (HSPCs) behavior (Ghiaur G et al. PNAS 2013, Schonberger K et al. Cell Stem Cell 2022). We generated Cyp26a1/ Cyp26b1 inducible knockout (CYP26KO) using ROSA26CreERT Cyp26a1/ Cyp26b1 conditional mice to study the impact on hematopoiesis and the bone marrow microenvironment (BME). Tamoxifen induces complete CYP26KO in these mice and subsequently higher retinoid levels and systemic inflammation (Snyder JM et al. FASEB J 2020). Here, we characterize the hematopoietic, bone and bone marrow (BM) phenotype of these mice. Compared to the CYP26 wild type controls (CTR), we observed leukocytosis and mild anemia as well as signs of extramedullary hematopoiesis in CYP26KO mice (Table). Spleen histology and flow cytometry showed an increased number of all mature elements, including megakaryocytes as well as increased absolute numbers of all hematopoietic progenitor populations (HPCs): LSK, CMP, GMP and MEP (Table). BM analysis showed decreased cellularity and myeloid biased in CYP26KO mice (Table). There was an increased frequency of colony forming unit cell (CFU-C) in the BM of CYP26KO mice but overall preserved absolute numbers when accounted for cellularity (Table). We observed increased frequency of LSK (Table) but decreased numbers of CMP, GMP and MEP in the BM of CYP26KO mice. Both CFU assay and flow cytometry showed myeloid biased HPCs found in the BM of CYP26KO mice (Table). When analyzing the stem cell compartment, short term stem cells and multipotent progenitor cells number were decreased in CYP26KO BM (p&amp;lt;0.01), but the long-term HSCs (LT-HSC) were relatively preserved (0.04 ± 0.01 vs. 0.17 ± 0.04 x10 6/femur, CTR vs CYP26KO, p&amp;lt;0.05). Both flow cytometry and CFU assay confirmed the presence of circulating HPCs in CYP26KO mice (Table). Thus, hematopoiesis of CYP26KO mice migrated away from the BM and into extramedullary tissues. The hematopoietic phenotype was most likely due to the cell extrinsic role of CYP26 since bone marrow transplantation experiments confirmed that a CYP26KO recipient (regardless of the genotype of the hematopoietic cells) and not CYP26 wild type recipient recapitulates the observed hematopoietic findings. This observation, together with necropsy findings of thin, frail bones in CYP26KO mice, prompted a closer look at the BM matrix/microenvironment of these mice. CYP26KO mice had smaller BM cavities, decreased trabecular bone and an increased fat/bone marrow volume ratio (Table). Consistent with these findings, CYP26KO mesenchymal stroma cells (MSC) had no osteoblastic differentiation potential in vitro but showed preserved adipogenic potential. The number of CFU-fibroblasts was decreased in the BM of CYP26KO mice but were found circulating in the PB and were dramatically increased in the spleen of these mice (Table). Single cell RNAseq showed quantitative and qualitative alterations in the BME populations with relative reduction of Cxcl12 abundant MSC2, which explained the qRT-PCR observation of lower levels of Cxcl12 mRNA in MSC isolated from CYP26KO mice. More so, Spp1 mRNA was downregulated in various MSC populations of CYP26KO bone marrow, consistent with their osteopenia and with potential implications for the observed HSPC phenotype. Receptor ligand analysis revealed that various components of the BME have impaired interactions with the extracellular matrix. Thus, the leading hypothesis is that absence of CYP26 leads to a dysregulated BME and to migration of a HSCs-niche unit out of the BM and into secondary hematopoietic organs. Interestingly, scRNAseq experiments uncovered upregulation of S100a8/9 in the CYP26KO neutrophil progenitors, which may be in response to systemic stress. The observed bone alterations (osteopenia and increased BM fat) coupled with noted changes in hematopoiesis (i.e. myeloid bias and inflammatory stress) may represent early signs of aging of the bone and bone marrow and suggest a central role of retinoid homeostasis in this process.

Identification of a Novel Gene Regulatory Element in Human Erythroid Progenitor Cells

Early erythropoiesis involves commitment of multi-lineage progenitors to erythroid progenitor cells and differentiation into erythroid burst-forming unit (BFU-E) cells, then erythroid colony-forming unit (CFU-E) cells. In contrast to terminal erythroid differentiation, regulation of gene expression in early erythropoiesis is poorly defined. To address this knowledge gap, we identified active enhancers in erythroid progenitor cells linked to known disease genes and genes associated with erythrocyte traits in genome wide association studies. Human CD34+ hematopoietic stem and progenitor cells (HSPCs) isolated from mobilized peripheral blood were cultured towards the erythroid lineage. From these cultures, FACS-based methods were utilized to purify four stages of erythroid progenitor cells, termed EP1 to EP4, and five populations of terminally differentiated erythroblasts, from proerythroblasts to orthochromatic erythroblasts. ChIP-seq was performed using antibodies to H3K4me1, H3K27Ac, H3K27me3, and H3K9me2/3. Chromatin accessibility was determined by ATAC-seq. Patterns of gene expression were determined by RNA-seq. Active enhancers were identified using the ModHMM algorithm. Numerous erythroid progenitor cell-specific active enhancers were identified near genes linked to known disease genes and GWAS-associated erythrocyte traits. rs162375, a single nucleotide polymorphism (SNP) linked to mean corpuscular hemoglobin, was in a 475bp Long Terminal Repeat Retrotransposon (LTR) element upstream of the RRP1B gene. The LTR enhancer near the RRP1B gene is a member of the LTR15 family. Transposable elements are a source of novelty in primate gene regulation when they acquire binding sites for critical tissue-specific transcription factors, and then these tissue-specific regulatory regions direct tissue-specific expression of nearby genes. The SNP-containing LTR15 erythroid progenitor-specific enhancer upstream of RRP1B contains 2 conserved GATA motifs and a conserved RUNX motif. The enhancer region had a peak of open chromatin determined by ATAC-seq, and H3K27 acetylation and strong GATA1 binding determined by ChIP-seq. These findings were present only in erythroid progenitor cells, not in earlier hematopoietic cells, terminally differentiating erythroid cells, or other non-erythroid cells. CRISPR-based gene editing was utilized to delete the LTR15 enhancer in CD34+ HSPCs cultured towards the erythroid lineage into erythroid progenitor cells. mRNA expression of RRP1B was decreased to 70.2% of wild type. Luciferase-based reporter gene studies demonstrated the enhancer increased expression of a minimal promoter 3.2-fold over background. Mutation of the GATA and RUNX sites individually or together decreased reporter gene activity to background levels. Bioinformatic analyses identified 291 additional erythroid progenitor cell specific LTR15 element active enhancers, with most containing two GATA motifs and a RUNX motif. The majority of the novel enhancer regions were also marked by progenitor cell-specific ATAC peaks and strong GATA1 binding in ChIP-seq. GATA1 and RUNX, both present in early erythropoiesis, are required to activate the regulatory elements in erythroid progenitor cells. Decreasing levels of RUNX in terminal erythroid differentiation parallels loss of enhancer activity. Since LTR15 first appeared in Catarrhine, old-world monkeys, these data indicate this novel regulatory element has recently evolved to contribute to regulation of gene expression in early erythropoiesis in primates.

The Antioxidant TEMPOL Protects Human Hematopoietic Stem Cells From Culture-Mediated Loss of Functions.

In a steady state, hematopoietic stem cells (HSC) exhibit very low levels of reactive oxygen species (ROS). Upon stress, HSC get activated and enter into proliferation and differentiation process to ensure blood cell regeneration. Once activated, their levels of ROS increase, as messengers to mediate their proliferation and differentiation programs. However, at the end of the stress episode, ROS levels need to return to normal to avoid HSC exhaustion. It was shown that antioxidants can prevent loss of HSC self-renewal potential in several contexts such as aging or after exposure to low doses of irradiation suggesting that antioxidants can be used to maintain HSC functional properties upon culture-induced stress. Indeed, in humans, HSC are increasingly used for cell and gene therapy approaches, requiring them to be cultured for several days. As expected, we show that a short culture period leads to drastic defects in HSC functional properties. Moreover, a switch of HSC transcriptional program from stemness to differentiation was evidenced in cultured HSC. Interestingly, cultured-HSC treated with 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (4-hydroxy-TEMPO or Tempol) exhibited a higher clonogenic potential in secondary colony forming unit cell (CFU-C) assay and higher reconstitution potential in xenograft model, compared to untreated cultured-HSC. By transcriptomic analyses combined with serial CFU-C assays, we show that Tempol, which mimics superoxide dismutase, protects HSC from culture-induced stress partly through VEGFα signaling. Thus, we demonstrate that adding Tempol leads to the protection of HSC functional properties during ex vivo culture.

Factors Influencing the Yield of Progenitor Cells in Bone Marrow Aspiration Concentrate—A Retrospective Analysis of 58 Patients

This study aims to identify the role of subjective factors (age, sex, and comorbidities) and procedure-specific factors (aspiration volume) in influencing the yield of progenitor cells in bone marrow aspiration concentrate (BMAC) harvested from the iliac crest. A retrospective analysis was conducted on 58 patients (male:female = 31:27; mean age: 52.56 ± 18.14 years) who underwent BMAC therapy between January 2020 and June 2021. The factors analyzed include individual factors such as age, sex, and comorbid conditions, and procedural factors such as aspirate volume. The mononuclear cell (MNC) count and colony-forming unit (CFU) assay were used to assess the yield of progenitors in the aspirate. Pearson's correlation test was performed for the age, aspirate volume, and outcome parameters, such as MNC and CFU. We used the chi-square test to analyze the role of sex and comorbidities on cellular yield. The mean volume of aspirate used for BMAC therapy was 66.65 (±17.82) mL. The mean MNC count of the BMAC was 19.94 (±16.34) × 106 cells, which formed 11 (±12) CFUs. Evidence of statistically significant positive associations was noted between the CFUs developed from the BMAC and the MNC count within them (r = 0.95, p < 0.001). The sex of the individual did not play any significant role in MNC count (p = 0.092) or CFUs formed (p = 0.448). The age of the individual showed evidence of a statistically significant negative association with the MNC count (r = -0.681, p < 0.001) and CFUs (r = -0.693, p < 0.001), as did the aspiration volume with the MNC count (r = -0.740, p < 0.001) and CFUs (r = -0.629, p < 0.001). We also noted a significant reduction in the MNC count (p = 0.002) and CFUs formed (p = 0.004) when the patients presented comorbidities. Individual factors such as age, comorbid conditions, and procedure factors such as aspirate volume significantly affected the yield of progenitor cells in the BMAC. The sex of the individual did not influence the yield of progenitor cells in BMAC.

Downregulation of MACC1 facilitates the reversal effect of verapamil on the chemoresistance to active metabolite of irinotecan in human colon cancer cells

The aim of this study is to investigate the reversal effect of verapamil (VER) on chemoresistance to irinotecan (CPT-11) in human colon cancer cells and relevant mechanisms. Cell counting kit-8 (CCK-8) test and colony-forming unit (CFU) experiment results show that VER strengthens the sensitivity of human colon cancer cell line HT29 to CPT-11 but has a small effect on SW480 cells. High-throughput transcriptome sequencing, RT-PCR, and Western blot results show that the inhibition of metastasis-associated in colon cancer-1 (MACC1) expression by VER is the key factor for reversal effect on chemoresistance to CPT-11. Transfection experiments further show that VER can reverse the resistance of human colon cancer cells to SN-38, the active metabolite of CPT-11, when MACC1 is overexpressed. The nude mouse transplantation tumor experiment provides an in vivo proof that VER can strengthen sensitivity to CPT-11 in drug-resistant human colon cancer cells, and the effect might be related to the inhibited expression of MACC1. In summary, VER might strengthen the reversal effect of VER on chemoresistance to CPT-11 in human colon cancer cells and facilitate the apoptosis of human colon cancer cells by downregulating MACC1 expression.

Study of the variation of the Malassezia load in the interdigital fold of dogs with pododermatitis

The yeast Malassezia pachydermatis is a common inhabitant of the skin and mucosae of dogs. However, under certain circumstances this yeast can overgrow and act as an opportunistic pathogen causing otitis and dermatitis in dogs. Canine pododermatitis is a common disorder in dogs in which M. pachydermatis acts as an opportunistic pathogen. In the present study, the presence of Malassezia yeasts was assessed and quantified in samples collected from the interdigital space of dogs with pododermatitis before and after treatment, and from healthy dogs. The samples were subjected to two different cytological examinations, culture on Sabouraud glucose agar and modified Dixon’s agar and a quantitative PCR targeting the internal transcribed spacer (ITS) genomic region. A selection of samples was analyzed by next generation sequencing (NGS) using the D1D2 domain of the large subunit of the ribosomal DNA as target. The pododermatitis samples before treatment showed higher cell counts, colony-forming units and ITS copies than the rest of samples. The NGS analysis revealed that Ascomycota was the main phylum in the healthy and post-treatment samples. However, Basidiomycota and M. pachydermatis was more abundant in the pododermatitis samples before treatment. These results support M. pachydermatis as an opportunistic agent in canine pododermatitis by a variety of methods, and demonstrate the correlation between cytologic and molecular methods for quantification.

Comparison of Cellular and Differentiation Characteristics of Mesenchymal Stem Cells Derived from Human Gingiva and Periodontal Ligament.

ABSTRACTObjectives:Dental tissues possess multipotent stem cells with varying biological properties. The present study was aimed to establish a primary culture of human gingiva-derived mesenchymal stem cells (GMSCs) and periodontal ligament-derived stem cells (PDLSCs) from periodontally healthy subjects and compare their biological characteristics.Materials and Methods:Gingival and periodontal ligament (PDL) tissues were collected from extracted premolar teeth of five healthy subjects and primary cultures were established. Basic biological characteristics, such as cell morphology, viability, proliferation capacity, and colony-forming units, and in vitro osteogenic and adipogenic differentiation potential were performed at passage 3 of GMSCs and PDLSCs. This was followed by immuno-phenotyping and flow cytometric analysis for identification of positive mesenchymal stem cell (MSC) markers, such as CD73, CD90, and CD105, and negative markers CD45 and CD34.Statistical Analysis Used:One-way analysis of variance (ANOVA).Results:Primary cultures of GMSCs and PDLSCs were successfully established. Cells exhibited a fibroblast-like morphology with a homogeneous population at passage 3. Cells derived from both tissues were highly viable (>95%), proliferative, and capable of forming colonies. Both cells did not exhibit any noticeable differences in cellular properties. Immunofluorescence and flow cytometric analyses showed positivity for MSC markers, CD73, CD90, and CD105, and negativity for CD34 and CD45. Furthermore, GMSCs and PDLSCs were capable of differentiating in vitro into osteocytes as evidenced by Alizarin red-S staining, and adipocytes as demonstrated by oil red O staining.Conclusions:The results of the present study indicate that both GMSCs and PDLSCs have similar cellular characteristics and mesenchymal differentiation potential. Therefore, they may serve as an equally potent source of stem cells for use in cell-based periodontal therapies.

Role of agglutinin-like sequence protein 3 (Als3) in the structure and antifungal resistance of Candida albicans biofilms.

Agglutinin-like sequence protein 3 (Als3) is a cell surface glycoprotein of Candida albicans that plays essential roles in the processes of adherence and biofilm formation in vitro. In this study, we focused on the contribution of Als3 to the structure and drug susceptibility of biofilms. The C. albicans wild-type (WT) strain DAY185, the als3Δ/Δ null strain and the als3Δ/Δ + pALS3 complemented strain were used. Colony-forming unit enumeration, crystal violet and cell surface hydrophobicity assays, scanning electron microscopy and confocal laser scanning microscopy coupled with analyses using COMSTAT software were performed to evaluate the biomass and architecture of the biofilms. The detailed architectural analysis showed a significant variation in the biofilm parameters of the als3Δ/Δ biofilms compared with those of the WT biofilms. Fluconazole, miconazole and amphotericin B were selected as the antifungal agents for the antimycotic susceptibility test, and increased susceptibility was found with the ALS3 deletion biofilms. A quantitative real-time polymerase chain reaction analysis showed downregulation of biofilm formation-related genes (ALS1, EFG1, HWP1 and CSH1) and drug resistance-related genes (ERG11, CDR1, CDR2 and MDR1) in the als3Δ/Δ biofilms. We concluded that Als3 contributes to biofilm formation by changing the biofilm architecture and is involved in the antifungal resistance of C. albicans biofilms.

Iron Overload Results in Poor Graft Function after Allogeneic Hematopoietic Stem Cell Transplantation By Impairing Hematopoiesis

Background:Clinical data suggest that pre-transplant iron overload due to high transfusion requirements may affect graft function after allogeneic hematopoietic stem cell transplantation (allo-HSCT) .OBJECTIVE:To determine the impact of iron overload and its underlying mechanism on poor graft function (PGF) .METHODS:In the setting of allo-HSCT, a BALB/C mice and a C57/BL6 (H-2b) were used as recipient and donor, respectively. Recipient mice were injected intraperitoneally with iron sucrose with the cumulative doses of 50 mg, 100 mg, and 200 mg in three experimental groups, while the control with phosphate-buffered saline. All mice received total body irradiation for HSCT. Plasma iron levels and Iron stores of liver, spleen and bone marrow (BM) were measured. Hematological parameter, CD34+ cells and the hematopoietic colony-forming capacity were monitored to evaluate the hematopoietic recovery. Bone marrow derived mesenchymal stem cells (BM-MSCs) were isolated and assessed for proliferation. Immunohistochemical analysis assessed the BM microenvironment, expression of haematopoietic chemokines and immunological parameters.RESULTS:Iron overload aggravated PGF as observed by the results that peripheral blood cells were significantly lower in number and recovered less rapidly with a dose-dependend manner, compared with that of controls (P<0.05). In the experimental groups, BM was hypocellular, CD34+ cell counts and colony-forming units were significantly decreased (P<0.05). BM-MSCs showed a longer double time than control (P<0.05). Furthermore, the immunohistochemical analysis demonstrated the immunological impairment and the low expression of stem cell factor -1 and vascular endothelial growth factor-1.CONCLUSION:Current evidence shows that iron overload is an important risk factor for PGF by damaging hematopoietic stem cells and the microenvironment, which highlights the need for optimizing iron management to improve the transplantation outcomes. DisclosuresNo relevant conflicts of interest to declare.

Effect of sodium chloride on the expression of genes involved in the salt tolerance of Bacillus sp. strain “SX4” isolated from salinized greenhouse soil

Abstract Salt stress is one of the important adverse conditions affecting bacterium growth. How bacteria isolated from greenhouse soil cope with salt stress and regulate the genes responsible for salt tolerance are still unclear. We conducted RNA transcriptome profiling of genes contributing to the salt tolerance of a Bacillus sp. strain (“SX4”) obtained from salinized soil. Results showed that NaCl effectively regulated the growth of “SX4” in terms of cell length and colony-forming unit number decrease. A total of 121 upregulated and 346 downregulated genes were detected under salt stress with reference to the control. The largest numbers of differential expression genes were 17 in carbon metabolism, 13 in the biosynthesis of amino acids, 10 in a two-component system, and 10 in ABC transporter pathways for adapting to salt stress. Our data revealed that cation, electron and transmembrane transport, and catalytic activity play important roles in the resistance of bacterial cells to salt ions. Single-nucleotide polymorphism and the mutation of base pair T:A to C:G play potential roles in the adaptation of “SX4” to high NaCl concentrations. The findings from this study provide new insights into the molecular mechanisms of strain “SX4” and will be helpful in promoting the application of salt-tolerant bacteria.

Telmisartan Inhibits the NLRP3 Inflammasome by Activating the PI3K Pathway in Neural Stem Cells Injured by Oxygen-Glucose Deprivation.

Angiotensin II receptor blockers (ARBs) have been shown to exert neuroprotective effects by suppressing inflammatory and apoptotic responses. In the present study, the effects of the ARB telmisartan on the NLRP3 inflammasome induced by oxygen-glucose deprivation (OGD) in neural stem cells (NSCs) were investigated, as well as their possible association with the activation of the PI3K pathway. Cultured NSCs were treated with different concentrations of telmisartan and subjected to various durations of OGD. Cell counting, lactate dehydrogenase, bromodeoxyuridine, and colony-forming unit assays were performed to measure cell viability and proliferation. In addition, the activity of intracellular signaling pathways associated with the PI3K pathway and NLRP3 inflammasome was evaluated. Telmisartan alone did not affect NSCs up to a concentration of 10μM under normal conditions but showed toxicity at a concentration of 100μM. Moreover, OGD reduced the viability of NSCs in a time-dependent manner. Nevertheless, treatment with telmisartan increased the viability and proliferation of OGD-injured NSCs. Furthermore, telmisartan promoted the expression of survival-related proteins and mRNA while inhibiting the expression of death-related proteins induced by OGD. In particular, telmisartan attenuated OGD-dependent expression of the NLRP3 inflammasome and its related signaling proteins. These beneficial effects of telmisartan were blocked by a PI3K inhibitor. Together, these results indicate that telmisartan attenuated the activation of the NLRP3 inflammasome by triggering the PI3K pathway, thereby contributing to neuroprotection.