Number Of Living Cells Research Articles

Effect of Seasonality for Optimization of Germ Cell Extraction from Mature Blue Catfish

AbstractXenogenesis is an innovative tool for hybrid catfish (female Channel Catfish Ictalurus punctatus × male Blue Catfish I. furcatus) seed production, accomplished by transplanting undifferentiated germ cells derived from a donor diploid fish into a sterile recipient, which then enables recipient fish to produce donor‐derived gametes. There is potential to collect donor‐derived germ cells from mature fish during certain times of the year depending upon seasonal temperature and serum sex steroid hormonal fluctuations. The objective of this study was to evaluate seasonal variations in germ cell counts and serum sex steroid hormonal profiles in mature Blue Catfish. Mature fish were collected monthly over the full annual cycle to quantify the number of live germ cells (spermatogonia A, oogonia), viability of germ cells, and levels of serum sex steroid hormones, including testosterone, 11‐ketotestosterone, and 17β estradiol. Extracted spermatogonia A counts were highest from April to June, whereas a significant decline was detected from July to November. Extracted live oogonia counts were highest in April and gradually decreased to zero over the months of May to August. Seasonal variations in serum testosterone, 11‐ketotestosterone, and 17β estradiol followed a similar pattern as the live spermatogonia A and oogonia counts. Even though spermatogonia A counts were relatively lower in mature than in immature Blue Catfish males, extracting spermatogonia A from mature Blue Catfish males during April to June provides an added advantage to the process of artificial fertilization, as it is required to sacrifice these fish to collect sperm.

Significant removal of bacterial biofilm induced by multiple-Short ranges of electric interventions

BackgroundBiofilm-related infections are serious problems in the Orthopedics field, and Staphylococcus aureus are the most popular causative agents of bacterial infections associated with arthroplasty. Several studies demonstrated a synergistic effect of the electric intervention (EI) and the antibiotic administration in killing bacteria in biofilm; however, a constant, long-time EI was needed. In the present study, the effective removal of biofilm formed with S. aureus on a titanium ring by multiple times of one minute-EI was observed and described. MethodsA methicillin-sensitive S. aureus clinical isolate was used to form biofilm on a titanium ring. After applying a series of EI with various combinations of the frequencies and timings, the amount and principal components of biofilms were assessed with crystal violet staining, live bacterial cell count, and fluorescence staining with confocal laser scanning microscopy. ResultsMore than 60% biofilm removal was observed in the 2-time EI applied at 24 (1) and 72 (3) h (days) post bacterial exposure (PBE) and in the 3-time EI at 0 (0), 24 (1), and 72 (3) h (days) PBE, or at 24 (1), 48 (2), and 72 (3) h (days) PBE. The live bacterial cell numbers, the proportion of live and dead cells, and the amount of extracellular polysaccharide substances (EPS) of biofilm were similar with or without EI. It was assumed that an excess amount of the biofilm removal shown in the several EI was not attributed to the effect of the electrolysis. ConclusionsThe effective removal of biofilm was observed when multiple times 1 min EI was applied without any changes in the proportion of live and dead bacteria or the amount of EPS. The mechanisms to explain extra biofilm removal remain to be elucidated.

The Properties of Activated Carbons Functionalized with an Antibacterial Agent and a New SufA Protease Inhibitor.

S. aureus is the cause of many diseases, including numerous infections of the skin. One way to help combat skin infections is to use bandages containing activated carbon. Currently, there are no dressings on the market that use the synergistic effect of activated carbon and antibiotics. Thus, in this study, we point out the adsorption level of an antimicrobial substance on three different active carbons of different origins; by examining the inhibition level of the growth of S. aureus bacteria, we determined the number of live cells adsorbed on activated carbons depending on the presence of gentamicin in the solution. In addition, we designed and synthesized a new antibacterial substance with a new mechanism of action to act as a bacterial protease inhibitor, as well as determining the antibacterial properties conducted through adsorption. Our results demonstrate that activated carbons with adsorbed antibiotics show better bactericidal properties than activated carbon alone or the antibiotic itself. The use of properly modified activated carbons may have a beneficial effect on the development and functioning of new starting materials for bacteria elimination, e.g., in wound-healing treatments in the future.

Inhibition of microbial extracellular electron transfer corrosion of marine structural steel with multiple alloy elements

The microbial corrosion of marine structural steels (09CrCuSb low alloy steel (LAS) and Q235 carbon steel (CS)) in Desulfovibrio vulgaris medium and Pseudomonas aeruginosa medium based on seawater was investigated. In the D. vulgaris medium, the weight loss and maximum pit depth of 09CrCuSb LAS were 0.59 and 0.56 times as much as those of Q235 CS, respectively. Meanwhile, in the P. aeruginosa medium, the values were 0.53 and 0.67 times, respectively. Compared to Q235 CS, 09CrCuSb LAS contains more alloy elements (Cr, Ni, Cu, Al and Sb), which led to obvious inhibition of sessile bacteria growth but had no effect on planktonic bacteria. The number of live sessile cells on the 09CrCuSb LAS surface was 23.4 % and 26.9 % of that on the Q235 CS surface in the D. vulgaris medium and P. aeruginosa medium, respectively. Fewer sessile cells on the steel surface led to a lower extracellular electron transfer (EET) rate so that less corrosion occurred. In addition, the combined effect of alloying elements on grain refinement and passive film formation also improved the anti-corrosion property of the steels.

The Assessment of the Phototoxic Action of Chlortetracycline and Doxycycline as a Potential Treatment of Melanotic Melanoma-Biochemical and Molecular Studies on COLO 829 and G-361 Cell Lines.

Melanoma is still one of the most dangerous cancers. New methods of treatment are sought due to its high aggressiveness and the relatively low effectiveness of therapies. Tetracyclines are drugs exhibiting anticancer activity. Previous studies have also shown their activity against melanoma cells. The possibility of tetracycline accumulation in pigmented tissues and the increase in their toxicity under the influence of UVA radiation creates the possibility of developing a new anti-melanoma therapy. This study aimed to analyze the phototoxic effect of doxycycline and chlortetracycline on melanotic melanoma cells COLO 829 and G-361. The results indicated that tetracycline-induced phototoxicity significantly decreased the number of live cells by cell cycle arrest as well as a decrease in cell viability. The simultaneous exposure of cells to drugs and UVA caused the depolarization of mitochondria as well as inducing oxidative stress and apoptosis. It was found that the combined treatment activated initiator and effector caspases, caused DNA fragmentation and elevated p53 level. Finally, it was concluded that doxycycline demonstrated a stronger cytotoxic and phototoxic effect. UVA irradiation of melanoma cells treated with doxycycline and chlortetracycline allows for the reduction of therapeutic drug concentrations and increases the effectiveness of tested tetracyclines.

Arginase-1 inhibition reduces migration ability and metastatic colonization of colon cancer cells

BackgroundArginase-1 (ARG1), a urea cycle-related enzyme, catalyzes the hydrolysis of arginine to urea and ornithine, which regulates the proliferation, differentiation, and function of various cells. However, it is unclear whether ARG1 controls the progression and malignant alterations of colon cancer.MethodsWe established metastatic colonization mouse model and ARG1 overexpressing murine colon cancer CT26 cells to investigate whether activation of ARG1 was related to malignancy of colon cancer cells in vivo. Living cell numbers and migration ability of CT26 cells were evaluated in the presence of ARG inhibitor in vitro.ResultsInhibition of arginase activity significantly suppressed the proliferation and migration ability of CT26 murine colon cancer cells in vitro. Overexpression of ARG1 in CT26 cells reduced intracellular l-arginine levels, enhanced cell migration, and promoted epithelial-mesenchymal transition. Metastatic colonization of CT26 cells in lung and liver tissues was significantly augmented by ARG1 overexpression in vivo. ARG1 gene expression was higher in the tumor tissues of liver metastasis than those of primary tumor, and arginase inhibition suppressed the migration ability of HCT116 human colon cancer cells.ConclusionActivation of ARG1 is related to the migration ability and metastatic colonization of colon cancer cells, and blockade of this process may be a novel strategy for controlling cancer malignancy.

Maintaining Antibacterial Activity against Biofouling Using a Quaternary Ammonium Membrane Coupling with Electrorepulsion.

Antibacterial modification is a chemical-free method to mitigate biofouling, but surface accumulation of bacteria shields antibacterial groups and presents a significant challenge in persistently preventing membrane biofouling. Herein, a great synergistic effect of electrorepulsion and quaternary ammonium (QA) inactivation on maintaining antibacterial activity against biofouling has been investigated using an electrically conductive QA membrane (eQAM), which was fabricated by polymerization of pyrrole with QA compounds. The electrokinetic force between negatively charged Escherichia coli and cathodic eQAM prevented E. coli cells from reaching the membrane surface. More importantly, cathodic eQAM accelerated the detachment of cells from the eQAM surface, particularly for dead cells whose adhesion capacity was impaired by inactivation. The number of dead cells on the eQAM surface was declined by 81.2% while the number of live cells only decreased by 49.9%. Characterization of bacteria accumulation onto the membrane surface using an electrochemical quartz crystal microbalance revealed that the electrorepulsion accounted for the cell detachment rather than inactivation. In addition, QA inactivation mainly contributed to minimizing the cell adhesion capacity. Consequently, the membrane fouling was significantly declined, and the final normalized water flux was promoted higher than 20% with the synergistic effect of electrorepulsion and QA inactivation. This work provides a unique long-lasting strategy to mitigate membrane biofouling.

Autocrine role of senescent cardiac fibroblasts-derived extracellular vesicles.

Cellular senescence is a highly stable state associated with cell cycle arrest, that is elicited in response to various stresses. The accumulation of senescent cells in tissues drives age-related diseases. Recent studies have shown that the cellular senescence enhances an extracellular vesicles (EV) secretion. EV are lipid-bilayer-capsuled particles released by various cells mediating cell-to-cell communication. It was recently reported that EV secreted by the senescent cells had several functions such as cancer cell proliferation and immune cell activation. In the present study, we investigated whether senescent cardiac fibroblasts-derived EV play an autocrine/paracrine role in the heart cells. Neonatal rat cardiac fibroblasts (NRCFs) were treated with doxorubicin (DOX) to induce cellular senescence. EV were isolated from NRCFs culture media. The vehicle-treated NRCFs-derived EV (D0-EV, 72 hr) increased a living cell number in NRCFs, which was attenuated by DOX (1,000 nM)-treated NRCFs-derived EV (D103-EV, 72 hr). While D0-EV did not affect protein concentration in NRCFs, D103-EV decreased it. Furthermore, D103-EV significantly increased a ratio of microtubule-associated protein 1 light chain 3 (LC3)-II to LC3-I in NRCFs, indicating an induction of autophagy. In addition, D103-EV increased phosphorylation of adenosine monophosphate-activated kinase (AMPK) α in NRCFs. In neonatal rat cardiomyocytes, however, NRCFs-derived EV (72 hr) had no effect on the living cell number, protein concentration, and ratio of LC3-II to LC3-I. In conclusion, we for the first time revealed that DOX-induced senescent NRCFs-derived EV induce autophagy in NRCFs perhaps partly through the activation of AMPKα.

Preclinical Demonstration of a Novel Treatment with High Efficacy and No Detectable Toxicity for Inflammatory Skin Conditions including Psoriasis.

Although the management options for psoriasis have progressed with the use of systemic agents, there are few efficacious nonsteroidal topical therapies for patients with limited or lower grade disease. The effects of allopurinol (Allo) and glutathione (GSH) were examined in two different in vitro models for psoriasis. In the first model, human immortalized keratinocytes (HaCaT) were treated with M5 cocktail (IL-17A, IL-22, oncostatin M, IL-1α, and TNF-α) in four interventional groups (control, Allo, oxypurinol (Oxy), and methotrexate (MTX)). The number of live and dead cells was determined after treatment for 48 and 72 hrs. Allo decreased cell proliferation (total cells) without increasing cell death compared to both its xanthine oxidase inhibiting metabolite Oxy and a standard agent in clinical use, MTX. In the second model, a human psoriatic skin equivalent (PSE) culture system, cells were treated with vehicle control, Allo and GSH (as monotherapies and in combination), and vitamin D (VitD) for 2 and 6 days followed by histological analysis and altered gene expression. The combined exposure to Allo and GSH was equivalent to a standard antipsoriasis agent VitD in the inhibition of both proliferative and replicative markers. Histologic examination of the tissue at 6 days of exposure to VitD resulted in loss of the integrity of the squamous/epithelial continuity whereas tissue integrity was preserved with Allo and GSH exposure. The additional exposure of GSH to Allo reversed the increased thickness of the dermis layer caused by Allo exposure alone. Taken together, this data shows that topical Allo and GSH may have a synergistic effect with low toxicity and constitute a therapeutic advantage over current nonsteroidal therapies in the treatment of inflammatory skin conditions marked by increased cell proliferation such as psoriasis.

Conception and Configuration of Biobank of CART Biospecimen Sets for Pediatrics Research

ABSTRACT Biospecimen sets usually consist of various biospecimens collected in continuous clinical stages, carefully designed in advance by experts with diverse backgrounds according to the potential research purposes of translational medicine. This article reviews the importance and configuration of collecting biospecimen sets from CART clinical trials. A well-designed biospecimen collection plan must be considered seriously before implementing a genetically engineered chimeric antigen receptor T (CART) clinical trial. CART cells have been applied as the last line of defense for children with B-cell malignancies. Although some patients have experienced complete remission after receiving autologous and allogeneic CART cell infusions, challenges remain, particularly regarding the individual differences in curative efficacy. Further studies are needed based on a well-preserved biospecimen set, including an in-depth systematic review of the available data. It is essential and significant to preserve a small number of live CART cells after patients' infusion and collect other traditional forms of biospecimen, such as DNA and serum, from the cases at different stages of treatment, to establish a biobank of CART biospecimen set. This approach can be achieved through the top design of administrative management by integrating teams from both clinical practice and basic research, thus preventing the omission of important data and improper biospecimen preservation. Establishing a biobank of CART biospecimen set, which contains various types of biospecimen at different stages of treatment and the relevant clinical information, are valuable resources for the long-term follow-up study of prognosis and exploring the underlying mechanisms of individual differences of curative efficacy of CART clinical trials.

Potentiation of novel porphyrin based photodynamic therapy against colon cancer with low dose doxorubicin and elucidating the molecular signalling pathways responsible for relapse.

Photodynamic therapy (PDT) is a promising non-invasive treatment modality for cancer and can be potentiated by combination with chemotherapy. Here, we combined PDT of novel porphyrin-based photosensitizers with low dose doxorubicin (Dox) to get maximum outcome. Dox potentiated and showed synergism with PDT under in vitro conditions on CT26.WT cells. The current colon cancer treatment strategies assure partial or even complete tumour regression but loco-regional relapse or distant metastasis is the major cause of death despite combination therapy. The spared cells after the treatment contribute to relapse and it is important to study their behaviour in host environment. Hence, we developed relapse models for PDT, Dox and combination treatments by transplanting respectively treated equal number of live cells to mice (n=5) for tumour formation. Most of the treated cells lost tumour forming ability, but some treatment resistant cells developed tumours in few mice. These tumours served as relapse models and Western blot analysis of tumour samples provided clinically relevant information to delineate resistance strategies of individual as well as combination therapies at molecular level. Our results showed that low dose Dox helped in increasing the tumour inhibiting effect of PDT in combination therapy, but still there are indeed possibilities of relapse at later stages due to chemoresistance and immune suppression that may occur post-treatment. We observed that the combination therapy may also lead to the development of multidrug resistant (MDR) phenotype during relapse. Thus, this study provided clinically relevant information to further strengthen and improve PDT-drug combination therapy in order to avoid relapse and to treat cancer more effectively.

SURVIVAL OF PROBIOTIC LACTOBACILLI IN THE COMPOSITION OF GRANULAR FEED DURING ITS LONG-TERM STORAGE

Bacteria of the genus Lactobacillus are a promising group of microorganisms that can be used as probiotics in aquaculture. An effective method of targeted delivery of these bacteria to the body of aquatic organisms, especially in large farms, is the addition of probiotic preparations directly to the feed. However, there is a problem of long-term storage of feed with added probiotics. In this study, the survival of probiotic microorganisms of the genus Lactobacillus in the composition of granular feed during its long-term storage was evaluated. The probiotic strains of lactobacilli Lactobacillus casei IMV B-7280, L. delbrueckii subsp. bulgaricus IMV B-7281 and L. acidophilus IMV B-7279, kindly provided by the D.K. Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine, were added to Aller Aqua Bronze feed samples, followed by pelletizing and drying and storage for 28 days. A gradual decrease in the number of probiotic microorganisms in all the studied feed samples during the experiment was found. Two weeks after the beginning of the study, the number of live cells of L. casei and L. bulgaricus did not differ statistically from each other and amounted to about 60% of the initial number of cells. In contrast, the number of viable L. acidophilus cells was 52%. The highest survival rate in the granular feed on the 28th day of its storage was observed for probiotic microorganisms L. delbrueckii subsp. bulgaricus IMV V-7281, which constituted 58% of the initial number of introduced bacteria. The lowest survival rate among the studied lactobacillus strains was observed for L. acidophilus. The optimal shelf life of granular feed enriched with probiotics is 14 days from the date of manufacture.

Bacterial decontamination of toothbrushes by immersion in a mouthwash containing 0.05% chlorhexidine and 0.05% cetylpyridinium chloride: A randomized controlled trial.

Toothbrushes are colonized by microorganisms, implying a risk of infection. That risk can be reduced by decreasing the microbial contamination of the filaments. Therefore, this study aimed to determine the antiseptic efficacy of a 0.05% chlorhexidine +0.05% cetylpyridinium chloride mouthwash on toothbrushes. A total of twelve toothbrushes used three times/day for 14 days by orally and systemically healthy people were randomly split into two groups, and their heads were immersed for 2h in PBS (control) or Perio·Aid Active Control (treatment). The microorganisms were recovered, and their number was calculated by culture, quantitative PCR, and viability PCR. Statistical differences were first assessed with a two-way mixed ANOVA and subsequently with Student's t-test. The results showed no statistical differences in the total number of cells for the treatment (mean ± CI95% of 7.27 ± 1.09 log10 bacteria/ml) and the control (7.62 ± 0.64 log10 bacteria/ml) groups, but a significantly lower number of live cells in the treatment group (4.58 ± 0.61 log10 viable bacteria/ml and 2.15 ± 1.42 log10 cfu/ml) than in the control group (6.49 ± 1.39 log10 viable bacteria/ml and 5.04 ± 0.93 log10 cfu/ml). Based on our findings, sanitization of toothbrushes with this mouthwash reduces the number of live microorganisms adhered to the filaments. Such decrease of the bacterial load could include bacteria from the oral cavity, from the environment, and from nearby toothbrushes since the quantification was not limited to any bacterial taxon.

Nitric oxide synthase is required for the pea aphid's defence against bacterial infection.

Compared to other insects, the pea aphid Acyrthosiphon pisum has a reduced immune system with an absence of genes coding for a lot of immunity-related molecules. Notably, nitric oxide synthase (NOS), which catalyses the synthesis of nitric oxide (NO), is present in the pea aphid. However, the role of NO in the immune system of pea aphid remains unclear. In this study, we explored the role of NO in the defence of the pea aphid against bacterial infections and found that the NOS gene of the pea aphid responded to an immune challenge, with the expression of ApNOS observably upregulated after bacterial infections. Knockdown of ApNOS using RNA interference or inhibition of NOS activity increased the number of live bacterial cells in aphids and the mortality of aphids after bacterial infection. Conversely, the increase in NO level in aphids using NO donor inhibited the bacterial growth, increased the survival of bacteria-infected aphids, and upregulated immune genes, such as Toll pathway and phagocytosis related genes. Thus, NO promotes immune responses and plays an important role in the immune system of pea aphid.

Phenotypic and functional characterisation of locally produced natural killer cells ex vivo expanded with the K562-41BBL-mbIL21 cell line.

We characterised the expansion, phenotype and functional activity of natural killer (NK) cells obtained for a clinical trial. Nineteen expansion procedures were performed to obtain NK cell products for 16 patients. NK cells were expanded ex vivo from haploidentical donor peripheral blood mononuclear cells in the presence of the locally generated feeder cell line K-562 with ectopic expression of 4-1BBL and mbIL-21. The median duration of expansion was 18days (interquartile range 15-19). The median number of live cells yielded was 2.26 × 109 (range 1.6-3.4 × 109) with an NK content of 96.6% (range 95.1-97.9%). The median NK cell fold expansion was 171 (range 124-275). NK cell fold expansion depended on the number of seeded NK cells, the initial level of C-myc expression and the initial number of mature and immature NK cells. The majority of expanded NK cells had the phenotype of immature activated cells (NKG2A + , double bright CD56 + + CD16 + + , CD57-) expressing NKp30, NKp44, NKp46, NKG2D, CD69, HLA-DR and CD96. Despite the expression of exhaustion markers, expanded NK cells exhibited high cytolytic activity against leukaemia cell lines, high degranulation activity and cytokine production. There was a noted decrease in the functional activity of NK cells in tests against the patient's blasts.In conclusion,NK cells obtained by ex vivo expansion with locally generated K562-41BBL-mbIL21 cells had a relatively undifferentiated phenotype and enhanced cytolytic activity against cancer cell lines. Expansion of NK cells with feeder cells yielded a sufficient quantity of the NK cell product to reach high cell doses or increase the frequency of cell infusions for adoptive immunotherapy. Registered at clinicaltrials.gov as NCT04327037.

Chicken Serum과 Horse Serum이 닭 근육위성세포의 증식과 분화에 미치는 영향

For efficient production of cultured meat, chicken breast muscle cells and chicken leg muscle cells were proliferated and differentiated using medium with various serums, and proliferation and differentiation ability were analyzed. Chicken breast muscle cells and chicken leg muscle cells were cultured with serum-free medium (NC), medium with 20% FBS (C), medium with 10% chicken serum (CS) and 5% horse serum (HS) (T1), medium with 10% CS (T2), medium with 5% HS (T3). Using the immunofluorescence staining, Pax7 and nuclei were stained in chicken breast muscle cells and chicken leg muscle cells to confirm that they were muscle satellite cells. As a result of cell counting, both cells cultured in T1 medium proliferated the most. Chicken breast muscle cells and chicken leg muscle cells were subcultured from passage 1 to passage 6 using T1 medium. In both cells, viability and number of live cells decreased with increasing passage, but chicken leg muscle cells had higher viability and number of live cells than chicken breast muscle cells in all passages. Cells were differentiated using differentiation medium containing 10% chicken serum and 5% horse serum for each passage. It was confirmed that the differentiation ability of chicken leg muscle cells was more maintained as passage increased than chicken breast muscle cells. Therefore, it is considered that the results of chicken cells will become the basic data for the development of cultured meat.

Novel ROCK Inhibitors, Sovesudil and PHP-0961, Enhance Proliferation, Adhesion and Migration of Corneal Endothelial Cells

The loss or dysfunction of human corneal endothelial cells (hCEnCs) is a leading cause of blindness due to corneal failure. Corneal transplantation with a healthy donor cornea has been the only available treatment for corneal endothelial disease. However, the need for way to regenerate the CEnCs has been increased due to the global shortage of donor corneas. The aim of the study is to investigate whether novel Rho-kinase (ROCK) inhibitors can induce the cultivation and regeneration of hCEnCs. Cultured hCEnCs were treated with Y-27632, sovesudil, or PHP-0961 for 24 h. Cellular responses, including cell viability, cytotoxicity, proliferation, and Ki67 expression with ROCK inhibitors were evaluated. We also evaluated wound healing and cell adhesion assays. Porcine corneas were used ex vivo to evaluate the effects of Y-27632, sovesudil, and PHP-0961 on wound healing and regeneration. We performed live/dead cell assays and immunofluorescence staining for SRY (sex determining region Y)-box 2 (SOX2), β-catenin, and ZO-1 on porcine corneas after ROCK inhibitor treatments. Cell viability, cell proliferation rate, and the number of Ki67-positive cells were higher in Y-27632, sovesudil and PHP-0961 treated cells compared to the control. There was no difference in LDH cytotoxicity test between any groups. Cells treated with Y-27632, sovesudil and PHP-0961 showed faster migration, wound healing, and cell adhesion. In the porcine ex vivo experiments, wound healing, the number of live cells, and SOX2-positive cells were higher in Y-27632, sovesudil and PHP-0961 treated corneas. In all experiments, sovesudil and PHP-0961, the novel ROCK inhibitors, were equal or superior to the results of the ROCK inhibitor positive control, Y-27632. In conclusion, sovesudil and PHP-0961, novel ROCK inhibitors have the capacity to regenerate hCEnCs by enhancing cell proliferation and adhesion between cells.

Efficacy of Combinatorial Treatment Approaches in CTCL

BACKGROUND. Mycosis fungoides/Sezary syndrome (MF/SS) are the most common subtypes of cutaneous T cell lymphoma. The patients (pts) with early-stage MF usually have an indolent course and are managed with skin directed therapies. In contrast, pts with advanced-stage disease, have poor prognosis and a median overall survival between 2 and 5 years. Over the past 20 years only six novel biological/targeted agents (bexarotene, denileukin diftitox, vorinostat, romidepsin, brentuximab vedotin, mogamulizumab) have been FDA approved for systemic therapy of relapsed/refractory MF/SS however, none render curative approach. Overall response rate across all agents is 30% with CR rate of only 10%. Classical chemotherapy has no advantage over less toxic novel targeted agents in MF/SS. The head-to-head comparison of active therapeutic agents in prospective randomized trials is mostly absent and systemic therapy sequencing strategies are usually derived from personal experience, expert opinion, or retrospective reviews. The biomarkers directing the selection of targeted agents for individual patients are not available or reliable. In this study, we tested sensitivity of autologous lymphoma cells obtained from peripheral blood to various therapeutic agents and combinations ex vivo, in order to predict response to tested agents in vivo and develop more sophisticated, personalized, cost-effective, and clinical approach for treatment of pts with advanced stage MF/SS. METHODS. A 384-well plate is coated with poly-L-ornithine to help anchor the cells and the CTCL cells collected from the patients at Moffitt Cancer Center are thawed and incubated over-night at 37C. The next day, isolated CTCL cells are stained with CytoLight Rapid Green Reagent from Essen Bioscience. Once the dye is neutralized and washed out from the cells CytoxRed from Essen Bioscience is added to the media with cells. The cells are then distributed in a 384-well plate. Next, the drugs are diluted and added to the plate. There is a 5-time serial dilution of the drug and if it is a drug combination, one drug is serial diluted and the second drug is constant. After plating, the cells are incubated at 37C for 30min to let them rest and equilibrate. The EVOS M7000 (Life Technologies) is used using the GFP and Texas Red cubes to detect live (green) and dead (red) cells at 0h, 6h, 12h, 18h and 24h. When analyzing the data, the Celleste 5 (Life Technologies) program is used, specifically the Live/Dead application. The intensity threshold is kept constant throughout the entire experiment. The total number of live cells are used to calculate the viability of the cells at each time point. All the time points are done in triplicates and media with DMSO is used as the control. RESULTS. Out of 11 MF/SS pts samples treated with single agent and/or combinatorial drugs, 4 pts samples had no appreciable responses to single or combination treatments. In this study, response to treatment is deemed favorable if there is a decrease of 50% or more in viable cells in the course of the experiment. Seven pts samples did have responses against either a single drug or a combination of drugs. Out of the 7 pts that did respond, the most prevailing treatment was the combination of Venetoclax and Duvelisib with 6 out of 7 pts samples having a response to this combination. The next noteworthy response was seen with Romidepsin alone in 4 out of 7 pts. CONCLUSION. Our observations on MF/SS pts samples thus far demonstrates various sensitivities to treatments for individual pts. This may account for genetic and epigenetic changes which highlight the unmet need for personalization of treatment and sensitivity screening for each patient, with the decisive goal to offer a personalized and efficacious treatment modalities.

Biological Activity of Picrorhiza kurroa: A Source of Potential Antimicrobial Compounds against Yersinia enterocolitica.

Yersiniosis, caused by Yersinia enterocolitica, is the third most rampant zoonotic disease in Europe; the pathogen shows high antibiotic resistance. Herbs have multiple anti-microbial components that reduce microorganism resistance. Therefore, an extract of Picrorhiza kurroa (P. kurroa) was evaluated for potential antimicrobial activity. We report that the ethanolic extract of P. kurroa showed effective antimicrobial activity (zone of inhibition: 29.8 mm, Minimum inhibitory concentration (MIC): 2.45 mg/mL, minimum bactericidal concentration (MBC): 2.4 mg/mL) against Yersinia enterocolitica. Potential bioactive compounds from P. kurroa were identified using LC-MS, namely, cerberidol, annonidine A, benzyl formate, picroside-1, and furcatoside A. P. kurroa showed effective antimicrobial potential in skim milk at different pH, acidity, and water activity levels. P. kurroa affected the physiology of Yersinia enterocolitica and reduced the number of live cells. Yersinia enterocolitica, when incubated with P. kurroa extract, showed lower toxin production. Picroside-1 was isolated and showed higher antimicrobial potential in comparison to the standard antibiotic. Picroside-1 lysed the Yersinia enterocolitica cells, as observed under scanning electron microscopy. Docking revealed that picroside-1 (ligand) showed both hydrophilic and hydrophobic interactions with the dihydrofolate reductase (DHFR) protein of Yersinia enterocolitica and that DHFR is a possible drug target. The high activity and natural origin of Picroside-1 justify its potential as a possible drug candidate for Yersinia enterocolitica.

Post cardiac arrest physical exercise mitigates cell death in the septal and thalamic nuclei and ameliorates contextual fear conditioning deficits in rats

A major concern for cardiac arrest (CA) survivors is the manifestation of long-term cognitive impairments. Physical exercise (PE) is a well-established approach to improve cognitive functions under certain pathological conditions. We previously showed that PE post-CA mitigates cognitive deficits, but the underlying mechanisms remain unknown. To define neuroprotective mechanisms, we analyzed whether PE post-CA protects neurons involved in memory. We first performed a contextual fear conditioning (CFC) test to confirm that PE post-CA preserves memory in rats. We then conducted a cell-count analysis and determined the number of live cells in the hippocampus, and septal and thalamic nuclei, all areas involved in cognitive functions. Lastly, we performed RNA-seq to determine PE post-CA effect on gene expression. Following CA, exercised rats had preserved CFC memory than sham PE animals. Despite this outcome, PE post-CA did not protect hippocampal cells from dying. However, PE ameliorated cell death in septal and thalamic nuclei compared to sham PE animals, suggesting that these nuclei are crucial in mitigating cognitive decline post-CA. Interestingly, PE affected regulation of genes related to neuroinflammation, plasticity, and cell death. These findings reveal potential mechanisms whereby PE post-CA preserves cognitive functions by protecting septal and thalamic cells via gene regulation.