Consecutive-Day Ventricular and Atrial Cardiomyocyte Isolations from the Same Heart: Shifting the Cost–Benefit Balance of Cardiac Primary Cell Research

Freshly isolated primary cardiomyocytes (CM) are indispensable for cardiac research. Experimental CM research is generally incompatible with life of the donor animal, while human heart samples are usually small and scarce. CM isolation from animal hearts, traditionally performed by coronary artery perfusion of enzymes, liberates millions of cells from the heart. However, due to progressive cell remodeling following isolation, freshly isolated primary CM need to be used within 4–8 h post-isolation for most functional assays, meaning that the majority of cells is essentially wasted. In addition, coronary perfusion-based isolation cannot easily be applied to human tissue biopsies, and it does not straightforwardly allow for assessment of regional differences in CM function within the same heart. Here, we provide a method of multi-day CM isolation from one animal heart, yielding calcium-tolerant ventricular and atrial CM. This is based on cell isolation from cardiac tissue slices following repeated (usually overnight) storage of the tissue under conditions that prolong CM viability beyond the day of organ excision by two additional days. The maintenance of cells in their near-native microenvironment slows the otherwise rapid structural and functional decline seen in isolated CM during attempts for prolonged storage or culture. Multi-day slice-based CM isolation increases the amount of useful information gained per animal heart, improving reproducibility and reducing the number of experimental animals required in basic cardiac research. It also opens the doors to novel experimental designs, including exploring same-heart regional differences.

Evaluating the Physicochemical Properties of Some Kosovo’s and Imported Honey Samples

This study evaluated the physicochemical properties (moisture, pH, electrical conductivity, free acidity, hydroxymethylfurfural (HMF), proteins, insoluble solids, and ash) of 45 Kosovo’s and imported honey samples, using methods provided by national and international standards. The moisture values of all honey samples analyzed were below 20%. The free acidity was above 50.0 meq kg−1 in 14 out of 33 samples (42%) collected in Kosovo, while 2 out of 12 imported honey samples (16.7%) showed higher values than 50 meq kg−1. In this study, 7 out of 33 honey samples (21%) from Kosovo and one out of 12 honey samples from imports had soluble solids content below 80 °Brix. In terms of HMF, 5 out of 33 Kosovo honey samples (15%) and 4 out of 12 imported honey samples (33%) exceeded 40 mg/kg, which is the maximum content of HMF set in standards. The values of some physicochemical parameters (free acidity, HMF, and soluble solids) of local and imported honey samples are not within the quality limits set in legislation. Further studies are needed to evaluate the properties of fresh honey produced in Kosovo and the stability of honey during prolonged storage.

Delayed processing of blood samples impairs the accuracy of mRNA-based biomarkers

Background: The transcriptome of peripheral white blood cells (PWBCs) contains valuable physiological information, thus making them a prime biological sample for investigating mRNA-based biomarkers. However, prolonged storage of whole blood samples can alter gene transcript abundance in PWBCs, compromising the results of biomarker discovery. Here, we designed an experiment to interrogate the impacts of delayed processing of whole blood samples on gene transcript abundance in PWBCs. We hypothesized that storing blood samples for 24 hours at 4°C would cause RNA degradation resulting in altered transcriptome profiles. Results: We produced RNA-sequencing data for 30 samples collected from five estrus synchronized heifers (Bos taurus). We quantified transcript abundance for 12,414 protein-coding genes in PWBCs. Analysis of parameters of RNA quality revealed no statistically significant differences (P>0.05) between samples collected from the jugular vein and coccygeal vein, as well as among samples processed after one, three, six, or eight hours. However, samples processed after 24 hours of storage had a lower RNA integrity number value (P=0.03) in comparison to those processed after one hour of storage. Next, we analyzed RNA-sequencing data between samples using those processed after one hour of storage as the baseline for comparison. Interestingly, evaluation of 3/5 prime bias revealed no differences between genes with lower transcript abundance in samples stored for 24 hours relative to one hour. In addition, sequencing coverage of transcripts was similar between samples from the 24-hour and one-hour groups. We identified four and 515 genes with differential transcript abundance in samples processed after storage for eight and 24 hours, respectively, relative to samples processed after one hour. Conclusions: The PWBCs respond to prolonged cold storage by increasing genes related to active chromatin compaction which in turn reduces gene transcription. This alteration in transcriptome profiles can impair the accuracy of mRNA-based biomarkers. Therefore, blood samples collected for mRNA-based biomarker discovery should be refrigerated immediately and processed within six hours post sampling.

USE OF ANALYSIS TECHNIQUE FOR IMMOBILIZED YEAST SACCHAROMYCES CEREVISIAE IN THE BIOTECHNOLOGICAL INDUSTRY

Article is devoted to the current state and problems of microbial cells immobilization and also prolonged storage of immobilized cells systems for the aims of biotechnological industry. In the experimental part immobilization conditions for the cells S. cerevisiae in alginate gel and vitality test, which had given high reproducibility of experimental results, were developed. Experimental results showed that viability of immobilized cells was higher than that of free yeast cells. It is possible that gel matrix has a protective effect on yeast cells during freezing. Comprehensive effect of cooling modes and preservation protective mediums, which contain sodium alginate, on viability of yeasts has been investigated. Advantage of yeast cells storage in immobilized state was shown experimentally. It was found that cooling mode and composition of preservation medium affect on the viability of S. cerevisiae cells during cryopreservation. In all freezing medium, both without protective components and with addition of a cryoprotective agent, the best results were obtained with cooling at a rate of 1°C/min. Viability indices in the samples were: 73.1 % – in distilled water; 90.8 % – in 1 % sodium alginate solution; 87.1 % – in 5 % DMSO solution and 86.1 % – in 1 % sodium alginate solution with the addition of 5 % DMSO. When cells were frozen in a 5 % DMSO solution and in a 1 % sodium alginate solution with the addition of 5 % DMSO, number of viable cells also decreased as cooling rate increased, but, probably, did not differ from the cell viability index in those samples that were frozen in 1 % sodium alginate solution. The highest results of viability for S. cerevisiae yeast cells were obtained during slow cooling for all cryoprotective mediums. For the first time, high cryoprotective properties of sodium alginate solution, were shown. Obtained results are enable to recommend the sodium alginate as a carrier for cryopreserved immobilized cells when using it in biotechnological processing for biologically active substances production.

Temporal Viability of Aedes aegypti and Aedes albopictus Eggs Using Two Hygroscopic Substances as Preservatives under a Sterile Insect Technique (SIT) Program in Southern Mexico

Dengue and other Aedes-borne diseases have dramatically increased over the last decades. The Sterile Insect Technique (SIT) has been successfully used as part of integrated pest strategies to control populations of insect-plant and livestock pests and is currently being tested as a potential method to reduce mosquito populations in an environmentally friendly approach. However, during the mass rearing steps needed to produce millions of mosquitoes, egg storage and preservation are essential for a certain amount of time. Eggs of Aedes aegypti have a chorionic pad that functions as a sticky substance to glue them onto the inner walls of larval breeding sites. The chorionic pad is chemically made of hyaluronic acid, a hygroscopic compound, responsible to protect them from desiccation over time. Two commercial products with hygroscopic properties, hydrolyzed collagen, and Hyalurosmooth®, both were tested to assess their ability to prolong egg life storage for A. aegypti and A. albopictus. Results showed that 85–95% of Ae. aegypti eggs were able to hatch up to week 8 after being treated with both hydrophilic compounds, compared with the control 66.3%. These two substances showed promising effects for keeping Ae. aegypti eggs viable during prolonged storage in mass rearing insect production focused on vector control SIT programs.

Influence of Floral Origin and Storage Conditions on Physicochemical Properties of Libyan Honeys

Aim: The effect of botanical origin and storage conditions on the quality of two Libyan monofloral honey samples (thyme and ziziphus honeys) were assessed during prolonged storage (12 months) at room temperature. Methodology: Physicochemical properties (moisture, viscosity, electrical conductivity, acidity pH, 5-(hydroxymethyl) furfural (HMF), diastase activity, sugars, and color) were monitored. Results: Generally, moisture, acidity, diastatic activity, and color values were significantly higher in thyme honey, whereas viscosity, electrical conductivity, pH, HMF, and sugars content were higher in ziziphus honeys. In comparison with the initial values, viscosity, acidity, and HMF values of all honey samples increased during storage. Storage period and containers did not affect the electrical conductivity and sucrose contents for the two honey types, which were below the stipulated limits. Moisture content, pH, diastatic activity, color (L*), fructose, and glucose content decreased during storage. Honey samples stored in opaque container showed significantly lower changes in all parameters during the storage period compared to those stored in transparent bottles. The results showed that both honey types stored for 12 months at room temperature could be considered safe for human consumption according to maintaining physicochemical parameters.

Effectiveness of Goji Berries (Lycium barbarum) Extract and Some Nanoparticles Loaded on Gelatin Film on Microbial Content of Labneh

This study was conducted to evaluate the effect of Lycium barbarum extract, Chitosan nanoparticles (ChNPs) and Nanotitanium dioxide (TiO2 NPs) loaded on Gelatin films on the microbial content of labneh during different storage periods. The samples were divided into seven treatments which included (T1) non-coated labneh, (T2) labneh coated with gelatin membranes, (T3) labneh coated with gelatin membranes and Lycium barbarum extract, (T4) labneh coated with gelatin films and ChNPs, (T5) labneh coated with gelatin films treated TiO2NPs, (T6) Labneh coated with gelatin films, Lycium barbarum and ChNPs, (T7) Labneh coated with gelatin films, Lycium barbarum and TiO2 NPs. The total number of bacteria decreased after loading with the membranes for each specific period of time, and the treatment with T7 was the best, as the total number of bacteria decreased to 9.93 log/gm compared to the two controls (T1, T2), which amounted to (15.58, 13.47 log/gm) after 14 days of storage, respectively. While the numbers of Lipolytic and Proteolytic bacteria, yeasts and molds did not show any growth at the time of one day, with the prolonged storage period, the gradual increase in the total count of bacteria occurred for all treatments, it reached the highest numbers at the time of 14 days. Treatment T7 was the best in reducing the numbers of both lipolytic and proteinolytic bacteria, as well as yeasts and molds.

Microorganisms in Whole Botanical Fermented Foods Survive Processing and Simulated Digestion to Affect Gut Microbiota Composition

Botanical fermented foods have been shown to improve human health, based on the activity of potentially beneficial lactic acid bacteria (LAB) and yeasts and their metabolic outputs. However, few studies have explored the effects of prolonged storage and functional spices on microbial viability of whole fermented foods from fermentation to digestion. Even fewer have assessed their impact on the gut microbiota. Our study investigated the effects of production processes on LAB and yeast microbial viability and gut microbiota composition. We achieved this by using physicochemical assessments and an in vitro gastrointestinal and a porcine gut microbiota model. In low-salt sauerkraut, we assessed the effects of salt concentration, starter cultures, and prolonged storage, and in tibicos, prolonged storage and the addition of spices cayenne, ginger, and turmeric. In both food matrices, LAB counts significantly increased (p<0.05), reaching a peak of 7–8 log cfu/g, declining to 1–1.5 log cfu/g by day 96. Yeast viability remained at 5–6 log cfu/g in tibicos. Ginger tibicos had significantly increased LAB and yeast viability during fermentation and storage (p<0.05). For maximum microbial consumption, tibicos should be consumed within 28days, and sauerkraut, 7weeks. Simulated upper GI digestion of both products resulted in high microbial survival rates of 70–80%. The 82% microbial survival rate of cayenne tibicos was significantly higher than other treatments (p<0.05). 16S rRNA sequencing of simulated porcine colonic microbiota showed that both spontaneously fermented sauerkraut and tibicos increase the relative abundance of Megasphaera 85-fold. These findings will inform researchers, producers, and consumers about the factors that affect the microbial content of fermented foods, and their potential effects on the gut.

Effect of Storage Time On the Hatchability of Eggs of Two-Line Dual-Purpose Combination for Production of Male Chickens for Meat

The aim of the study was to assess the effect of the storage time on the hatching traits of eggs obtained from a two-line dual-purpose combination for production of male chickens that will be utilized for meat. The trial was carried out in the experimental poultry farm of the Institute of Animal Science-Kostinbrod, with a total of 150 Lohmann Brown Classic layers, at the age of 54 weeks. Hatching eggs were gathered for three weeks and were stored for 5, 10 and 15 days at 15-18°C ambient temperature prior incubation. The storage time had no effect on the fertility of the set eggs, however, it adversely affected the hatchability. Prolonged storage led to significant decline in the total hatchability (P=0.0027) and the hatchability of the fertile eggs (P<0.0001) which was lowest after 15 days of storage prior incubation. The viability of the chickens was influenced by the storage of the eggs prior incubation (P<0.0001), and decreased considerably when the chicks were hatched from eggs stored for 15 days.

Effects of abiotic factors on the stability and infectivity of polyvalent coliphage

Bacteriophage has attracted growing interest as a promising therapeutic agent for pathogenic bacteria, especially for antibiotic-resistant bacteria. However, the various abiotic conditions could impact the stability of phages and further threat host-virus interactions. Here, we investigated the stability and lytic activity of virulent polyvalent coliphage (named PE1) by double-layer plaque assay. PE1 can efficiently infect both the drug-sensitive Escherichia coli K12 and multidrug-resistant E. coli NDM-1 even after prolonged storage at 4 °C up to two months. Results showed that PE1 exhibits an outstanding stability to infect E. coli strains under a wide range of thermal (4 °C–60 °C) and pH (4–11) conditions, which covers the thermal and pH variations of most wastewater treatment plants. Moreover, PE1 exhibited high resistibility to heavy metals exposure including Cu2+, Cd2+, Co2+, and Cr3+ at the concentrations below 0.5 mM, and an excellent resistant ability to the variation of ionic strength, which still retained strong infectious ability even treated with saturated sodium chloride solution (350 g/L). This work shows that polyvalent phage PE1 has a strong adaptive capacity to various abiotic factors and should be a good candidate of being an antibacterial agent, especially for antibiotic-resistant bacteria control in sewage.