Hours Of Incubation Research Articles

Exometabolites of streptomycetes isolated from the Odesa bay exhibit a toxic effect against human cancer cell lines

The aim of the work was to determine the cytotoxic activity of marine actinobacteria exometabolites on the human larynx adenocarcinoma Hep-2 and rhabdomyosarcoma RD cell lines. Exometabolites of 6 strains of Streptomyces sp. were extracted with ethyl acetate and their composition was determined with UltiMate™ 3000 BioRS UPLC System coupled to mass spectrometer. Cytotoxic activity of exometabolites against cancer cells was determined after 24 hours of incubation by microscopic examination of cell morphological changes and the degree of cell monolayer degeneration. Exometabolites from Streptomyces sp. Myt7b strain showed the highest cytotoxic activity and at concentration of 500.0 μg/ml caused up to 90.0% death of RD and Hep-2 cells. The presence of 53 identified compounds with potential cytotoxic activity, including enterocin, 6-prenyltryptophol, medelamine A and turbinaric acid was detected in Streptomyces sp. Myt7b exometabolome. It is concluded that this strain is a promising producer of compounds with anticancer activity. Keywords: cytotoxic activity, exometabolites, marine streptomycetes, RD and Hep-2 cancer cells

Antimicrobial activity of silver-copper coating against aerosols containing surrogate respiratory viruses and bacteria

The transmission of bacteria and respiratory viruses through expelled saliva microdroplets and aerosols is a significant concern for healthcare workers, further highlighted during the SARS-CoV-2 pandemic. To address this issue, the development of nanomaterials with antimicrobial properties for use as nanolayers in respiratory protection equipment, such as facemasks or respirators, has emerged as a potential solution. In this study, a silver and copper nanolayer called SakCu® was deposited on one side of a spun-bond polypropylene fabric using the magnetron sputtering technique. The antibacterial and antiviral activity of the AgCu nanolayer was evaluated against droplets falling on the material and aerosols passing through it. The effectiveness of the nanolayer was assessed by measuring viral loads of the enveloped virus SARS-CoV-2 and viability assays using respiratory surrogate viruses, including PaMx54, PaMx60, PaMx61 (ssRNA, Leviviridae), and PhiX174 (ssDNA, Microviridae) as representatives of non-enveloped viruses. Colony forming unit (CFU) determination was employed to evaluate the survival of aerobic and anaerobic bacteria. The results demonstrated a nearly exponential reduction in SARS-CoV-2 viral load, achieving complete viral load reduction after 24 hours of contact incubation with the AgCu nanolayer. Viability assays with the surrogate viruses showed a significant reduction in viral replication between 2–4 hours after contact. The simulated viral filtration system demonstrated inhibition of viral replication ranging from 39% to 64%. The viability assays with PhiX174 exhibited a 2-log reduction in viral replication after 24 hours of contact and a 16.31% inhibition in viral filtration assays. Bacterial growth inhibition varied depending on the species, with reductions ranging from 70% to 92% for aerobic bacteria and over 90% for anaerobic strains. In conclusion, the AgCu nanolayer displayed high bactericidal and antiviral activity in contact and aerosol conditions. Therefore, it holds the potential for incorporation into personal protective equipment to effectively reduce and prevent the transmission of aerosol-borne pathogenic bacteria and respiratory viruses.

Cell pleomorphism and changes in the enzymatic profile of selected Candida albicans strains in interaction with Escherichia coli – pilot study

Interactions between C. albicans and the microbiota play an important role in maintaining the balance between commensal and pathogenic organisms. Although the exact role of bacteria in reducing the pathogenicity of yeast remains poorly understood, a few examples have been documented so far: probiotics administration effectively reduces the formation of biofilm and bacterial metabolites inhibit the formation of hyphae.The aim of the study was to analyze C. albicans virulence levels based on the changes in the morphological structure and enzymatic profile in experimental cultures mixed with Escherichia coli. Viable cell abundance, cell pleomorphism and enzymatic profile were analyzed in single and mixed cultures (C. albicans + E. coli).The microscope analysis showed a large decrease in the number of viable C. albicans cells in mixed cultures with E. coli from 485.3±132.1 immediately after the establishment of the culture to 238.1±71.2 after an hour of incubation and 24.4±5.4 after 24 h. The length of C. albicans cells differed significantly between the single-species cultures and the mixed cultures for 24 h. Our present findings indicate a significant reduction in the secretion of several enzymes by fungi following contact with E. coli, including acid phosphatase, N-acetyl-β-glucosaminidase, naphthol-AS-BI-phosphohydrolase and leucine arylamidase.The interactions between fungi and bacteria appear to be extremely complex. On the one hand, during C. albicans with E. coli co-incubation, the bacteria stimulated the elongation of yeast cells, leading to the formation of a filamentous form; however, the number of yeast cells and their enzymatic activity decreased significantly. Therefore, it can be concluded that while E. coli stimulates some pathogenic properties, e.g. cell elongation, it also inhibits other virulence features, e.g. enzymatic activity of C. albicans.

The Potential Amoebicidal Effect of Some Milk forms as an Inhibitor for Entamoeba histolytica Viability Isolated from Diarrhea

Milk includes numerous compounds such as lacto-peroxidase, lactoferrin, immunoglobulin G, secretory immunoglobulin A, and Lysozymes The invasive trophozoites of E. histolytica are capable to inhabit host tissues and provoke diseases such as intestinal colitis. The objective of the study was to compare different forms of milk’s effect on E. histolytica trophopzoite by measuring protein concentration in vitro using a spectrophotometer and number of trophozoite by microscopy. For this purpose, E. histolytica trophozoites were exposed to several types of milk (human, sheep, cattle, goats, and formula) at concentrations of 6, 12, 18, and 24% for each type after 8, 16, and 24 hours of incubation. According to the findings, sheep and goat milk had lower total protein concentrations after 8 hours than other types of milk when compared to the control group. After 16 hours of incubation, raise the milk concentration to 24% leading to a reduction in the formula milk's total protein concentration to 5g/l. after 8 and 16 hours of incubation, sheep milk and cattle have significantly lower total protein concentrations (24% vs. 8%), respectively. The action of formula and human milk is increased by 24-hour incubation. Increase the trophozoite inhibition percentage compared to metronidazole in human milk, which was followed by sheep and cattle milk with respective values of 60.9%, 56.1%, and 46.3% respectively. Human milk, goat and formula milk had the greatest inhibitory activity after incubation 24hrs. at 24%.the extent of amoebicidal activity of milk was shown to depend on incubation period and concentration of milk.

Purification and characterization of L-asparaginase produced from Bacillus sp.

The objective of this study was to isolate and identify the asparaginase-producing bacteria, then purify and characterize the enzyme in order to investigate their properties in the future. Fifteen local bacterial isolates were isolated from various sites in the city of Baghdad, identified by conventional morphological and biochemical procedures, and confirmed using vitek 2 methods, and submitted to primary screening processes for asparaginase production. For secondary screening, eight isolates with the greatest yellow zone ability on a specific solid medium were chosen. Bacillus sp. was reported to have the highest enzyme production (7.5 U/mg proteins). After 24 hours of incubation, submerged fermentation yielded optimal conditions for the production of L-asparaginase (L-ASNase) by the chosen isolate, with medium (2) serving as the optimal medium for production and fructose serving as the optimal source of carbon. In pH 6 at 40°C, Sephadex G-150 gel filtration chromatography was used to purify the enzyme. The final purification folds were increased by 2.5 times, resulting in an enzyme yield of 93.7%. It also showed the highest purified enzyme activity and stability was at 37°C. Also it revealed the highest activity and stability at pH 7.0 and pH 8.0 respectively. Enzyme lost activity when exposed to several metallic ions at concentrations of 1, 5, and 10 mM.

Screening of protease-producing lactic acid bacteria from Indonesian fermented fish products and its application in chitin extraction from vannamei shrimp (Litopenaeus vannamei) shell

This study aimed to screen lactic acid bacteria (LAB) from traditional Indonesian fermented fish products, namely wadi and jambal, with high protease activity for chitin production from Litopenaeus vannamei shrimp shells. Twenty-eight colonies were successfully isolated and identified through biochemical tests such as catalase and Gram staining. Seven out of the twenty-eight colonies were identified as lactic acid bacteria based on their Gram-positive and catalase-negative results. These strains from wadi samples were named GWB 1.3, GWB 1.7, and GWB 2.5, while those from jambal samples were named GMJB 1.3, GMJB 1.6, GMJB 2.6, and GMJB 2.7. The protease activity of these strains was tested using the well-diffusion method on skim milk agar to assess their deproteinized activity. The diameter of the clear zone formed after 24, 48, and 72 hours of incubation was measured. GWB 1.7 and GMJB 2.6 showed the largest clear zone diameter with 21.3 mm and 22.5 mm, respectively, and were selected for the single step-fermentation of shrimp shells. The fermentation process was conducted in 6 days with different inoculum ranging from 0%, 5%, 10%, 15%, and 20% (v/v) as treatments. All treatments, including moisture, ash, and protein, were evaluated for proximate content in the final days. The results indicate that the 20% inoculum showed the highest deproteinization and demineralization for both bacterial strains. GWB 1.7 exhibited a demineralization of 43.7% and deproteinization of 25.2%, while GMJB 2.6 showed a demineralization of 39% and deproteinization of 16.1%. This study demonstrated that lactic acid bacteria from wadi and jambal samples could produce chitin from shrimp shells, a valuable biopolymer with various applications.

ВЛИЯНИЕ ФАКТОРОВ РАДИАЦИОННОЙ И НЕРАДИАЦИОННОЙ ПРИРОДЫ НА КОНЦЕНТРАЦИЮ ТBX21 В ЛИЗАТАХ МОНОНУКЛЕАРОВ, СТИМУЛИРОВАННЫХ МИТОГЕНОМ

Purpose: To evaluate the effect of radiation and non-radiation factors on the intracellular concentration of the ТBX21 transcription factor in peripheral blood PHA-stimulated mononuclear cells of chronically exposed residents of the Techa Riverside settlements in the long-term period after the start of exposure. Material and methods: The main group consisted of 30 people aged 67–80 years with the mean dose to the red bone marrow 867±136 mGy, to thymus and peripheral lymphoid organs 125±20 mGy. The comparison group included 10 unexposed people aged 63–82 years. The main and the comparison groups had similar sex and ethnic composition. Peripheral blood mononuclear cells were PHA-stimulated for 24 hours. Cellular lysates were normalized by total protein concentration prior to being used for ELISA test. Results: After 24 hours of incubation the median and interquartile range of the intracellular TBX21 concentration in mononuclear cells was 34.2 (6.6–86.0) pg/ml after the mitogen stimulation, and 0 (0–24.9) pg/ml – without mitogen stimulation (р=0.001). In the comparison group these values made up 24.8 (0.2–47.6) and 13.0 (0–19.2) pg/ml, respectively Conclusion: The intracellular TBX21 concentration after 24-hour mitogen stimulation did not differ statistically significantly in chronically exposed and unexposed people, as well as in people from different dose groups. Statistically significant increase in TBX21 concentration in the lysates of mononuclear cells that were PHA-stimulated for 24 hours relative to non-mitogen stimulated cells was noted in chronically exposed people with medium and high doses to the red bone marrow. No correlation was revealed between the TBX21 concentration and dose characteristics, sex, and ethnicity of the studied individuals. The results are preliminary.

The Effect of Adding Thymol on Rumen Fermentations, the Digestibility of Nutrients and Bacterial Count of Arabi Lamb’s Rumen Fluid in Vitro

This study aimed to investigate the microbial responses to thymol supplementation in the rumen fluid of Arabi lambs and determine the effective dose of thymol on rumen fermentation. Thymol was added at different concentrations (0, 200, 300, 400 mg thymol/L of rumen fluid) with four replicates for each treatment during a 24-hour laboratory incubation period. The Real-Time PCR-DNA technique was used to count the bacteria in the rumen fluid, and the fermentations of the rumen fluid were examined. The results indicated that thymol did not have an effect on the pH and ammonia nitrogen concentration of the rumen fluid. However, the addition of thymol at concentrations of 200, 300, and 400 mg/L showed significant improvements in metabolic energy, total fatty acids, and total gas production compared to the control treatment (0 mg thymol). Methane gas concentration significantly decreased when thymol was added at a concentration of 400 mg/L. The digestibility of organic matter and fiber improved at concentrations of 300 and 400 mg/L, respectively. The digestibility of crude protein was significantly higher (P<0.05) in the control and 200 mg/L treatments compared to the 300 and 400 mg/L treatments. Regarding the number of bacteria, there were no significant differences between the treatments in the average total number of bacteria and the number of fiber-degrading bacteria of the genus Ruminococcus flavefaciens in the rumen fluid. However, the addition of thymol led to a significant increase in the number of fibrous bacteria from the genus Ruminococcus albus. The third treatment (300 mg/L) showed the highest number of Ruminococcus albus bacteria (5.86 × 710 CFU/mL rumen fluid) compared to the first and second treatments (4.55 × 710, 4.71 × 710 CFU/mL rumen fluid, respectively). In conclusion, thymol improved metabolic energy, total fatty acids, digestibility of organic matter and total gas production, while reducing methane gas concentration. The addition of thymol did not significantly affect the total number of bacteria but increased the number of fibrous bacteria from the genus Ruminococcus albus.

#386 : Warming Vitrified Oocytes in a Fraction of the Current Required Time Results in Superior Survival Rates

Background and Aims: Oocyte vitrification and warming is very time consuming. Oocyte vitrification protocols are different from that of embryos or blastocysts, while warming protocols allow all to be warmed the same. For blastocysts, frequently only one device is warmed per patient; however, numerous devices are required in oocyte warming. We aim to evaluate whether vitrified oocytes can successfully survive a warming protocol involving only Thawing Solution (TS) with a total elapsed warming time of a mere 90 seconds. Methods: 58 vitrified donor oocytes (Treatment Group) were prospectively warmed in January 2023. 71 vitrified donor oocytes (Control Group) warmed in 2022 were retrospectively analyzed. Both groups were vitrified in Kitazato or Irvine Scientific Vit Kit with identical protocols (elapsed time approximately 17-minutes). In both groups the TS was equilibrated to 37-degrees for a minimum of 2-hours. The Control Group was warmed in Irvine Scientific Vit Kit (1-minute TS, 3-minute DS, 8-minute WS) for a total warming time of 12-minutes. Warming for the Treatment Group was in Irvine Scientific Vit Kit Warm-NX, TS only, for 90-seconds. Results: After Chi-square analysis, [Formula: see text] value &lt; 0.05 reflected significance. The Control Group warmed with 90.1% survival. The Treatment Group resulted in 98.3% survival, ([Formula: see text] -value = 0.056). Survival at 3-hours post warm in the Control Group was 100%. At this point for the Control Group, ICSI was performed, and the oocytes placed back into the incubator for resulting embryo culture. For the Treatment Group, oocyte survival was re-assessed at 1-hour and 24-hours, and 100% survival was maintained over the course of 24-hour incubation, with little-to-no visible cryo-damage. Conclusions: Vitrified oocytes exhibit outstanding survival when warmed with a highly modified and considerably faster protocol than current practice. The extended oocyte survival of at least 24-hours shows a very favorable outcome for the accelerated warming protocol and gives credibility to the survival assessment.

Pengaruh Infusa Batang Pisang Kepok Kuning terhadap Daya Hambat Pertumbuhan Bakteri Escherichia Coli secara In Vitro

Diarrheal disease is one of the main causes of morbidity and death worldwide. Diarrheal disease is an infection caused by pathogenic bacteriaEscherichia Coli. Kepok banana stems have antibacterial properties that can inhibit bacterial growthE. coli. The purpose of this study was to determine the diameter of the inhibition zone on bacterial growthE. coli. This study used the infusion method by taking the kepok banana stem as the research sample. The inhibition of kepok banana stem infusion was tested by the methodKirby Bauer diffusion using disc paper. This study used kepok banana stem infusion with various concentrations. The concentration used starts from 60%, 70%, 80%, 90% and 100% with 4 repetitions. The positive control used chloramphenicol and distilled water as negative controls. This research is included in the type of researchTrue experimentalby design Posttest-Only With Control group Design. The results in this study stated that no inhibition zone was formed against bacterial growthEscherichia coli.These results are shown by the results of measuring the diameter of inhibition after 24 hours of incubation which is 0 mm and chloramphenicol as a positive control has an average inhibition of 27.6 mm and it is stated that chloramphenicol is sensitive to bacteria E.Coli. This can be caused by the small content of antibacterial substances that can dissolve in aquadest (infusion method). The conclusion is that the infusion of kepok banana stems does not have the effectiveness of inhibiting the growth of bacteria Escherichia Coli.Suggestions for future researchers to conduct this research using samples from other parts of the banana tree and can also use a different extraction method.

Pengaruh Infusa Batang Pisang Kepok Kuning (Musa Paradisiaca Formatypica) Terhadap Daya Hambat Pertumbuhan Bakteri Escherichia coli Secara In Vitro

Diarrheal disease is one of the main causes of morbidity and death worldwide. Diarrheal disease is an infection caused by pathogenic bacteriaEscherichia Coli. Kepok banana stems have antibacterial properties that can inhibit bacterial growthE. coli. The purpose of this study was to determine the diameter of the inhibition zone on bacterial growthE. coli. This study used the infusion method by taking the kepok banana stem as the research sample. The inhibition of kepok banana stem infusion was tested by the methodKirby Bauer diffusion using disc paper. This study used kepok banana stem infusion with various concentrations. The concentration used starts from 60%, 70%, 80%, 90% and 100% with 4 repetitions. The positive control used chloramphenicol and distilled water as negative controls. This research is included in the type of researchTrue experimentalby design Posttest-Only With Control group Design. The results in this study stated that no inhibition zone was formed against bacterial growthEscherichia coli.These results are shown by the results of measuring the diameter of inhibition after 24 hours of incubation which is 0 mm and chloramphenicol as a positive control has an average inhibition of 27.6 mm and it is stated that chloramphenicol is sensitive to bacteria E.Coli. This can be caused by the small content of antibacterial substances that can dissolve in aquadest (infusion method). The conclusion is that the infusion of kepok banana stems does not have the effectiveness of inhibiting the growth of bacteria Escherichia Coli.Suggestions for future researchers to conduct this research using samples from other parts of the banana tree and can also use a different extraction method.

Study of the Effect of CD34+ Umbilical Cord Blood Stem Hematopoietic Cell Microparticles on the Induction of Oxidative Stress in HL-60 Promyelocytic Cells and in Umbilical Cord Blood Mononuclear Cells

Objective Oxidative stress is defined as a disturbance between prooxidant-antioxidant balance and its main cause are molecules or ions such as reactive oxygen species. Antioxidant enzymes have a primary role against oxidative stress, while its effects can be tracked from the oxidation of biological molecules such as fatty acids. Microparticles are small membrane vesicles released from different cell types upon activation or during apoptosis such as platelets, erythrocytes, leucocytes and endothelial cells. Microparticles consist of cytosolic molecules and membrane protein antigens. They can interact with other cells activating multitude functions that are associated with many pathological conditions. The present study aims to investigate a possible correlation between oxidative stress and hematopoietic stem cell derived microparticles in HL-60 and mononuclear cells (MNCs). Methodology The source of MNCs as well as CD34 + microparticles (CD34+MPs) is the umbilical cord blood (UCB). The units (n=17) that were used in this study, were rejected as not appropriate for processing due to low volume. The isolation of the mononuclear cells is accomplished after density gradient centrifugation on lymphoprep. CD34+MPs were isolated from the plasma of UCB after centrifugation and were purified using magnetic bead MACS (Miltenyi Biotec). The number of CD34+ MPs was estimated by flow cytometry using CD34-PE and Annexin V-FITC Abs. Cells are then co-incubated with the CD34+MPs for 24 and 48 hours. Finally, the activity of the antioxidant enzymes superoxide dismutase (SOD), glutathione reductase (GR) and glutathione S-transferase (GST) as well as the concentration of malondialdehyde (MDA) produced by lipid peroxidation are measured in the samples using spectrophotometry. Results The results from the measurements that were conducted, displayed a statistically significant increase in the antioxidant activity of the samples in the presence of CD34+MPs in both ΗL60 and MNCs cells after 24 and 48 hours of incubation. Specifically SOD activity was significantly increase in CD34+MPs compared to the control in HL60 after 24 hrs incubation (4.38x10 -1 vs.2.24x10 -1units/mg) and in MNCs (5.51x10 -1 vs. 3.52x10 -1units/mg) The GR activity was increased significantly in HL60 treated with CD34+MPs compared to the control (4.96x10 -2 vs.3.09x10 -2units/mg) as well as in MNCs (5.25x10 -2 vs.3.23x10 -2 units/mg). Similarly the GST ativity increased in the presence of CD34+MPs compared to the control (6.3x10 -3 vs. 2.96x10 -3units/mg) in HL60 and in MNCs (5.34x10 -3 vs.3.28x10 -3units/mg).The MDA concentration produced by lipid peroxidation was increased in HL60 after CD34+MPs treatment compared to the control (5.59 vs.2.84μΜ after 24 hrs and 10.08 vs.3.43 after 48hrs) and in MNCs (6.34 vs.3.04 after 24 hrs and 8.76 vs.3.52μΜ after 48 hrs incubation). Conclusions In this study we have shown the induction of oxidative stress in the leukemic cell line ΗL60 and in the mononuclear cells from cord blood by CD34 +MPs. Therefore the CD34+ microparticles can promote oxidative stress resulting in the activation of inflammation or apoptosis.

Pyruvate Kinase Thermostability Is Associated with Red Blood Cell Adhesion, Deformability and Oxygen Affinity in Patients with Sickle Cell Disease

Introduction: Sickle cell disease(SCD) is caused by a mutation in the beta globin gene that causes formation of abnormal hemoglobin S (HbS). Red blood cells (RBCs) from patients with SCD are less deformable and take on the shape of sickle upon deoxygenation. This results in multiple downstream effects including RBC adhesion, microvascular occlusion and chronic hemolysis. RBCs rely solely on anaerobic glycolysis to produce adenosine triphosphate (ATP), which is needed to maintain the cell's structure and function. Pyruvate kinase (PK) is a key enzyme involved in the last step of glycolysis. We have previously demonstrated a decrease in PK activity and stability as a novel pathophysiological feature of SCD. Currently there are several clinical trials ongoing investigating the efficacy of PK activation by small molecules such as mitapivat (NL8517, NCT04000165, NCT05031780), AG-946 (NCT04536792) and etavopivat (NCT04624659, NCT04987489). In this study we aim to investigate how PK properties correlate to sickle RBC properties in order to explore if impaired PK function is associated with specific features of the complex SCD pathophysiology. Methods: Homozygous HbS (HbSS) and HbS/ β0 thalassemia patients were eligible to participate. Patients who received blood transfusion (&amp;lt;3 months) were excluded. Dense RBCs (MCHC &amp;gt;41 g/dL, Advia 120/2120 Siemens) and HbF and HbS levels (Tosoh G8) were measured. PK and hexokinase (HK) activity were measured on purified RBCs. PK thermostability (reflected by % residual PK activity) was measured on purified RBCs lysates after one hour of incubation (53°C). RBC adhesion to laminin (Biolamina) was measured using a microfluidic device (IBIDI µ-Slide I 0.4). RBC hydration (Ohyper), RBC deformability (EImax) and point of sickling (PoS) were assessed by osmotic and oxygen gradient ektacytometry using the Laser-Optical Rotational Red Cell Analyzer (Lorrca, RR Mechatronics). Oxygen affinity (P50) was measured with the Hemox-Analyzer (TCS). Statistical analysis was performed in Graphpad Prism (Pearson's correlation). Results: Fifty-seven patients with SCD (53 HbSS, 4 HbS/β 0 thalassemia) were included of which 17 were children (median age 14 years [range 6-17]) and 40 were adults (median age 36 years [range 18-58]). Baseline characteristics are depicted in Table 1. Figure 1 shows a heatmap with correlation coefficients of all laboratory parameters from Table 1 as measured in this cohort. When focusing on PK properties we found that patients with SCD and lower PK thermostability had significantly higher absolute reticulocyte counts (ARC, p&amp;lt;0.0001), lower hemoglobin (p=0.012) and HbF (p=0.020) levels and increased RBC adhesion to laminin (p=0.007). Furthermore, PK thermostability was significantly correlated with EImax (p=0.017) and inversely correlated with PoS (p=0.011) and P50 (p=0.003), indicating that patients with less stable RBC PK had less deformable RBCs which sickled at a higher oxygen tension. RBC adhesion to laminin was significantly correlated with both ARC (p=0.0003) and dense RBCs (p=0.019). To explore whether our findings were a reflection of reticulocytosis we fractionated whole blood samples of HbSS patients according to cell density. We found that more RBCs from the most dense fraction adhered to laminin than from the less dense, reticulocyte enriched, fraction, even though RBCs from this less dense fraction had reduced PK-thermostability. Additionally, the most dense RBCs had the lowest PK activity, were less deformable and had a higher PoS (data not shown). Altogether these findings indicate that the reduced PK thermostability we found in our SCD cohort is associated with an overall functionally impaired RBC population with increased RBC adhesion and ARC. We found no correlation of markers of RBC dehydration (Ohyper and % dense RBCs) with PK thermostability which suggests that RBCs with reduced PK thermostability are not more dehydrated. Discussion/conclusion: This study shows for the first time a significant correlation between PK thermostability in sickle RBCs and RBC functions, such as adhesion, deformability and oxygen affinity. Our results suggest that enhancing the activity and stability of PK, with PK activators, is an attractive target in SCD that might improve other pathophysiological targets outside RBC metabolism. Future studies are needed to explore how activation of PK will affect these altered sickle RBC properties in vivo.

Achromobacter sp. Strain BUKˍBCHˍTQ1: A Potential Paraquat-Degrading Bacterium Isolated from Pesticide Contaminated Agricultural Soil

Paraquat (1, 1′-dimethyl-4, 4′-bipyridinium dichloride) is one of the most frequently used herbicide in agriculture. It is a cationic non-systematic, non-selective contact compound that instantaneously interferes with the photosynthetic processes of plants. It has an immediate effect, once the compound comes into contact with the plants’ leaves, where the reaction occurs. However, the contamination of paraquat residue in soil can harm soil microbes, flora and fauna, farmer health and also soil ecology, which affects the soil fertility. The objective of this study was to isolate and characterize bacteria with the ability to break down and utilize paraquat as the primary carbon source. The isolation process involved the enrichment of mineral salt media (MSM) using serial dilution. The isolated bacterium underwent morphological, biochemical, and molecular identification following characterization. Results showed the isolate was identified as Achromobacter sp. with the accession number OQ372943 based on partial 16S rRNA gene sequence and phylogenetic analysis. The growth and degradation of paraquat by this isolate were optimum at a pH of 6.5, 276 mgL-1 of the substrate (paraquat), temperature of 35 °C, 200 µL of biomass size, and 48 h of incubation. The degradation efficiency of the isolate after 120 hours of incubation under optimal conditions was 91.01%. Hence, these results demonstrated a very high efficiency of paraquat degradation. Consequently, this isolate holds significant promise for paraquat degradation and could serve as a viable contender for remediating paraquat-contaminated environments.

Antibacterial Activity of Cinnamomum burmannii Extract Against Escherichia coli

This study aims to determine the antibacterial activity of the cinnamon extract in inhibiting the growth of Escherichia coli. The research design was a laboratory experiment consisting of three phases, namely the preparation phase of cinnamon extract using the maceration method using 96% ethanol, the characterization phase consisting of qualitative analysis with phytochemical tests and quantitative analysis with UV-Vis spectrophotometry, and the application phase including antibacterial activity test by disc diffusion method (Kirby-Bauer Test). The concentration series were 20%, 40%, 60%, 80%, and 100%, chloramphenicol as the positive control and methanol as the negative control. The results showed that cinnamon extract positively contained flavonoids with a total content of 1.310 mg QE/g or 0.131%. Cinnamon extract is effective as an antibacterial in the medium category with the optimum concentration to inhibit Escherichia coli is 40% indicated by the diameter of the inhibition zone of 9.55 mm at 12 hours of incubation. Therefore, the cinnamon extract has potential activity against Escherichia coli.

Renin and renin blockade have no role in complement activity

Renin, an aspartate protease, regulates the renin-angiotensin system by cleaving its only known substrate angiotensinogen to angiotensin. Recent studies have suggested that renin may also cleave complement component C3 to activate complement or contribute to its dysregulation. Typically, C3 is cleaved by C3 convertase, a serine protease that uses the hydroxyl group of a serine residue as a nucleophile. Here, we provide seven lines of evidence to show that renin does not cleave C3. First, there is no association between renin plasma levels and C3 levels in patients with C3 Glomerulopathies (C3G) and atypical Hemolytic Uremic Syndrome (aHUS), implying that serum C3 consumption is not increased in the presence of high renin. Second, in vitro tests of C3 conversion to C3b do not detect differences when sera from patients with high renin levels are compared to sera from patients with normal/low renin levels. Third, aliskiren, a renin inhibitor, does not block abnormal complement activity introduced by nephritic factors in the fluid phase. Fourth, aliskiren does not block dysregulated complement activity on cell surfaces. Fifth, recombinant renin from different sources does not cleave C3 even after 24 hours of incubation at 37 ⁰C. Sixth, direct spiking of recombinant renin into sera samples of patients with C3G and aHUS does not enhance complement activity in either the fluid phase or on cell surfaces. And seventh, molecular modeling and docking place C3 in the active site of renin in a position that is not consistent with a productive ground state complex for catalytic hydrolysis. Thus, our study does not support a role for renin in the activation of complement.

Rapid detection of antimicrobial resistance in methicillin-resistant Staphylococcus aureus using MALDI-TOF mass spectrometry.

Antimicrobial resistance is a growing problem in modern healthcare. Most antimicrobial susceptibility tests (AST) require long culture times which delay diagnosis and effective treatment. Our group has previously reported a proof-of-concept demonstration of a rapid AST in Escherichia coli using deuterium labeling and MALDI mass spectrometry. Culturing bacteria in D2O containing media incorporates deuterium in newly synthesized lipids, resulting in a mass shift that can be easily detected by mass spectrometry. The extent of new growth is measured by the average mass of synthesized lipids that can be correlated with resistance in the presence of antimicrobials. In this work, we adapt this procedure to methicillin-resistant Staphylococcus aureus using the Bruker MALDI-TOF Biotyper, a low-cost instrument commonly available in diagnostic laboratories. The susceptible strain showed a significant decrease in average mass in on-target microdroplet cultures after 3 hours of incubation with 10 µg/mL methicillin, while the resistant strain showed consistent labeling regardless of methicillin concentration. This assay allows us to confidently detect methicillin resistance in S. aureus after only 3 hours of culture time and minimal sample processing, reducing the turn-around-time significantly over conventional assays. The success of this work suggests its potential as a rapid AST widely applicable in many clinical microbiology labs with minimal additional costs.

Antifungal susceptibility of Aspergillusgenus determined by the Etest® method: eleven years of experience at the Instituto Médico La Floresta. Caracas, Venezuela.

Abstract. This research aimed to determine the susceptibility of Aspergil-lus spp. to four antifungal agents using the Etest® method in several clinical samples (respiratory samples, soft tissue, otic tissue, and ocular tissue, among others) from a private health center in Venezuela. Thirty-three strains were evaluated: 11 Aspergillus section Flavi, eight Aspergillus section Fumigati, six Aspergillus section Nigri, four Aspergillus section Terrei, and four Aspergillus spp. A 0.5 McFarland standard suspension of a 5-day culture of each Aspergillusstrain was prepared on Potato Dextrose agar and then inoculated on Sabouraud agar plates with 2% glucose. Voriconazole (VCZ), amphotericin B (AMB), caspo-fungin (CAS), and posaconazole (PCZ) were tested. Minimal inhibitory concen-trations (MIC) in μg/mL were determined after 24 and 48 hours of incubation at 35 °C and th range (R), geometric mean (GM), MIC50, and MIC90 were calcu-lated. The results for the 33 Aspergillus spp. tested after 24 h were the follow-ing: VCZ (R = 0.031- 16; GM = 0.145; MIC50 = 0.125 and MIC90 = 0.5), AMB (R = 0.031-16; GM = 0.644; MIC50 = 0.5 and MIC90 = 8), CAS (R = 0.031-16; GM = 0.1076; MIC50 = 0.063 and MIC90 = 1), PCZ (R =0.031 - 0.5; GM = 0.0755; MIC50 = 0.063 and MIC90 = 0.25). This investigation allowed assessing the an-tifungal susceptibility profiles of Aspergillus spp. isolated from clinical samples by the Etest® method, which is practical, reproducible and easy to perform in microbiology laboratories.

Enantioselective metabolism of fenpropathrin enantiomers by carboxyl/choline esterase 6 in Tetranychus cinnabarinus.

Tetranychus cinnabarinus is a polyphagous pest mite commonly found in agriculture. As an excellent acaricide, fenpropathrin (FEN) is frequently used to control T. cinnabarinus in agriculture. However, commercial FEN is a racemate with two enantiomers, R-FEN and S-FEN. Considering that investigations on the metabolism of FEN by T. cinnabarinus are based on racemate FEN, it is important to investigate the enantioselective metabolism of FEN in T. cinnabarinus. S-FEN was more toxic to T. cinnabarinus than R-FEN by more than 68.8-fold. Moreover, the synergist bioassay revealed that carboxylesterase and cytochrome P450 were the primary enzymes engaged in the detoxification of FEN in T. cinnabarinus, with carboxylesterase playing a leading role. Seven genes were substantially different after the induction of S-FEN and R-FEN. TcCCE06 was screened and selected as a key gene that related to FEN metabolism in T. cinnabarinus. The metabolic results showed that the recombinant TcCCE06 effectively metabolized 32.1% of the R-FEN and 13.8% of the S-FEN within 4 h of incubation. Moreover, R-FEN was demonstrated to have a higher affinity for the TcCCE06 protein than S-FEN based on molecular docking. Our results indicated that TcCCE06 mediates the enantioselective metabolism of FEN in T. cinnabarinus. Our findings will contribute to a more comprehensive understanding of the mechanisms underlying the differential toxicity of the FEN enantiomers against T. cinnabarinus. Furthermore, they also provide a new perspective for the development of enantiomer-enriched acaricides with higher activity and lower pesticide dosage and pollution risks. © 2023 Society of Chemical Industry.

Expression of the TR53 oncosuppressor gene modified with benzo[a]pyrene and the SARS-COV-2 vaccine antigen in an in vitro experiment

Introduction. The impact of chemical and biological environmental factors is associated with the risk of a genetic predisposition to the development of cardiovascular and cancer-associated diseases, which determines the relevance of the search for genetic indicator markers of early disorders in the mRNA structure.&#x0D; Materials and methods. The analysis of TP53 rs1042522 gene polymorphism, as well as the relative normalized expression level of TP53 hs1034249_m1 transcript, in whole blood cell culture in healthy volunteers, both spontaneous and induced by 24-hour incubation with benzo[a]pyrene and SARS-CoV-2 vaccine antigen &#x0D; (at concentrations of 0.006 mg/kg). Ml), was conducted.&#x0D; Results. Comparative analysis of spontaneous and antigen-induced levels of TP53 hs1034249_m1 mRNA expression allowed establishing individual and group values of relative expression associated with the polymorphism features of the TP53 rs1042522 gene. Benzo[a]pyrene and SARS-CoV-2 were found to have opposite effects on the expression of hs1034249_m1 TP53 genes in the case of the CG rs1042522 genotype, while the combined effect of benzo[a]pyrene and SARS-CoV-2, which reflected the inhibition of the expression of hs00900055_m1 of the TP53 gene was associated with the GG genotype.&#x0D; The limitations of the study are the use of a relatively small sample and a limited number of whole blood samples. &#x0D; Conclusion. The ability of benzo[a]pyrene and SARS-CoV-2 at concentrations of 6 µg/L to modify the expression of the TP53 apoptosis gene in vitro has been shown, which makes it possible to consider the increase in the expression of hs00900055_m1 of the TP53 gene induced by benzo[a]pyrene as one of the mechanisms for aggravating the course of viral infections (SARS-CoV-2) in connection with loss of p53-controlling for the development of inflammation (its proliferative phase) for owners of the heterozygous variant of CG TP53 rs1042522. In the case of variant monozygotic polymorphism GG TP53 rs1042522, the combination of benzo[a]pyrene and SARS-CoV-2 leads to inhibition of the expression of hs00900055_m1 mRNA of the TP53 gene, which is phenotypically reflected by the formation of asthenia, immunosuppression and onco-proliferative complications. The hs00900055_m1 transcript of the TP53 gene is recommended as an indicator for the tasks of diagnosing early disorders associated with the combination of SARS-CoV-2+ benzo[a]pyrene. The experiment simulates the natural conditions of real combinations of influencing factors.