Growth In Medium Research Articles

A novel alkali and thermotolerant protease from Aeromonas spp. retrieved from wastewater

Enzymes are integral to numerous industrial processes, with a growing global demand for various enzyme types. Protease enzymes, in particular, have proven to be cost-effective, stable, and compatible alternatives to traditional chemical processes in both industrial and environmental applications. In this study, an alkaline protease-producing strain of Aeromonas spp. was isolated from a wastewater treatment plant in Iran. The protease production was confirmed by culturing the strain on casein agar medium. The bacterium was identified through morphological, biochemical, and 16 S rRNA sequencing analyses. The optimal culture medium for bacterial growth and enzyme production was obtained using peptone, salt, yeast extract, galactose, and CaCl₂ at an initial pH of 8. Maximum protease production was achieved after 20 h of incubation at 40 °C. To partially purify the enzyme, the supernatant of the bacterial culture medium was first centrifuged, and the enzyme was precipitated using ammonium sulfate, followed by dialysis. Zymography revealed the production of one type of protease during bacterial growth. The partially purified protease exhibited optimal activity at pH 8.5 and maximum stability at pH 9. The optimum temperature for maximum enzyme activity was observed at 50 °C, with 100% residual activity retained for 1 h at 0 °C. The effect of metal ions on enzyme activity was assessed, revealing that KCl induced the most significant effects (p < 0.0001) on enzyme activity. Chemical amino acid modifiers and inhibitors, such as EDTA, DEPSI, and IAA, did not exhibit significant inhibition. In contrast, PMSF and HNBB significantly (p < 0.0001) reduced enzyme activity, suggesting that the enzyme could be classified as a serine protease. The protease also demonstrated high stability in the presence of 2% SDS, showing no signs inactivation. The alkaline pH optimum, thermal stability, and resistance to SDS exhibited by the protease produced by the Aeromonas strain are particularly promising characteristics that warrant further investigation. Based on preliminary tests and the enzyme’s characteristics, this protease can be recommended for various applications, pending further studies.

Attenuation of endoplasmic reticulum stress improves invitro growth and subsequent maturation of bovine oocytes

Endoplasmic reticulum (ER) stress interferes with developmental processes in oocyte maturation and embryo development. Invitro growth (IVG) is associated with low developmental competence, and ER stress during IVG culture may play a role. Therefore, this study aimed to examine the effect of tauroursodeoxycholic acid (TUDCA), an ER stress inhibitor, on the IVG of bovine oocytes to understand the role of ER stress. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles (1.5–1.8 mm) and allowed to grow in vitro for 5 days at 38.5 °C in a humidified atmosphere containing 5 % CO2. Basic growth culture medium was supplemented with TUDCA at various concentrations (0, 50, 100, 250, and 500 μM). After IVG, oocyte diameters were similar among groups, but the antrum formation rate tended to be higher in the TUDCA 100 μM group. The mRNA expression levels of ER stress-associated genes (PERK, ATF6, ATF4, CHOP, BAX, IRE1, and XBP1) in OGCs were downregulated in the TUDCA 100 μM group than those in the control group. Moreover, the TUDCA 100 μM group exhibited reduced ROS production with higher GSH levels and improved in vitro-grown oocyte maturation compared with those in the control group. In contrast, no difference in the developmental competence of embryos following invitro fertilization was observed between the control and TUDCA 100 μM groups. These results indicate that ER stress could impair IVG and subsequent maturation rate of bovine oocytes, and TUDCA could alleviate these detrimental effects. These outcomes might improve the quality of oocytes in IVG culture in assisted reproductive technology.

Separation and identification of bacteria producing protease from the intestine of Sillago sihama and Rastrelliger kanagurta

The importance of marine enzymes, especially protease enzymes, made us investigate the presence of these enzymes in the intestines of two fish species of the Persian Gulf (Sillago sihama and Rastrelliger kanagurta). Our investigations showed that initially 29 strains of bacteria were isolated of which 13 colonies had a specific growth in culture medium, and finally only seven strains were capable of producing a strong aura. T1/2 HR08 and S6B1 HR09 are two strains with the largest diameter of the halo around the bacteria, along with 5 other bacterial strains that were selected for more research. The results from the absorption of the supernatant by the spectrophotometer showed that the two strains T1/2 HR08 and S6B1 HR09 had the highest level of enzyme activity and were selected for identification accordingly. Also, the studies conducted with the 16S rRNA gene showed that these bacteria belonged to two genera, Kocuria and Aeromonas, from the Micrococcaceae and Aeromonadaceae families, and the highest similarity of HR08 and HR09 strains, with a similarity of 99% was shown to Kocuria rhizophila strain 8d-S5 (MN062066.1) and Aeromonas salmonicida strain SHY16- (CP038102.1), respectively. Generally, based on the results, these bacterial strains could be a good and ideal source for the production of the protease enzyme, which is used in many different industries.

Active Media Perfusion in Bioprinted Highly Concentrated Collagen Bioink Enhances the Viability of Cell Culture and Substrate Remodeling.

The bioprinting of high-concentrated collagen bioinks is a promising technology for tissue engineering and regenerative medicine. Collagen is a widely used biomaterial for bioprinting because of its natural abundance in the extracellular matrix of many tissues and its biocompatibility. High-concentrated collagen hydrogels have shown great potential in tissue engineering due to their favorable mechanical and structural properties. However, achieving high cell proliferation rates within these hydrogels remains a challenge. In static cultivation, the volume of the culture medium is changed once every few days. Thus, perfect perfusion is not achieved due to the relative increase in metabolic concentration and no medium flow. Therefore, in our work, we developed a culture system in which printed collagen bioinks (collagen concentration in hydrogels of 20 and 30 mg/mL with a final concentration of 10 and 15 mg/mL in bioink) where samples flow freely in the culture medium, thus enhancing the elimination of nutrients and metabolites of cells. Cell viability, morphology, and metabolic activity (MTT tests) were analyzed on collagen hydrogels with a collagen concentration of 20 and 30 mg/mL in static culture groups without medium exchange and with active medium perfusion; the influence of pure growth culture medium and smooth muscle cells differentiation medium was next investigated. Collagen isolated from porcine skins was used; every batch was titrated to optimize the pH of the resulting collagen to minimize the difference in production batches and, therefore, the results. Active medium perfusion significantly improved cell viability and activity in the high-concentrated gel, which, to date, is the most limiting factor for using these hydrogels. In addition, based on SEM images and geometry analysis, the cells remodel collagen material to their extracellular matrix.

97 Use of genomic testing for predicting performance of finishing steers

Abstract Genomic testing is a commercially available tool that has the potential to be beneficial in selecting cattle for retained ownership, as well as making management and marketing decisions after finishing. The objectives of this study were to determine if commercially available genomic tests are predictive of differences in finishing performance of steers from sires selected based on divergent growth and milk EPDs. The commercial genomic testing of milk and average daily gain (ADG) scores used in the current experiment were based on Neogen Igenity Beef test results. Two trials were conducted at the OSU Willard Sparks Beef Research Center in Stillwater, Oklahoma. These experiments used two sets of steers from the OSU Range Cow Research Center (400 ± 13 kg, n = 154) from a spring calving cowherd (South Range Unit) and a fall calving cowherd (North Range Unit) of primarily Angus ancestry. Following preconditioning and a wheat pasture stocker program (South Range) or dry lot growing period (North Range), steers were allocated into pens (n = 5 animals/pen) by Igenity ADG Score: low growth (LG) had scores ranging from 1 to 4, medium growth (MG) had scores from 5 to 6, and high (HG) had scores from 7 to 10. Steers were blocked by initial BW and fed a step-up diet for 30 d before being switched to a finishing ration diet (8% prairie hay, 20% Sweet Bran, 62% dry-rolled corn, 5% liquid supplement, and 5% dry supplement [DM basis]). Cattle were fed ractopamine hydrochloride for 30 d before slaughter. The heavy block was on feed for an average of 136 d, while the light block was on feed for an average of 160 d. Data were analyzed as a randomized complete block design using the mixed procedure of SAS (SAS Inst. Inc., Cary NC) with pen as the experimental unit and finishing pen within block as the random statement. During the feeding period, HG steers gained more rapidly than MG or LG steers, with ADG of 2.08, 1.94, and 1.90 kg/d, respectively (P = 0.04). Because gains were greater, BW at slaughter for the HG steers was greater than MG and LG, averaging 690, 669, and 660 kg, respectively (P = 0.04) and hot carcass weights were greater for HG than MG and LG, averaging 438, 426 and 418 kg, respectively (P = 0.02). There were no other differences (P &amp;gt; 0.19) in carcass characteristics among steers. These data suggests that the genomic testing is accurate predictor of performance and can be used by producers can make better marketing decisions in selecting cattle for the retained. Genomic testing could be very beneficial for producers looking to minimize risk and maximize performance in their herd.

Influence of Different Culture Media on Growth and Sporulation of Colletotrichum capsici

Aim: To know the best culture media for growth and development of Colletotrichum capsici causing chilli anthracnose.&#x0D; Study Design: Twelve different culture media were selected and three replications were maintained.&#x0D; Place and Duration of Study: Department of Plant Pathology, University of Agricultural Sciences, Raichur, Karnatka, which was carried out in the year 2021-2023.&#x0D; Methodology: The laboratory study was conducted to know the best nutrient media for the growth and sporulation of the pathogen. The pathogen was isolated from infected chilli fruit using tissue isolation technique and pure culture was maintained. Twelve different nutrient media were selected and tested for the growth and sporulation of the pathogen in three replications.&#x0D; Results: Among twelve media tested, Red chilli dextrose agar have promoted the maximum growth of C. capsici (90.0 mm), which was followed by carrot dextrose agar and potato carrot agar (89.50 mm). Green chilli extract agar significantly promoted minimum growth of C. capsici (33.50 mm), which was followed by red chilli extract agar (48.00 mm) and Sarbouraud’s agar (48.50 mm). The cultural characters were varied among the media.&#x0D; Conclusion: Potato dextrose agar and carrot dextrose agar media were proved to be best media for growth and sporulation of Colletotrichum capsica.

Production of iron-rich biomass using Chlorella vulgaris cultivation under iron stress

Fertilizers play a crucial role in enhancing the yield of agricultural crops and their market value. Therefore, it is essential to have large-scale, low cost, eco-friendly, broad-spectrum, and clean production of fertilizers. This study aims to prepare an iron-rich biomass, which holds the potential to serve as a biofertilizer, through the cultivation of Chlorella vulgaris microalgae under iron stress conditions. To attain this objective, three different iron sources were examined by supplementing them to the microalgal growth culture medium, namely ammonium ferric citrate, FeCl3, and EDDHA-Fe, each at varying concentrations of iron. The experimental findings revealed that, under iron stress conditions, the addition of ammonium ferric citrate as the iron source to the microalgal growth culture medium resulted in a microalgal biomass with a maximum iron content of 4.88 % by weight. However, the iron content of the obtained biomass from the culture media sourced from FeCl3 and EDDHA-Fe reached a maximum of only 2.01 % and 3.55 % by weight, respectively. Ultimately, the efficacy of the iron-rich microalgal biomass was evaluated by utilizing it during corn cultivation, in conjunction with regular microalgal biomass, ammonium ferric citrate, and the iron-deficient soil (as control) samples. The dry weight of the corn stem cultured with iron-rich biomass was 13 %, 55 %, and 152 % higher, compared to those cultured with regular microalgal biomass, ammonium ferric citrate, and the iron-deficient soil, respectively.

To Check Efficacy of Different Disinfectants by Kelsey Sykes Method

The incidence of hospital and community acquired infections has globally increased worldwide. The concentration of the disinfectants is important to give its higher efficacy against pathogens or microorganisms. Microorganisms are part of or environment cause major or severe diseases to reduce the risk of these diseases we generally clean surrounding by using cleaning agents like soaps, antiseptics or disinfectants. So to check weather these disinfectants are performs there work against specific bacteria or microorganism we performed this test to check the effectiveness of disinfectants against specific microorganism and fungus. Five bacterial types that are frequently acquired in hospitals from which two are gram positive organism, two are gram negative organism and single strain of fungus ‘(Pseudomonas aeruginossa, Klebsiella pneumoniae, Staphylococcus aureus (MRSA), Acinetobacter baumannii, Candida tropicalis) (clinical isolates) were examined for their susceptibility against 3 commonly used disinfectants hospitals Chlorine releasing agent, Quaternary ammonium compound and Aldehyde free disinfectant). The turbidity in the nutrient broth and the growth in culture media showed that all three disinfectants had lowered the microbial growth of every clinical strain. In general, the reference strains have lower growth and turbidity than the clinical strains. When combined, our results demonstrated that every product we examined had a high disinfection killing rate against microorganisms from various sources, indicating the excellent caliber of these goods. In response to rising disinfectant usage, hand sanitizer production has expanded to keep up with demand. Normal flora or microorganisms are part of our body in everyday life in which some are necessary some are unnecessary and can cause major or severe diseases to avoid from these diseases we generally clean our self and surrounding by using cleaning agents like soaps, antiseptics or disinfectants. So, to check weather these sterilizing agents are performed their work against specific bacteria or microorganism we performed this test to check the effectiveness of disinfectants against specific microorganism and fungus.

Screening for Antimycobacterial Activity of Actinomycetes Collected in Vietnam – Isolation and Activity of Metabolites from Streptomyces alboniger (A121)

Objective Actinomycetes have been known to be the great natural sources to explore antibiotics for the treatment of tuberculosis (TB). The isolation of actinomycetes from the samples in Vietnam followed by the screening of their antimycobacterial activity was performed in this study. The metabolites isolated from the most active strain were further evaluated for their antimycobacterial, antimicrobial and cytotoxic activity. Methods Actinomycetes were growth in culture media, isolated and identified by colony, spore chain morphology and 16S rRNA gene sequencing. Agar diffusion assay was used for the screening of the isolated strains against Mycobacterium smegmatis, a safety surrogate for Mycobacterium tuberculosis. The metabolites produced from the most active strain were investigated by actinomycete fermentation, extraction and isolation from biomass and cultures. The structures of the isolated compound were elucidated by spectral data and comparison with the reported literatures. Results 181 strains were isolated from nine regions along the north to central Vietnam. The five most active strains against Mycobacterium smegmatis were detected. Following the bioassay-guided result, the strain A121 ( Streptomyces alboniger) was selected for further isolation of the bioactive metabolites. As a result, obscurolide B2β (1) and chartreusin (2) were obtained and evaluated for their antimycobacterial activity against M. smegmatis. Compound 2 displayed potential antimycobacterial activity, antimicrobial effect against the Gram positive bacteria Staphylococcus aureus, Bacillus subtilis, Lactobacillus fermentum and cytotoxicity against four cancer cell lines KB, HepG-2, Lu-1 and MCF-7. Conclusions Five strains possessing potential antimycobacterial activity were identified from the samples collected in Vietnam. Two compounds including obscurolide B2β (1) and chartreusin (2) were isolated from the most active strain A121 ( Streptomyces alboniger). This is the first time these compounds have been isolated from this strain. Chartreusin (2) exhibited notable antimycobacterial, antimicrobial and cytotoxic activity, making its worthy attention for further drug development, particularly for antituberculosis therapeutic agents.

Towards the Standardization of Mesenchymal Stem Cell Secretome-Derived Product Manufacturing for Tissue Regeneration.

Mesenchymal stem cell secretome or conditioned medium (MSC-CM) is a combination of biomolecules and growth factors in cell culture growth medium, secreted by mesenchymal stem cells (MSCs), and the starting point of several derived products. MSC-CM and its derivatives could be applied after injuries and could mediate most of the beneficial regenerative effects of MSCs without the possible side effects of using MSCs themselves. However, before the clinical application of these promising biopharmaceuticals, several issues such as manufacturing protocols and quality control must be addressed. This review aims to underline the influence of the procedure for conditioned medium production on the quality of the secretome and its derivatives and highlights the questions considering cell sources and donors, cell expansion, cell passage number and confluency, conditioning period, cell culture medium, microenvironment cues, and secretome-derived product purification. A high degree of variability in MSC secretomes is revealed based on these parameters, confirming the need to standardize and optimize protocols. Understanding how bioprocessing and manufacturing conditions interact to determine the quantity, quality, and profile of MSC-CM is essential to the development of good manufacturing practice (GMP)-compliant procedures suitable for replacing mesenchymal stem cells in regenerative medicine.

Identification and characterization of two CRISPR/Cas systems associated with the mosquito microbiome.

The microbiome profoundly influences many traits in medically relevant vectors such as mosquitoes, and a greater functional understanding of host-microbe interactions may be exploited for novel microbial-based approaches to control mosquito-borne disease. Here, we characterized two novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems in Serratia sp. Ag1, which was isolated from the gut of an Anopheles gambiae mosquito. Two distinct CRISPR/Cas systems were identified in Serratia Ag1, CRISPR1 and CRISPR2. Based on cas gene composition, CRISPR1 is classified as a type I-E CRISPR/Cas system and has a single array, CRISPR1. CRISPR2 is a type I-F system with two arrays, CRISPR2.1 and CRISPR2.2. RT-PCR analyses show that all cas genes from both systems are expressed during logarithmic growth in culture media. The direct repeat sequences of CRISPRs 2.1 and 2.2 are identical and found in the arrays of other Serratia spp., including S. marcescens and S. fonticola , whereas CRISPR1 is not. We searched for potential spacer targets and revealed an interesting difference between the two systems: only 9 % of CRISPR1 (type I-E) targets are in phage sequences and 91 % are in plasmid sequences. Conversely, ~66 % of CRISPR2 (type I-F) targets are found within phage genomes. Our results highlight the presence of CRISPR loci in gut-associated bacteria of mosquitoes and indicate interplay between symbionts and invasive mobile genetic elements over evolutionary time.

Enhanced cultivation of chicken primordial germ cells

The cultivation and expansion of chicken primordial germ cells (cPGCs) are of critical importance for both biotechnological applications and the management of poultry genetic biodiversity. The feeder-free culture system has become the most popular approach for the cultivation and expansion of cPGCs. However, despite some success in the cultivation of cPGCs, the reproducibility of culture conditions across different laboratories remains a challenge. This study aimed to compare two defined and enriched media for the growth of cPGCs originating from the Hubbard JA57 broiler. To this end, cPGCs were isolated from the embryonic blood of Hamburger–Hamilton (HH) stages 14–16 and cultured at various time points. The Growth properties and characteristics of these cells were evaluated in two different culture conditions (the defined or enriched medium) and their migratory properties were assessed after genetic engineering and injection into the vasculature of 2.5-day-old chicken embryos. The main finding of this study was that the use of an enriched medium (the defined medium with Knock-Out Serum Replacement; KOSR) resulted in improved growth properties of cPGCs originating from the Hubbard JA57 broiler compared to a defined medium. The ability to cultivate and expand cPGCs is crucial for the generation of both genetically engineered birds and breeds of interest from local or commercial origins. Therefore, these results highlight the importance of choosing an appropriate culture medium for cPGCs growth and expansion.

Acrylates-based hydrophilic co-polymeric nanobeads as nanocarriers for imaging agents

Acrylates-based co-polymeric nanoparticles (PNPs) are widely used in nanomedicine applications due to their tunable hydrophilic surface, physical and chemical versatility. Particularly attracting is their use as nanocarriers for imaging agents. Herein, methyl methacrylate (MMA) and N,N-dimethylacrylamide (DMAA) monomers were used to synthesize hydrophilic p(MMA-co-DMAA) nanoparticles in the 200–600 nm size range via surfactant-free radical emulsion polymerization technique. Different MMA/DMAA molar ratios, temperatures, and reaction times were investigated to evaluate their role in determining the average particle size, polydispersity, and optimizing surface properties of PNPs. Nanoparticles formation, stability (in water and culture medium for cell growth), swelling behavior, structural features and molecular weights were assessed by spectroscopic, non-spectroscopic, and chromatographic techniques. Morphological profiles confirming spherical-shaped NPs were obtained at solid state via microscopies (FESEM, AFM). To use such colloids as potential imaging agents, PNPs were loaded with Y3+(aq) ions by the addition of aqueous solutions of YCl3 at different concentrations, and results compared with p(MMA-co-AA)-DTPA NPs (AA = acrylic acid) functionalized with DTPA chelating agent. Yttrium ions loading percentage was ca. 90% for both p(MMA-co-DMAA) and p(MMA-co-AA)-DTPA, with negligible release (<15%) over a month. Parallelly, optical imaging nanoprobes were obtained by physical encapsulation of fluorescein isothiocyanate isomer I (FITC) dye during the synthesis process, and the spontaneous FITC incorporation was evaluated by spectroscopic studies and fluorescence microscopy. Cytotoxicity studies on pristine and yttrium-loaded nanoparticles were done in vitro on human glioblastoma T98G cell line within 24 h of treatment. Transmission electron microscopy (TEM) studies on cancer cells treated with NPs confirmed an active uptake of PNPs through multiple endocytic pathways to reach the perinuclear region of the cell. Overall, this work elucidated the role of synthetic parameters for a rational design of hydrophilic PNPs as nanocarriers for imaging agents with potential applications in theranostics.

151 Effect of Neogen Igenity Beef Index Scores on Performance of Steers During Finishing

Abstract Genomic testing of feeder cattle is reported to be beneficial in selecting cattle for the feedlot, as well as making management and marketing decisions. The objective of this study was to determine differences in performance and efficiency of finishing steers with a range of Neogen Igenity Beef scores for average daily gain (ADG) and milk. Spring born steer calves from the Oklahoma State University Range Cow Research Center (n = 83, BW = 419 ± 31.9) of Angus ancestry were placed on feed at the Willard Sparks Beef Research Center on May 5, 2022, after grazing wheat pasture for 155 d. The steers were grouped based on Igenity ADG Score [low (1-4), medium (5-6), or high (7-10)] and Milk Score [low (1-5) or high (6-10)] based on the Igenity results. Steers were allocated into pens (n = 5 steers/pen) in heavy and light BW blocks. Steers were fed a step-up diet for 30 d before being switched to a finishing ration diet (8% prairie hay, 20% Sweet Bran and 62% dry-rolled corn). Cattle were fed ractopamine for 30 d before slaughter. The heavy block were on feed for 119 d, while the light block were on feed for 148 d. Data were analyzed as a randomized complete block design using the mixed procedure of SAS (SAS Inst. Inc., Cary NC) with pen as the experimental unit. Steers with high growth (HG) scores gained weight more rapidly during finishing and weighed more at slaughter than medium growth (MG) and low growth (LG; P &amp;lt; 0.01); while steers with high milk (HM) gained faster (P &amp;lt; 0.01) and tended (P = 0.06) to be heavier at slaughter than low milk (LM). Growth score was shown to have an effect on dry matter intake (P &amp;lt; 0.01) with HG consuming more feed than MG or LG. Cattle with HM scores were less efficient than LM scores (P = 0.01), but there was no difference (P = 0.37) in feed efficiency due to ADG score. Those with high growth and milk scores had a higher average daily gain (P &amp;lt; 0.01). These data indicate that Igenity ADG scores can be used to select cattle with higher performance and bodyweight at slaughter with no adverse effect on feed efficiency. Steers with higher Igenity Milk scores were less efficient than those with LM scores. Igenity Milk scores can be used to predict efficiency, according to these data.

Analysis of demand, generation, and emission for long-term sustainable power system planning using LEAP: The case of Bangladesh

The availability of quality power is a foremost need for a nation's sustainable development. The government of Bangladesh has the vision to be a high-income country by 2041. To meet the power challenges in the near future associated with the vision, there should be a well-planned master plan for the power system. Bangladesh has a power system master plan (PSMP) up to 2041. However, it is unclear whether the PSMP is the most adaptable plan considering different power generation scenarios by considering the demand, generation, and emissions. Hence, the long-range energy alternative planning (LEAP) tool is employed for scenario analyses of Bangladesh's electricity sector from 2022 to 2041. On the demand side, the final electricity demand has been projected as 335.25, 314.76, 376.59, and 398.10 TWh in 2041 for business-as-usual (BAU), low growth (LG), medium growth (MG), and high growth (HG) scenarios, respectively. Considering technical and environmental parameters, eight generation scenarios are also analyzed on the supply side. The analysis projected 58,230 MW capacity for BAU and LG under P1 to P8 generation scenarios and 68,830 MW capacity for MG and HG under Q1–Q8 generation scenarios in 2041. In terms of emission in 2041, 167.4 and 165 MMt CO2 equivalent are found for the P8 scenario in the case of BAU and LG. In Q8, for MG and HG, the emissions are found to be 206.5 and 209.4 MMt CO2 equivalent, respectively. The generation scenarios of P8 for BAU and LG and Q8 for MG and HG are found to be suitable ones with respect to energy reliability and reduced emission. A similar analysis could also be performed to identify suitable power generation plans for other developing countries.

Standardized ready-to-use laboratory protocol to isolate primary human wound associated fibroblasts from infected wound samples for clinical applications

Owing to the importance of fibroblasts in healing of wounds, it is necessary to isolate and culture them under in vitro conditions for the purpose of understanding the wound biology, drug discovery and development of personalized treatment. Although, several fibroblast cell lines are commercially available, they fail to represent the patient associated parameters. However, establishing a primary fibroblast culture, especially from infected wound samples, is challenging as the sample is more prone to contamination and number of live cells will be minimum in heterogeneous population. Also, it takes lot of efforts and resources for optimization of the protocol to get good quality cell lines from wound samples necessitating multiple trials, resulting in large number of clinical samples to be processed. To the best of our knowledge, for the first time we are reporting the standardized protocol to isolate primary human fibroblasts from acute and chronic wound samples. In this study, various parameters such as explant size (1–2 mm), explant drying time (2 min), transportation and growth culture media (antibiotics (working concentrations 1–3) and serum concentration (10%)) have been optimised. This can be altered for specific needs of cell in terms of both quality and quantity. Outcome of the work provides a ready-to-use protocol, which is very useful to those who want to initiate primary fibroblasts cell culture from infected wound samples either for clinical or research purpose. Further, these cultured primary wound associated fibroblasts have various clinical and biomedical applications in tissue grafting, treatment of burns and scars and wound regeneration especially in non-healing chronic wounds.

In vitro expansion of human mesenchymal stem cells and hepatocytes using purified platelet derived growth factors, a potential therapeutic modality for liver fibrosis

Mesenchymal stem cells (MSCs) and hepatocytes are considered valuable candidates for cell-based therapy. The use of free zoonotic media, as purified platelets derived growth factors (L-GF) and human platelet lysate (hPL), instead of using fetal bovine serum (FBS) to support the growth and proliferation of these cells could be used as a promising therapeutic tool in hepatic regeneration. This study aimed to evaluate the usage of purified platelet derived growth factors and platelet lysate in both MSCs and hepatocyte cultures and to compare them with the usage of FBS. MSCs and hepatocytes were cultured in growth media supplemented with L-GF or hPL and compared them to their culture in growth media supplemented with FBS. Cells were subjected to population doubling (PD) and generation time (GT) calculations. The best result for MSCs was that obtained by using 10% hPL or 10% FBS with the highest cell count, highest viability and shortest incubation time needed to reach confluency compared to supplementation with 10%, 20% or 30% L-GF. As for hepatocyte culture, the use of 10% FBS for supplementation of media used for hepatic cell proliferation showed better performance regarding cell count, viability, and incubation time to reach confluency compared to the use of either hPL or L-GF. In conclusion, our study showed that 10% hPL had the best results in MSCs culture which suggests that hPL could be a better alternative for the development of xenofree stem cell culture that can be used for many clinical applications. On the other hand, 10% FBS showed the best results in hepatocyte culture.

CCCP Facilitates Aminoglycoside to Kill Late Stationary-Phase Escherichia coli by Elevating Hydroxyl Radical.

Improving the efficacy of existing antibiotics is significant for combatting antibiotic resistance that poses a major threat to human health. Carbonyl cyanide m-chlorophenylhydrazine (CCCP), a well-known protonophore for dissipating proton motive force (PMF), has been widely used to block the PMF-dependent uptake of aminoglycoside antibiotics and thus suppress aminoglycoside lethality. Here, we report that CCCP and its functional analog FCCP, but not other types of protonophores, unprecedently potentiate aminoglycosides (e.g., tobramycin and gentamicin) by 3-4 orders of magnitude killing of Escherichia coli, Staphylococcus aureus, Shigella flexneri, and Vibrio alginolyticus cells in stationary phase but not these cells in exponential phase nor other 12 bacterial species we examined. Overall, the effect of CCCP on aminoglycoside lethality undergoes a gradual transition from suppression against E. coli exponential-phase cells to potentiation against late stationary-phase cells, with the cell growth status and culture medium being crucial. Consistently, disturbance of the PMF by changing transmembrane proton gradient (ΔpH) or electric potential (ΔΨ) also potentiates tobramycin. Nevertheless, CCCP neither increases the intracellular concentration of tobramycin nor decreases the MIC of the antibiotic, thus excluding that CCCP acts as an efflux pump inhibitor to potentiate aminoglycosides. Rather, we show that the combined treatment dramatically enhances the cellular level of hydroxyl radical under both aerobic and anaerobic culturing conditions, under which the antioxidant N-acetyl cysteine fully suppresses both hydroxyl radical accumulation and cell death. Together, these findings open a new avenue to develop certain protonophores as aminoglycoside adjuvants against pathogens in stationary phase and also illustrate an essential role of hydroxyl radical in aminoglycoside lethality regardless of aerobic respiration.

Revisiting the Design of the Long-Term Evolution Experiment with Escherichia coli.

The long-term evolution experiment (LTEE) with Escherichia coli began in 1988 and it continues to this day, with its 12 populations having recently reached 75,000 generations of evolution in a simple, well-controlled environment. The LTEE was designed to explore open-ended questions about the dynamics and repeatability of phenotypic and genetic evolution. Here I discuss various aspects of the LTEE's experimental design that have enabled its stability and success, including the choices of the culture regime, growth medium, ancestral strain, and statistical replication. I also discuss some of the challenges associated with a long-running project, such as handling procedural errors (e.g., cross-contamination) and managing the expanding collection of frozen samples. The simplicity of the experimental design and procedures have supported the long-term stability of the LTEE. That stability-along with the inherent creativity of the evolutionary process and the emergence of new genomic technologies-provides a platform that has allowed talented students and collaborators to pose questions, collect data, and make discoveries that go far beyond anything I could have imagined at the start of the LTEE.

In Vitro Screen of Lactobacilli Strains for Gastrointestinal and Vaginal Benefits

Traditional probiotics comprise mainly lactic acid bacteria that are safe for human use, tolerate acid and bile, and adhere to the epithelial lining and mucosal surfaces. In this study, one hundred commercial and non-commercial strains that were isolated from human feces or vaginal samples were tested with regards to overall growth in culture media, tolerance to acid and bile, hydrogen peroxide (H2O2) production, and adhesion to vaginal epithelial cells (VECs) and to blood group antigens. As a result, various of the tested lactobacilli strains were determined to be suitable for gastrointestinal or vaginal applications. Commercial strains grew better than the newly isolated strains, but tolerance to acid was a common property among all tested strains. Tolerance to bile varied considerably between the strains. Resistance to bile and acid correlated well, as did VEC adhesion and H2O2 production, but H2O2 production was not associated with resistance to bile or acid. Except for L. iners strains, vaginal isolates had better overall VEC adhesion and higher H2O2 production. Species- and strain-specific differences were evident for all parameters. Rank-ordered clustering with nine clusters was used to identify strains that were suitable for gastrointestinal or vaginal health, demonstrating that the categorization of strains for targeted health indications is possible based on the parameters that were measured in this study.