Cell Clumps Research Articles

Distinctive Biological Properties between Mesenchymal Stem Cell Spheroids and Clumps of Mesenchymal Stem Cells/Extracellular Matrix Complexes in 3D Culture Systems

Background: Cells typically function and behave within a three-dimensional (3D) environment. Mesenchymal stem cells (MSCs), known for their self-renewal, multi-lineage differentiation capabilities, and paracrine effects, have garnered significant medical interest. MSC spheroid culture is widely adopted to study the biological properties of MSCs in a 3D context. In contrast, we previously developed 3D clumps of MSC/ECM complexes termed C-MSCs. C-MSCs consisted of cells and self-produced ECM proteins, allowing grafting into tissue defects without any artificial scaffolds. This present study aimed to elucidate the fundamental biological distinctions between 3D MSC spheroids and C-MSCs. Methods: MSC spheroids and C-MSCs are generated from human bone-marrow-derived MSCs. The physical properties, histological structures, and gene expression patterns were compared in vitro. Results: Macroscopic and histological examinations revealed that, whereas MSC spheroids are dense cell clusters primarily formed through Cadherin-mediated cell–cell interactions, C-MSCs are cell aggregates anchored by the ECM component COL1, enabling them to form larger structures. Furthermore, transcriptome analysis showed that C-MSCs possess enhanced capacities to produce immunomodulatory and cytoprotective factors, a prominent biological characteristic of MSCs. Conclusion: Recognizing the distinct attributes of each cell aggregate offers insights into the potential evolution of 3D cell culture techniques and possible therapeutic implications.

In vitro evaluation of the antimicrobial properties of terpinen-4-ol on apical periodontitis-associated bacteria

Manuka oil and tea tree oil are essential oils with known antibacterial properties that are believed to be caused by one main component: terpinen-4-ol. Terpinen-4-ol has potent antibacterial activity against caries-related microorganisms. However, few studies have investigated the antimicrobial effects of terpinen-4-ol on bacteria in apical periodontitis. Thus, the objective of the present study was to evaluate the antibacterial and antibiofilm potential of terpinen-4-ol against Enterococcus faecalis, Porphyromonas gingivalis, Prevotella intermedia, and Fusobacterium nucleatum, which have all been detected in apical periodontitis. The minimum inhibitory and minimum bactericidal concentrations of terpinen-4-ol were determined to assess its activity against biofilms. The minimum inhibitory concentration of terpinen-4-ol was 0.25% against E. faecalis and F. nucleatum, 0.05% against P. gingivalis, and 0.1% against P. intermedia. The minimum bactericidal concentration of terpinen-4-ol was 1.0% against E. faecalis, 0.2% against P. gingivalis and P. intermedia, and 0.5% against F. nucleatum. In the biofilm evaluations, all terpinen-4-ol-treated bacteria had significant reductions in biofilm viability compared with controls in experiments assessing attachment inhibitory activity. Furthermore, structural alterations and decreased bacterial cell clumping were observed under scanning electron microscopy, and significantly decreased cell survival was noted using fluorescence microscopy. Together, these results suggest that terpinen-4-ol is a potential antibacterial agent with bactericidal properties, and can also act on established biofilms.

Cytological changes in respiratory epithelium in association with marijuana smoking

Marijuana is one of the commonly used illicit drugs in Nigeria. This study evaluated the cytological changes in the respiratory epithelium associated with marijuana smoking in Calabar Municipality, Cross River State, Nigeria. This study was a cross- sectional study involving 100 marijuana smokers and 50 marijuana non-smokers, aged 15 years and above. Sputum samples were aseptically collected, and smears prepared. The smears were fixed in ethyl alcohol and were stained using Giemsa and Papanicolaou staining techniques. The stained smears were examined using x10 and x40 objectives of light microscope. Photomicrographs of stained smears were obtained. Data were represented in percentages in tables. Chi-square was used to determine statistical difference between two categorical variables. P-values < 0.05 were considered significant. Marital status, level of education and age of subjects significantly influenced the smoking of marijuana (p=0.001). There was a significant increase in the number of marijuana smokers between the age of 15 – 24(p=0.001) when compared with other age groups. There were also significant abnormalities (p=0.001) observed in the respiratory epithelium of marijuana smokers when compared with non-marijuana smokers. Respiratory cytology smears from marijuana smokers who have smoked for at least 10 years showed mild to moderate metaplasia, numerous acute and chronic inflammatory infiltrates while those who have smoked for more than 10 years showed giant epithelial cells, multinucleated cells and clumps of dysplastic cells. Data from this study showed that marijuana smoking causes inflammatory and dysplastic changes in the respiratory epithelium.

Rapid arsenite oxidation by Paenarthrobacter nicotinovorans strain SSBW5: unravelling the role of GlpF, aioAB and aioE genes.

A novel arsenite resistant bacterial strain SSBW5 was isolated from the battery waste site of Corlim, Goa, India. This strain interestingly exhibited rapid arsenite oxidation with an accumulation of 5mM arsenate within 24h and a minimum inhibitory concentration (MIC) of 18mM. The strain SSBW5 was identified as Paenarthrobacter nicotinovorans using 16S rDNA sequence analysis. Fourier-transformed infrared (FTIR) spectroscopy of arsenite-exposed cells revealed the interaction of arsenite with several important functional groups present on the cell surface, possibly involved in the resistance mechanism. Interestingly, the whole genome sequence analysis also clearly elucidated the presence of genes, such as GlpF, aioAB and aioE encoding transporter, arsenite oxidase and oxidoreductase enzyme, respectively, conferring their role in arsenite resistance. Furthermore, this strain also revealed the presence of several other genes conferring resistance to various metals, drugs, antibiotics and disinfectants. Further suggesting the probable direct or indirect involvement of these genes in the detoxification of arsenite thereby increasing its tolerance limit. In addition, clumping of bacterial cells was observed through microscopic analysis which could also be a strategy to reduce arsenite toxicity thus indicating the existence of multiple resistance mechanisms in strain SSBW5. In the present communication, we are reporting for the first time the potential of P. nicotinovorans strain SSBW5 to be used in the bioremediation of arsenite via arsenite oxidation along with other toxic metals and metalloids.

Surprising Effects of Rocking Motion on Leishmania tarentolae Behavior in Culture and Implications for Cell Stress

Leishmania are an understudied genus of parasitic protozoans causing significant health problems for people, particularly in tropical climates. To better understand the growth of Leishmania and potential drug sensitivity implications, the effects of motion on cells grown in vitro were probed. Using a stock Leishmania tarentolae cell culture, cells were placed in 10 mL of a Brain–Heart Infusion medium in either a non-moving (static) environment or on a flat platform of one of two lab rockers (set at a minimal speed) in a dark environment for 13 days. Also, the addition of 0.5 M of L-Proline was evaluated. Microscopy, cell clumping, cell viability, and secreted acid phosphatase (SAP) activity data were collected. Results show that a constant slow rocking motion changed cell growth, clumping behavior, and detectable SAP activity relative to the no-motion cultures, but this change was dependent on which rocker was used, indicating a complexity in the growth of these cells in culture. Thus, continuous motion affects the stresses placed on the cells during a growth curve under some conditions. The implications of this study lead to questions about the effects of motion on the efficacy of pharmaceutical testing in vitro. Further study of the effects of motion on Leishmania is important.

Luteolin photo-protects zebrafish from environmental stressor ultraviolet radiation (UVB)

ABSTRACT Ultraviolet B wavelength ray radiation (UVB) is an environmental stressor with detrimental effects to the aquatic and human systems but also enhances adverse effects when combined with several other environmental factors such as temperature and pollution. UV rays induce cellular oxidative damage and impair motility. This study aimed to examine the photo-protective activity of flavonoid luteolin against UV-B irradiation-induced oxidative stress and cellular damage using zebrafish. An in-vivo photoaging model was established using UV-B irradiation in zebrafish larvae exposed to 100 mJ/cm2. Data demonstrated that UV-B irradiation of swimming water enhanced production of ROS and superoxide anions as well as depleted total glutathione levels in zebrafish larvae. UV-B irradiation also triggered cellular damage and membrane rupture in zebra fish. Further, 100 mJ/cm2 of UV-B radiation exposure to adult-wild type zebrafish co-exposed with intraperitoneally (ip) injected luteolin upregulated the local neuroendocrine axes by activating vascular endothelial growth factor (VEGF) and elevating levels of pro-inflammatory cytokines IL-1β and TNF-α. Histologically, UV-B irradiation induced skin lesions and locomotory defects with clumping and degeneration of brain glial cells. However, luteolin effectively inhibited the excess production of reactive oxygen species (ROS) and decreased superoxide anion levels induced by UV-B irradiation. Luteolin restored the depleted glutathione levels. In addition, luteolin blocked apoptosis and lipidperoxidation. Luteolin protected adult zebrafish by downregulating the pro-inflammatory cytokine protein expression levels and diminishing VEGF activation. Luteolin also alleviated locomotory defects by inhibiting activation of microglia and inflammatory responses by preventing accumulation of glial cells and vacuolation. Data demonstrate that luteolin may protect zebrafish from UV-B-induced photodamage through DNA-protective, antioxidant and anti-inflammatory responses.

Gene expression and metabolic activity of Streptococcus mutans during exposure to dietary carbohydrates glucose, sucrose, lactose, and xylitol.

Recent RNA sequencing studies have given us a deeper insight into the cariogenic impact of carbohydrate sources in the bacterium Streptococcus mutans, the principal microbial agent in dental caries etiopathogenesis. The process of dental caries development is facilitated by the ability of this bacterium to ferment some carbohydrates into organic acids contributing to a pH decrease in the oral cavity and the demineralization of the hard tissues of the tooth. Furthermore, in dental caries progression, biofilm formation, which starts and ends with free planktonic cells, plays an important role and has several unique properties called virulence factors. The most cariogenic carbohydrate is sucrose, an easily metabolizable source of energy that induces the acidification and synthesis of glucans, forming typical bacterial cell clumps. We used multifaceted methodological approaches to compare the transcriptomic and metabolomic profiles of S. mutans growing in planktonic culture on preferred and nonpreferred carbohydrates and in fasting conditions. Streptococcus mutans in a planktonic culture with lactose produced the same pH drop as glucose and sucrose. By contrast, xylitol and lactose showed high effectiveness in regulating intracellular polysaccharide metabolism, cell wall structure, and overall virulence involved in the initial phase of biofilm formation and structure but with an opposite pattern compared with sucrose and glucose. Our results confirmed the recent findings that xylitol and lactose play a vital role in biofilm structure. However, they do not reduce its formation, which is related to the creation of a cariogenic environment.

Differential Impact of Spread Through Air Spaces on Subtypes of Early-stage Lung Cancer.

To investigate the prognostic significance of STAS (Spread through air spaces) and its effect on survival in the various types of non-small cell lung cancer (NSCLC). Descriptive analytical study. Place and Duration of the Study: Kartal Dr. Lutfi Kirdar City Hospital, Istanbul, Turkiye,between 2018 and 2021. Early-stage lung cancer patients who underwent lobectomy were included. STAS was defined as presence of tumour cell clumps, solid nests or set of single cells located in airway spaces apart from the main tumour border and determined by pathological work-up. The clinical significance of STAS was investigated by means of histopathological subtype, tumour size, and maximum standardised uptake value (SUVmax) on PET-CT scan in early-stage lung cancer by grouping it as adenocarcinoma and non-adenocarcinoma. Five-year overall and disease-free survival, and recurrence were the outcome measures. A total of 165 patients were included in the study. No recurrence was observed in 125 patients, 40 patients developed recurrence. Five-year overall survival (OS) was 69.6% in STAS (+) cohort and 74.5% in STAS (-) cohort (p=0.88). Five-year disease-free survival (DFS) was 51.1% in STAS (+) cohort and 73.1% for STAS (-) cohort (p=0.034). While the absence of STAS in the adenocarcinoma group was associated with better DFS, lower SUVMax and smaller tumour size, similar results were not found to be at statistically significant level in the non-adenocarcinoma group. STAS positivity makes a difference in DFS, tumour size and SUVmax, especially in adenocarcinoma, however, it does not create a significant difference in survival or clinic pathological features in the non-adenocarcinoma. Lung Cancer, Lobectomy, Spread through air spaces, Survival, Prognosis.

Lung Cancer Detection and Classification using Machine Learning Algorithms

Lung cancer is a clump of cells in the lung that are multiplying uncontrollably and improperly. Lung cancer is the deadliest disease, and its cure should be the primary focus of all scientific research. Although it cannot be prevented, we can lessen the danger. Thus, a patient's chance of life depends on the early identification of lung cancer. Several machine learning methods, such as Support Vector Machine, Logistic Regression, Artificial Neural Networks, and Naive Bayes, have been used for the investigation and prognosis of lung cancer. In this paper, Lung cancer prediction is finished by gathering the dataset from the survey and applying machine learning methods such as Support Vector Machine, Nave Bayes, K-Nearest Neighbors, Decision Tree, and Random Forest. With this result, it is revealed that Decision Tree attained the maximum accuracy of 100% as compared to the others.

Evaluation of a Hydroxyethylstarch Solution for Dimethyl Sulfoxide Removal from Mobilized Peripheral Blood using an Automated System

Background and objectives: This study evaluates the efficacy of synthetic colloid hydroxyethyl starch for use as a washing solution to remove DMSO from hematopoietic stem cells cryopreserved grafts in comparison to a crystalloid based solution. 
 Materials and methods: We evaluated samples of cryopreserved mobilized peripheral blood (MPB) from 6 (six) patients that had not been used for transplant. For comparison, we used two equal bags of the same collection procedure, allowing the analysis of two different solutions simultaneously. Washing solutions were used: a crystalloid solution (solution 1, sodium chloride 0.9%) and a colloidal solution (solution 2, hydroxyethyl starch 6%), both added with human albumin 2.5%. The washes were performed using the SEPAX2™ (Biosafe) system automated methodology, using the CS-600.1 kit (Biosafe), according to the washing protocol established by the manufacturer. 
 Results: The washing solution containing HES showed a statistically significant increase in the recovery of CNT and CD34+/CD45+ cells (p = 0.0313, both), in addition to a greater number of CFU-GM colonies (without statistical significance) when compared to the 0.9% sodium chloride solution. Furthermore, the wash solution containing HES also prevented significant clumping, contrary to what was observed in the wash with 0.9% sodium chloride solution. 
 Conclusion: This work shows that the colloidal washing solution containing hydroxyethyl starch is a good option for DMSO removal procedures in samples of cryopreserved mobilized peripheral blood, maintaining the CD34+ cells viability and functionality and reducing the cell clumping.

Modeling the effect of acquired resistance on cancer therapy outcomes

Adaptive therapy (AT) is an evolution-based treatment strategy that exploits cell–cell competition. Acquired resistance can change the competitive nature of cancer cells in a tumor, impacting AT outcomes. We aimed to determine if adaptive therapy can still be effective with cell’s acquiring resistance. We developed an agent-based model for spatial tumor growth considering three different types of acquired resistance: random genetic mutations during cell division, drug-induced reversible (plastic) phenotypic changes, and drug-induced irreversible phenotypic changes. These three resistance mechanisms lead to different spatial distributions of resistant cells. To quantify the spatial distribution, we propose an extension of Ripley’s K-function, Sampled Ripley’s K-function (SRKF), which calculates the non-randomness of the resistance distribution over the tumor domain. Our model predicts that the emergent spatial distribution of resistance can determine the time to progression under both adaptive and continuous therapy (CT). Notably, a high rate of random genetic mutations leads to quicker progression under AT than CT due to the emergence of many small clumps of resistant cells. Drug-induced phenotypic changes accelerate tumor progression irrespective of the treatment strategy. Low-rate switching to a sensitive state reduces the benefits of AT compared to CT. Furthermore, we also demonstrated that drug-induced resistance necessitates aggressive treatment under CT, regardless of the presence of cancer-associated fibroblasts. However, there is an optimal dose that can most effectively delay tumor relapse under AT by suppressing resistance. In conclusion, this study demonstrates that diverse resistance mechanisms can shape the distribution of resistance and thus determine the efficacy of adaptive therapy.

The Effect of Inoculum Size on Antimicrobial Susceptibility Testing of Mycobacterium tuberculosis.

Phenotypic drug susceptibility testing (DST) requires a standardized amount of inoculum to produce reproducible susceptibility results. The most critical step in the application of DST in Mycobacterium tuberculosis isolates is the preparation of the bacterial inoculum. In this study, the effect of bacterial inoculum prepared in various McFarland turbidities on primary antituberculosis drug susceptibility of M. tuberculosis strains was investigated. Five standard ATCC strains (ATCC 27294 [H37Rv], ATCC 35822 [izoniazid-resistant], ATCC 35838 [rifampicin-resistant], ATCC 35820 [streptomycin-resistant], ATCC 35837 [ethambutol-resistant]) were tested. Inoculums of McFarland standard of 0.5, 1, 2, 3, and 1:100 dilutions of 1 McFarland standard of each strain were used. The effect of inoculum size on DST results was determined by the proportion method in Lowenstein-Jensen (LJ) medium and nitrate reductase assay (NRA) in the LJ medium. In both test methods, the increase in inoculum size did not affect the DST results of the strains. On the contrary, DST results were obtained more rapidly as a result of the use of dense inoculum. DST results obtained in all McFarland turbidities were found to be 100% compatible with the recommended amount of inoculum, 1:100 dilution of 1 McFarland standard (inoculum size of gold standard method). In conclusion, the use of a high amount of inoculum did not change the drug susceptibility profile of tuberculosis bacilli. Minimizing manipulations during the inoculum preparation phase of susceptibility testing, this outcome will decrease the need for equipment and make the test application easier, particularly in developing countries. IMPORTANCE During DST application, it can be challenging to evenly homogenize TB cell clumps with lipid-rich cell walls. These experiments must be carried out under Biosafety Level-3 (BSL-3) laboratory conditions with personal protective equipment and taking safety precautions because the procedures applied at this stage cause the formation of bacillus-laden aerosols and carry a serious risk of transmission. Considering this situation, this stage is important given that it is not possible to establish a BSL-3 laboratory in poor and developing countries. Reducing the manipulations to be applied during the preparation of bacterial turbidity will minimize the risk of aerosol formation. Perhaps there will be no need to do these steps for susceptibility tests in these countries or even in developed countries.

A Cartilaginous Construct with Bone Collar Exerts Bone-Regenerative Property Via Rapid Endochondral Ossification.

Three-dimensional clumps of mesenchymal stem cells (MSCs)/extracellular matrix (ECM) complexes (C-MSCs) can be implanted into tissue defects with no artificial scaffolds. In addition, the cellular properties and characteristics of the ECM in C-MSCs can be regulated in vitro. Most bone formation in the developmental and healing process is due to endochondral ossification, which occurs after bone collar formation surrounding cartilage derived from MSCs. Thus, to develop a rapid and reliable bone-regenerative cell therapy, the present study aimed to generate cartilaginous tissue covered with a mineralized bone collar-like structure from human C-MSCs by combining chondrogenic and osteogenic induction. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free (XF) growth medium. Confluent cells that formed cellular sheets were detached from the culture plate using a micropipette tip. The floating cellular sheet contracted to round clumps of cells (C-MSCs). C-MSCs were maintained in XF-chondro-inductive medium (CIM) and XF-osteo-inductive medium (OIM). The biological and bone-regenerative properties of the generated cellular constructs were assessed in vitro and in vivo. C-MSCs cultured in CIM/OIM formed cartilaginous tissue covered with a mineralized matrix layer, whereas CIM treatment alone induced cartilage with no mineralization. Transplantation of the cartilaginous tissue covered with a mineralized matrix induced more rapid bone reconstruction via endochondral ossification in the severe combined immunodeficiency mouse calvarial defect model than that of cartilage generated using only CIM. These results highlight the potential of C-MSC culture in combination with CIM/OIM to generate cartilage covered with a bone collar-like structure, which can be applied for novel bone-regenerative cell therapy.

Abstract 5824: Human ovarian cancer cell seeding density as a mechanism of cisplatin resistance associated with expression of anti-apoptotic and autophagy proteins

Abstract Drug effects may vary depending on cancer cell density. We investigated discrepancies in phenotypes for high density and low-density cultured cancer cells and elucidated relevant variables that influence them. We assessed chemosensitivity as a function of tumor cell density by plating TOV-21G and OVMANA ovarian cancer cell lines in 96-well plates at various seeding densities per well and treated them with various concentrations of cisplatin. CellTiterGlo assays were performed at 72 hours of treatment to determine cisplatin dose response curves. We used ethidium homodimer fluorescence staining and imaging to quantify extent of cell death at differing cell densities of TOV-21G and OVMANA cells treated with cisplatin for 48 and 72hours in 48-well plates. We performed western blot analyses of apoptotic and autophagy proteins from TOV-21G and OVMANA cells that were seeded at high and low cell densities in 24-wellplates. TOV-21G and OVMANA cells plated at higher cell seeding densities displayed less cisplatin sensitivity than TOV-21G and OVMANA cells plated at lower seeding densities, as shown by cisplatin dose response curves. Ethidium homodimer staining of TOV-21G and OVMANA cells treated with cisplatin revealed that cells seeded at lower densities experience a greater amount of cell death that cells seeded at higher densities. Western blot analyses showed that high density TOV-21G cells may express greater amounts of BCL2 and c-FLIP and lower amounts of p62 and NOXA compared to low density cells when normalized for protein expression. Similarly, high density OVMANA cells express greater amounts of c-FLIP andBCL2 and lower amounts of NOXA compared to low density cells when normalized for protein expression. Our experiments reveal that human ovarian cancer cells display different phenotypic changes depending on cell density, causing higher density cells to require increased cisplatin to achieve the same level of effect seen in lower density cells. Ovarian cancer cell resistance to cisplatin is associated with alterations in autophagy and anti-apoptotic protein expression at high cell seeding density. These alterations may also affect chemosensitivity in vivo in settings of differential tumor density, such as single circulating tumor cells compared to clumps of tumor cells, and other settings, such as metastasis. Citation Format: Thomas W. Brown, Ujwal Punyamurtula, Wafik S. El-Deiry. Human ovarian cancer cell seeding density as a mechanism of cisplatin resistance associated with expression of anti-apoptotic and autophagy proteins. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5824.

Resilience and bioresponse of two marine algae to petroleum fuel pollution

ABSTRACT Petroleum pollution can upset the ecological stability and health of a marine ecosystem. The physiological, biochemical and morphological responses of Nannochloropsis oculata and Porphyridium cruentum to three different petroleum fuels, kerosene, diesel and gasoline were examined. The effect of water soluble fractions (WSFs) of the three petroleum fuels was investigated at 0%, 25%, 50% and 100%. The growth response of both species was monitored optically every two days for 14 days using a 721 visible spectrophotometer. Chlorophyll a, morphology and antioxidant enzyme activity of the algae were examined using prescribed methods. In both algae, minimum growth was obtained with 100% WSF of the petroleum fuels. In N. oculata, there was growth stimulation and the maximum growth was obtained at different concentrations (25% and 50%) depending on the test fuels. The maximum growth of P. cruentum was obtained at 10% WSF in all the fuels. ANOVA (p < 0.05) showed significant differences in algal growth with changes in concentration of the test fuels. Unpaired t-tests showed that in all the fuels, there was a significant difference (p < 0.05) between the growth of N. oculata and P. cruentum. N. oculata showed more tolerance to petroleum fuel pollution than P. cruentum. Morphological studies showed that petroleum fuel pollution altered the size of N. oculata and caused severe cell clumping in P. cruentum. Antioxidant concentration assessment showed that whereas N. oculata produced high levels of superoxide dismutase, catalase and peroxidase, P. cruentum produced high levels of superoxide dismutase but was less efficient in catalase and peroxidase production. Clumping and inefficiency in antioxidant production affect the physiological and biochemical response of algae. This study showed that the severity of petroleum fuel pollution is reflected in physiological, morphological, and biochemical responses of the test algae. This research provides baseline information that can be used for the evaluation of the effect of petroleum fuel pollution in marine environment and policy making.

Blood Groups and Behaviour

Early in the twentieth century, a most important discovery was made in blood transfusion when Karl Landsteiner showed that by cross-testing one blood sample with another, some samples would mix successfully with no visual signs of reaction while others would react strongly, causing agglutination, which is a massive clumping of the red cells.

Enhancement of the activity of the antimicrobial peptides HNP1 and LL-37 by bovine pancreatic ribonuclease A.

Background: HNP1, LL-37, and HBD1 are antimicrobial against Escherichia coli ATCC 25922 at the standard inoculum but less active at higher inocula. Methods: The virtual colony count (VCC) microbiological assay was adapted for high inocula and the addition of yeast tRNA and bovine pancreatic ribonuclease A (RNase). 96-well plates were read for 12 hours in a Tecan Infinite M1000 plate reader and photographed under 10x magnification. Results: Adding tRNA 1:1 wt/wt to HNP1 at the standard inoculum almost completely abrogated activity. Adding RNase 1:1 to HNP1 at the standard inoculum of 5x10 5 CFU/mL did not enhance activity. Increasing the inoculum to 6.25x10 7 CFU/mL almost abrogated HNP1 activity. However, adding RNase 25:1 to HNP1 enhanced activity at the highest tested concentration of HNP1. Adding both tRNA and RNase resulted in enhanced activity, indicating that the enhancement effect of RNase overwhelms the inhibiting effect of tRNA when both are present. HBD1 activity at the standard inoculum was almost completely abrogated by the addition of tRNA, but LL-37 activity was only slightly inhibited by tRNA. At the high inoculum, LL-37 activity was enhanced by RNase. HBD1 activity was not enhanced by RNase. RNase was not antimicrobial in the absence of antimicrobial peptides. Cell clumps were observed at the high inoculum in the presence of all three antimicrobial peptides and at the standard inoculum in the presence of HNP1+tRNA and HBD1+tRNA. Conclusions: Antimicrobial peptide-ribonuclease combinations have the potential to be active against high cell concentrations, conditions where the antimicrobial agent alone is relatively ineffective.

Microbial reduction of Fe(III) in nontronite: Role of biochar as a redox mediator

Biochar naturally occurs in soils and sediments as a result of wildfire, but its role in Fe redox cycling is poorly known. In this study, bioreduction experiments were conducted with lactate as the electron donor, Fe(III) in nontronite (NAu-2) or ferric citrate as the electron acceptor, and Shewanella putrefaciens CN32 as the Fe(III)-reducing bacterium in the absence and presence of biochar. High biochar concentrations increased but low concentrations decreased the reduction rate and extent. When NAu-2 was replaced with ferric citrate [aqueous Fe(III)-citrate complex], while the rate was enhanced, the extent of bioreduction remained unchanged at low biochar concentration but declined at high biochar concentration. These different redox behaviors of biochar can be explained by the interplay among four factors: (1) the electron shuttling role of biochar; (2) the electron buffering capacity of biochar; (3) the effect of biochar surface on cell distribution; (4) the effect of biochar on cell attachment and growth. In the absence of biochar, the electron transfer pathway is from cells directly to Fe(III) (Pathway 1). However, in the presence of biochar, there is a second pathway (Pathway 2), i.e., from attached cells to biochar and from biochar to Fe(III). At low biochar concentrations, the presence of biochar decreases the efficiency of Pathway 1 because some cells are diverted to biochar surface, but this deficiency is not made up by Pathway 2 because of physical separation between negatively-charged biochar and NAu-2 particles. Therefore, even though biochar particles receive electrons from attached cells, they are not able to transfer them to NAu-2, thus retaining a proportion of electrons. Possible cell clumping on biochar surface may decrease effective cell concentration and thus pose additional inhibition of NAu-2 bioreduction. However, in the treatment of soluble Fe(III)-citrate complex, Pathway 2 is still effective, because there is no physical barrier between biochar and soluble Fe(III). The electron shuttling role of biochar actually increases the rate of Fe(III) bioreduction.As biochar concentration increases, large surface area of biochar spreads out cells and increases the probability of encounter between cells and NAu-2 particles. In this case, both pathways are effective. Increasing biochar concentrations result in fewer electron retention per gram of biochar. Possible cell growth on biochar surface would further enhance Pathway 2. Therefore, the overall result is the enhanced rate and extent of Fe(III) bioreduction in NAu-2. However, for soluble Fe(III)-citrate complex, both pathways may be impaired because high concentration of biochar would accumulate toxic substances and decrease concentration of cells, which would inhibit Fe(III) bioreduction. As biochar accumulates in soils, the role of biochar is expected to greatly impact Fe redox reactions and other environmental processes.

Molecular targeting of prodigiosin against anti-inflammatory genes cyclooxygenase-1 and -2

The prodigiosin produced by the bacteria Serratia marcescens (SM) has been isolated and optimized with the streak plate method from different natural sources, such as human urine (SM1), soil (SM2), vegetable matter (Cucurbita maxima, SM3) and water (SM4). The isolated prodigiosin was confirmed as SM by its morphological characteristics, genomic size (1.5 kb), identification, and protein purification (4 kDa) using SDS-PAGE. Further, the total cell protein was confirmed and purified through thin layer chromatography (TLC) and high performance liquid chromatography (HPLC). The prodigiosin exhibited good microbial activity against selected Gram-positive bacteria and fungi. The binding efficiency of prodigiosin against cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2) genes was assessed using an in silico molecular docking method that suggested higher binding efficiency for COX-2 than COX-1. Further, prodigiosin was nontoxic to rat islet (RIN-5F) cells in an assay with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylteterazolium bromide) in vitro, and its morphological characteristics of cell shape, clumping, chromatin, and nuclear morphology were normal when viewed under bright light microscopy. Finally, prodigiosin was tested for anti-inflammatory properties against lipopolysaccharide-induced RIN-5F cells. The tests revealed that prodigiosin protected against elution of inflammatory markers like NO, interleukin-6 (IL6), and IL8 in a dose-dependent manner.

S-38-6: DYSFUNCTION OF ATP2B1, ONE OF HYPERTENSION ASSOCIATED GENES, IN CD4+ T CELLS INDUCE COLITIS THROUGH THE CD4+ T-CELL DYSREGULATION

Introduction: ATP2B1 is known as one of the key genes associated with hypertension developments, which were derived from SNPs analysis. ATP2B1 gene encodes the calcium pumps, which excreting calcium ions from cells and maintaining intracellular calcium homeostasis. Lack of ATP2B1 in CD4-positive (CD4+) T-cells increase an intracellular calcium levels, and which results in the activation of immune responses such as differentiation, cytokine production and proliferation. Immune system of gastrointestinal tract plays an important role in preventing viral and bacterial infection from the foreign environment. Especially, CD4+T-cells play critical roles in the fine tune of an intestinal inflammatory response. We hypothesized that ATP2B1 dysfunction in CD4+T-cell may contribute to the gastrointestinal inflammation. We aimed to clarify the role of ATP2B1 in the CD4+T-cell to the gastrointestinal tract. Methods: CD4+T-cell specific knockout of ATP2B1 mice (CD4+T-ATP2B1-KO) were created using the Cre-loxP system (deletion site; exon-10). Lymphocytes were separated from the peripheral blood, spleen, and thymus, and thereafter CD4+T-cells were isolated by the FACS method. Gel electrophoresis-based DNA-sequencing was performed. To quantify mRNA levels, realtime-PCR was performed. Results: ATP2B1 gene in CD4+T-cell of thymus, spleen and blood were amplified by PCR, and DNA-sequencing revealed that the exon-10 of ATP2B1 gene was definitely deleted in the CD4+T-ATP2B1-KO mice. Percentages of CD4+T-cell in the thymus were same between KO and control mice, while those in both spleen and blood of the KO mice decreased significantly compared to control mice. Incidentally, blood pressure in KO mice was lower than that in control. Expression levels of both T-bet and GATA3 in the blood of KO mice were significantly increased more than those of control mice, which possibly indicating that Th1 and Th2 are activating. Interestingly, this KO mice show diarrhea and colon wall thickening while control mice did not. There were mucosal changes such as a reduction of intestinal crypts, thickening of intestinal epithelium, and cell clumps in intestinal epithelium (appearing to be inflammatory cells) in the KO mice colon. Surprisingly, colon length of KO mice was significantly shorter than that of controls even before the diarrhea occurred, and TNF-alpha and gp91 expression was increased more in the colon of KO mice than in controls. Conclusion: Lack of ATP2B1 in CD4+T-cells lead to the reduction of CD4+T-cells and shift to Th1 and Th2 activation, which may contribute to colitis via the activation of TNF-alpha and/or oxidative stress.