Viability Of Cultures Research Articles

A new strategy for the decellularization of whole organs by hydrostatic pressure.

Tissue engineering has been able to develop novel decellularization-recellularization techniques, which facilitates the research for the generation of functional organs. This is based in the initial obtention of the organ's extracellular matrix (ECM). Therefore, any improvement in the decellularization process would have a positive impact in the results of the recellularization process. Nevertheless, commonly the methods and equipment employed for this process are expensive and thus limit the access of this technique to various research groups globally. To develop a decellularization technique with the exclusive use of hydrostatic pressure of detergent solutions, to have an easily accessible and low-cost technique that meets the basic requirements of acellularity and functionality of the ECM. This experimental study was performed in 10 male Wistar rats, obtaining the liver to carry out serial washes, with 1%, 2%, and 3% Triton X-100 solutions and 0.1% SDS. The washes were performed by using a gravity perfusion system (GPS), which assured us a continuous hydrostatic pressure of 7.5 mmHg. The obtained ECM was processed using stains and immunostaining to determine the residual cell content and preservation of its components. The staining showed a removal of cellular and nuclear components of approximately 97% of the acellular ECM, with an adequate three-dimensional pattern of collagen and proteoglycans. Furthermore, the acellular ECM allowed the viability of a primary hepatocyte culture. The use of the GPS decellularization technique allowed us to obtain an acellular and functional ECM, drastically reducing experimentation costs.

Fresh tissue procurement and preparation for multicompartment and multimodal analysis of the prostate tumor microenvironment.

BackgroundProstatic cancers include a diverse microenvironment of tumor cells, cancer‐associated fibroblasts, and immune components. This tumor microenvironment (TME) is a known driving force of tumor survival after treatment, but the standard‐of‐care tissue freezing or fixation in pathology practice limit the use of available approaches/tools to study the TME's functionality in tumor resistance. Thus, there is a need for approaches that satisfy both clinical and laboratory endpoints for TME study. Here we present methods for clinical case identification, tissue processing, and analytical workflow that are compatible with standard histopathology while enabling molecular and functional interrogation of prostate TME components.MethodsWe first performed a small retrospective review to identify cases where submission of alternate prostate tissue slices and a parallel live tissue processing protocol complement traditional histopathology and enable viable multicompartment analysis of the TME. Then, we tested its compatibility with commonly employed methods to study the microenvironment including quantification of components both in situ and after tissue dissociation. We also evaluated tissue digestion conditions and cell isolation techniques to aid various molecular and functional endpoints.ResultsWe identified Gleason Grade Group 3+ clinical cases where tumor volume was sufficient to allow slicing of unfixed tissue and distribution of alternating tissue slices to standard‐of‐care histopathology and viable multi‐modal TME analyses. No single method was found that preserved cellular sub‐types for all downstream readouts; instead, tissues were further divided so techniques could be catered to each endpoint. For instance, we show that incorporating the protease dispase into tissue dissociation improves viability for culture and functional analyses but hinders immune cell analysis by flow cytometry. We also found that flow activated cell sorting provides highly pure cell populations for quantitative reverse‐transcription polymerase chain reaction and RNA‐seq while isolation using antibody‐labeled paramagnetic particles facilitated functional coculture experiments.ConclusionsThe identification of candidate cases and use of these techniques enable translational research and the development of molecular and functional assays to facilitate prostate TME study without compromising standard‐of‐care histopathological diagnosis. This allows bridging clinical histopathology and further interrogation of the prostate TME and promises to advance our understanding of tumor biology and unveil new predictive and prognostic markers of prostate cancer progression.

Cytotoxicity and Effects on the Synapsis Induced by Pure Cylindrospermopsin in an E17 Embryonic Murine Primary Neuronal Culture in a Concentration- and Time-Dependent Manner.

Cylindrospermopsin (CYN) is a cyanotoxin whose incidence has been increasing in the last decades. Due to its capacity to exert damage at different levels of the organism, it is considered a cytotoxin. Although the main target organ is the liver, recent studies indicate that CYN has potential toxic effects on the nervous system, both in vitro and in vivo. Thus, the aim of the present work was to study the effects of this cyanotoxin on neuronal viability and synaptic integrity in murine primary cultures of neurons exposed to environmentally relevant concentrations (0–1 µg/mL CYN) for 12, 24, and 48 h. The results demonstrate a concentration- and time-dependent decrease in cell viability; no cytotoxicity was detected after exposure to the cyanotoxin for 12 h, while all of the concentrations assayed decreased this parameter after 48 h. Furthermore, CYN was also demonstrated to exert damage at the synaptic level in a murine primary neuronal culture in a concentration- and time-dependent manner. These data highlight the importance of studying the neurotoxic properties of this cyanotoxin in different experimental models.

Formulation, Processing and Characterization of Fermented Probiotic Mango Juice Using Selected Starter Cultures

Probiotics are live microorganisms added to food products to confer health benefits such as gut health, cancer protection and improved immunity when consumed. Fruits juices are considered as potential channels for delivering probiotics as they contain essential nutrients and low pH suitable for the growth of probiotics. The study was undertaken to formulate, process and characterize fermented probiotic mango fruit juice using three different mixed lactic acid bacteria cultures: Abt-5® culture (Streptococcus thermophillus, Bifidobacterium and Lactobacillus acidophilus); Fiti® culture (Lactobacillus rhamnosus GR-1) and Yo-mix® culture (Streptoc.occus thermophillus and Lactobacillus delbruikii ssp bulgaricus). The viability of the cultures was also assessed. The mango juice was formulated using mango pulp to water ratios of 100:0, 25:75, 50:50 and 75:25 (v/v) and adjusted to 17° brix by adding cane sugar. The formulated mango juices were pasteurized at 90 °C for 1 minute and cooled to 10 °C. Sensory evaluation was carried out to determine the most preferred formulation of the fruit juice before fermentation. The preferred mango juice samples were cooled to 402 °C and incubated with the three different mixed bacteria cultures (1 g/l) for 0, 12, 16, 24, 36, 48 and 72 hours. The final product stored at 4 °C. Analysis for sensory evaluation, culture viability, pH, total soluble solids (TSS) and total titratable acidity (TTA) was done. The 50:50 ([mango pulp: water) mango fruit juice formulation was significantly different (p<0.05) from the other samples in consistency and was rated the best in overall acceptability. There was general increase in cell count in all cultures with mango juice fermented with Lactobacillus rhamnosus GR-1 for 48 hours recording the highest viable cell count of log10 9.14 CFU/ml. Highest pH decrease was observed in 50:50 formulation with significant (p<0.05) increase in TTA from 0.15% to 0.69% using Lactobacillus rhamnosus GR-1 within 72 hours. Subsequently, the TSS decreased from 17° brix to 14.47° brix. The study showed that the three mixed were able to utilize nutrients present in the formulated mango juice during fermentation as demonstrated by decrease in pH and TSS. The study therefore recommends that further research should be done on the probiotic fermented mango juice to determine the change in physco-chemical characteristics and culture viability during storage.

Technical-economic viability of white snook Centropomus viridis culture in floating cages in a coastal lagoon in northwestern Mexico

Snooks from the genus (Centropomus spp.) are an important fishery resource in the Americas and are a new species for marine aquaculture. Nevertheless, their production potential remains unknown. This study assessed the viability to grow-out Centropomus viridis juveniles raised in marine floating cages. At the nursery stage, 15,000 juveniles (0.8 ± 0.2 g) were stocked in one 235 m3 cage at a mean water temperature of 21 ± 3.4 °C. After 120 days, the juveniles reached an average weight of 43.6 ± 20.6 g. Subsequently, the juveniles were harvested and graded by weight to continue the grow-out process. Three groups were formed for the initial grow-out period: 30 g (G30); 40 g (G40); 60 g (G60). After a period of 270 days, the mean weights of G30, G40, and G60 were 522 ± 84, 565 ± 72, 600 ± 85 g, respectively. The average grow from the three groups showed 0.77 ± 0.18 g day−1 when they were cultured at temperatures < 24 °C, while at temperatures > 26 °C the mean growth rate was 3.07 ± 0.76 g day−1. Economic simulations, using sensitivity analyses showed that 10 USD/kg is the only final sale price that generates profitability with a production of 20 tons and survival of 60%. Our work suggests that the grow-out site should be located in an area with a tropical-subtropical latitude, in which water temperatures oscillate between 26 and 32 °C, and that 30 g juveniles should be used in order to improve survival during the rearing conditions in the initial grow-out stage. The results showed that C. viridis culture could be both technically and economically viable.

Cytoprotective Effect of Idebenone through Modulation of the Intrinsic Mitochondrial Pathway of Apoptosis in Human Retinal Pigment Epithelial Cells Exposed to Oxidative Stress Induced by Hydrogen Peroxide.

Idebenone is a ubiquinone short-chain synthetic analog with antioxidant properties, which is believed to restore mitochondrial ATP synthesis. As such, idebenone is investigated in numerous clinical trials for diseases of mitochondrial aetiology and it is authorized as a drug for the treatment of Leber’s hereditary optic neuropathy. Mitochondria of retinal pigment epithelium (RPE) are particularly vulnerable to oxidative damage associated with cellular senescence. Therefore, the aim of this study was to explore idebenone’s cytoprotective effect and its underlying mechanism. We used a human-RPE cell line (ARPE-19) exposed to idebenone pre-treatment for 24 h followed by conditions inducing H2O2 oxidative damage for a further 24 h. We found that idebenone: (a) ameliorated H2O2-lowered cell viability in the RPE culture; (b) activated Nrf2 signaling pathway by promoting Nrf2 nuclear translocation; (c) increased Bcl-2 protein levels, leaving unmodified those of Bax, thereby reducing the Bax/Bcl-2 ratio; (d) maintained the mitochondrial membrane potential (ΔΨm) at physiological levels, preserving the functionality of mitochondrial respiratory complexes and counteracting the excessive production of ROS; and (e) reduced mitochondrial cytochrome C-mediated caspase-3 activity. Taken together, our findings show that idebenone protects RPE from oxidative damage by modulating the intrinsic mitochondrial pathway of apoptosis, suggesting its possible role in retinal epitheliopathies associated with mitochondrial dysfunction.

Storage stability of fluid bed dried solid state fermented (SSF) lactic cultures inmilk

The present investigation was intended to study theeffect of storage at ambient temperature on the fluid beddried powder of Solid State Fermented (SSF) lactic cultures such as dahi, yoghurt and probiotic cultures and also made an attempt to check the performance in milk. The SSF cultures were prepared from various dhals and screened for the survival of bacterial aerobic spore, the raw black gram dhal showed lowest spore count of 1.52 log10cfu/g. Combination of hot air oven exposure of black gram dhal at 1000C/1 h and sterilization at 1210C/30 min completely destroyed the spores. On fermentation the maximum viable counts of dahi (8.41log10cfu/g), yoghurt (8.98log10cfu/g) andprobiotic (9.47log10cfu/g) cultures were obtainedon black gram dhal containing 1:0.8 ratio of moisture was supplemented with 1% level of skim milk powder, ash gourd, carrot and tomato juices as they provided growth promoting agents. The fermented SSF cultures were subjected for fluid bed drying for 1.5 h at ambient temperature (250C).The SSF culture viability upon fluid drying decreased from 8.65 to 8.33, 9.01 to 8.85 and 9.65 to 9.34log10cfu/g in case of dahi, yoghurt and probiotic cultures, with the moisture content of 9.4%. When these dried powders of SSF cultures of dahi, yoghurt and probiotic cultures were inoculated at 1,0.5 and 3% to heated milk, the cultures took 5:00, 3:30 h and 9:30 h to set the heat treated milk with acidity of 0.68, 0.71 and 0.59% lactic acid respectively. The dried SSF lactic cultures possessed viability of 6 log10cfu/g till 24th days of storage at ambient temperature (28±10C) and these cultures showed absence for the contaminants like coliforms, aerobic spores and yeast and molds. With respect to performance, the curdling time increased with theincreased storage days at ambient temperatures due to the reduction in viable lactic counts.

Exploring the Impact of Climate Change on Arctic Shipping through the Lenses of Quadruple Bottom Line and Sustainable Development Goals

Climate change is everywhere, and the Arctic is no exception. The melting sea ice has caused renewed interest in expanding maritime shipping for potentially more accessible ocean routes. Canada emerges as a natural land bridge for trade between Asia, Europe, and the Americas. Plausibly, it is not a choice but an imperative to properly integrate the stakeholders (the environment, countries, remote communities, industrial partners) in opening the Arctic Circle to the global economy while considering the challenges. Keeping sustainability front and center and drawing on the extant literature and government policies, this interdisciplinary study offers a Canadian perspective on Arctic transportation routes over tribal lands and their quadruple bottom line (QBL) impacts on the environment, economy, society, and Indigenous cultures. Unlike the arguable premise that new transport corridors will increase trade traffic and enhance the economy in Northern Canada, the QBL approach enables a more holistic and realistic strategy for the Arctic region’s sustainable development regarding regional economies, rural logistics, supply chain efficiency, and social licensing. Drawing on an integrative literature review as methodology, we highlight the QBL framework and the United Nations Sustainable Development Goals as crucial policy tools. Such a holistic perspective helps stakeholders and decision makers frame better policies in identifying, assessing, adapting, and mitigating risks for transportation infrastructure exposed to climate change. We recap the impacts of Arctic Shipping (ArSh) on QBL pillars in an interaction matrix and emphasize that while ArSh may be complementary to economic development, it poses threats to the viability of the Indigenous cultures.

A Three-Dimensional Organoid Model of Primary Breast Cancer to Investigate the Effects of Oncolytic Virotherapy

Background: Although several oncolytic viruses have already been tested in early-stage clinical studies of breast cancer, there is still an urgent need to develop patient-derived experimental systems that mimic the response of breast cancer to oncolytic agents in preparation of testing different oncolytic viruses in clinical trials. We addressed this need by developing a protocol to study the effects of oncolytic viruses in stable organoid cell cultures derived from breast cancer tissue. Methods: We used an established three-dimensional organoid model derived from tissue of 10 patients with primary breast cancer. We developed an experimental protocol for infecting organoid cultures with oncolytic viruses and compared the oncolytic effects of a measles vaccine virus (MeV) and a vaccinia virus (GLV) genetically engineered to express either green fluorescent protein (MeV-GFP) and red fluorescent protein (GLV-0b347), respectively, or a suicide gene encoding a fusion of cytosine deaminase with uracil phosphoribosyltransferase (MeV-SCD and GLV-1h94, respectively), thereby enabling enzymatic conversion of the prodrug 5-fluorocytosine (5-FC) into cytotoxic compounds 5-fluorouracil (5-FU) and 5-fluorouridine monophosphate (5-FUMP). Results: The method demonstrated that all oncolytic viruses significantly inhibited cell viability in organoid cultures derived from breast cancer tissue. The oncolytic effects of the oncolytic viruses expressing suicide genes (MeV-SCD and GLV-1h94) were further enhanced by virus-triggered conversion of the prodrug 5-FC to toxic 5-FU and toxic 5-FUMP. Conclusions: We were able to develop a protocol to assess the effects of two different types of oncolytic viruses in stable organoid cell cultures derived from breast cancer tissue. The greatest oncolytic effects were observed when the oncolytic viruses were engineered to express a suicide gene (MeV-SCD and GLV-1h94) in the presence of the prodrug 5-FC. The model therefore provides a promising in vitro method to help further testing and engineering of new generations of virotherapeutic vectors for in vivo use.

Simple assessment of viability in 2D and 3D cell microarrays using single step digital imaging.

Simple and rapid imaging and analysis of 2D and 3D cell culture compatible with miniaturized arrays of nanoliter droplets are essential for high-throughput screening and personalized medicine applications. In this study, we have developed a simple one-step, cost-effective and sensitive colorimetric method for the analysis of cell viability in 2D and 3D cell cultures on a nanoliter droplet microarray. The method utilizes a flatbed document scanner that detects a color change in response to cell metabolism in nanoliter droplets with high sensitivity in a single step without the need for expensive specialized equipment. This new nanoliter-based method is faster and more sensitive than equivalent methods using multi-well plate assays. The method detects quantifiable signal from as few as 10 cells and requires only 5min. This is 2.5 to 10-fold more sensitive and 12 times faster than the same assay in multi-well plates. The method is simple, affordable, fast and sensitive. It can be used for various applications including high-throughput cell-based and biochemical screenings.

Analysis of fouling and breakthrough of process related impurities during depth filtration using confocal microscopy.

Titer improvement has driven process intensification in mAb manufacture. However, this has come with the drawback of high cell densities and associated process related impurities such as cell debris, host cell protein (HCP), and DNA. This affects the capacity of depth filters and can lead to carryover of impurities to protein A chromatography leading to early resin fouling. New depth filter materials provide the opportunity to remove more process related impurities at this early stage in the process. Hence, there is a need to understand the mechanism of impurity removal within these filters. In this work, the secondary depth filter Millistak+ X0HC (cellulose and diatomaceous earth) is compared with the X0SP (synthetic), by examining the breakthrough of DNA and HCP. Additionally, a novel method was developed to image the location of key impurities within the depth filter structure under a confocal microscope. Flux, tested at 75, 100, and 250 LMH was found to affect the maximal throughput based on the max pressure of 30 psi, but no significant changes were seen in the HCP and DNA breakthrough. However, a drop in cell culture viability, from 87% to 37%, lead to the DNA breakthrough at 10% decreasing from 81 to 55 L/m2 for X0HC and from 105 to 47 L/m2 for X0SP. The HCP breakthrough was not affected by cell culture viability or filter type. The X0SP filter has a 30%–50% higher max throughput depending on viability, which can be explained by the confocal imaging where the debris and DNA are distributed differently in the layers of the filter pods, with more of the second tighter layer being utilized in the X0SP.

The Compound (E)-2-Cyano-N,3-diphenylacrylamide (JMPR-01): A Potential Drug for Treatment of Inflammatory Diseases.

The compound (E)-2-cyano-N,3-diphenylacrylamide (JMPR-01) was structurally developed using bioisosteric modifications of a hybrid prototype as formed from fragments of indomethacin and paracetamol. Initially, in vitro assays were performed to determine cell viability (in macrophage cultures), and its ability to modulate the synthesis of nitrite and cytokines (IL-1β and TNFα) in non-cytotoxic concentrations. In vivo, anti-inflammatory activity was explored using the CFA-induced paw edema and zymosan-induced peritonitis models. To investigate possible molecular targets, molecular docking was performed with the following crystallographic structures: LT-A4-H, PDE4B, COX-2, 5-LOX, and iNOS. As results, we observed a significant reduction in the production of nitrite and IL-1β at all concentrations used, and also for TNFα with JMPR-01 at 50 and 25 μM. The anti-edematogenic activity of JMPR-01 (100 mg/kg) was significant, reducing edema at 2–6 h, similar to the dexamethasone control. In induced peritonitis, JMPR-01 reduced leukocyte migration by 61.8, 68.5, and 90.5% at respective doses of 5, 10, and 50 mg/kg. In silico, JMPR-01 presented satisfactory coupling; mainly with LT-A4-H, PDE4B, and iNOS. These preliminary results demonstrate the strong potential of JMPR-01 to become a drug for the treatment of inflammatory diseases.

Effect of antioxidants and growth regulators on shoot organogenesis in the apical meristem culture of &lt;i&gt;Fragaria × ananassa&lt;/i&gt; (Duchesne ex Weston) Duchesne ex Rozier

The initiation of strawberries into in vitro culture is known to be complicated by the inhibition of organogenesis by phenolic oxidation products. An important role in this process is given to the selection of growth regulators that increase meristematic cell activity and shoot proliferation at the stage of organogenesis induction. The present study aims to obtain a viable apical meristem culture of garden strawberry and to study the effect of different antioxidants (reduced glutathione (RG); a new preparation, i.e., a mechanical composite (MC) on the basis of biogenic silicon and green tea catechins and plant growth regulators (6-benzylaminopurine; thidiazuron) on the initiation of axillary shoot formation in strawberry meristem culture. Terminal buds containing an apical meristem and two leaf primordia isolated from the stolons of two garden strawberry cultivars (Sunny Meadow and Festival Chamomile) were used as primary explants for the initiation of strawberries into in vitro culture. It was found for the first time that the MC exhibits higher antioxidant activity as compared to reduced glutathione, reduces darkening of initial explants, as well as enhancing regeneration up to 13.0% at p ≤ 0.05. Furthermore, the best effect on the formation of microshoots per explant is observed toward the end of material introduction into in vitro culture when combining the MC with growth regulators in the culture medium. Here, the effect of strawberry cultivar on explant regeneration and the number of microshoots per explant are insignificant. It is concluded that the procedure for using the MC as an effective antioxidant during material initiation into the culture can be applied to the large-scale in vitro propagation of garden strawberries. Moreover, the technology for obtaining the MC from plant waste is environmentally friendly, which is a significant advantage for its use in in vitro technologies.

Bax and Bak knockout apoptosis-resistant Chinese hamster ovary cell lines significantly improve culture viability and titer in intensified fed-batch culture process.

In the field of therapeutic protein production, process intensification strategies entailing higher starting cell seeding densities, can potentially increase culture productivity, lower cost of goods and improve facility utilization. However, increased cell densities often trigger apoptotic cell death at the end of the cell culture process and thus reduce total viable cell count. Apoptosis-resistant Chinese hamster ovary cell lines may offer the possibility to diminish this undesired outcome of the intensified production process. In this study, we have generated and tested Bax/Bak double-knock-out (DKO) apoptosis resistant hosts to express standard and bispecific antibodies, as well as complex molecules in intensified production processes both as pools and single cell clones, and at different scales. In all cases, therapeutic proteins expressed from clones or pools generated from the Bax/Bak DKO hosts showed not only better viability but also enabled extended productivity in the later stages of the 14-day intensified production process. The product qualities of the produced molecules were comparable between Bax/Bak DKO and wild type cells. Overall, we showed that Bax/Bak DKO apoptosis-resistant host cell lines significantly improve viability and volumetric productivity of the intensified production cultures without altering product qualities.

Technical efficiency and economic viability of fish culture under cold water production environment of Kashmir

This study examines the progress and economic viability of trout and crap fish culture under cold water environment of Kashmir valley. Results revealed a significant progress in the encouragement of fish culture especially in Kashmir. Trout and carp culture is capital intensive but their production is highly profitable compared to cultivation of commercial crops. Regression estimates revealed that the expenditure on fingerlings has contributed significantly in the improvement of yield levels of fishes while as the feed cost has been a positive and significant determinant of trout yield. There are wide technological gaps in the input use in both trout and carp culture though the gap was relatively lesser for farm category who have participated in extension events. A majority of fish farmers falls between 31 to 75% technical efficiency level. Regression analysis indicated important role of literacy, extension participation and female workers in the enhancement of technical efficiency in fish culture. Fish farmers brought out number of pushing and pulling factors associated with fish culture in Kashmir valley. Based upon findings, this study put forth few policy options for the encouragement of aquaculture in the valley including need for HRD and institutional role for the creation of marketing logistics.

Trial productions of freeze-dried Lactobacillus plantarum culture using dairy by-products as cryoprotectants: Viability and characterization of cultures

This study is a research on the utilization of dairy industry by-products in the practical production and long-term preservation of industrial microbial cultures. Accordingly, lyophilized cultures were obtained by freeze-drying of Lactobacillus plantarum bacteria in large quantities and for a constant period of time in skimmed milk-based medium containing 5% acid casein, rennet casein, whey and demineralized whey. The cryoprotectants did not have significant protection against freeze-drying process, moreover, the use of rennet casein significantly reduced viability. The efficiency of the preservatives in preserving the viability during storage was very successful compared to the control culture. Casein-enriched cultures had high water activity and moisture, but high viability was maintained during storage. The powder morphologies of the cultures were differentiated by the use of different cryoprotectants. Particle sizes of the cultures were parallel to the moisture content. Particle size and distribution increased with storage. By thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) analysis, it was determined that the addition of casein protein increased the degradation temperature of the cultures. The production of freeze-dried cultures became difficult with the use of casein. However, it was generally concluded that the protective properties of skimmed milk used for the preservation of L. plantarum could be improved by the addition of dairy by-products as a protective agent.

Impact of High-Intensity Ultrasound on Fermentation, Viability and Predictive Growth of Lactic Acid Cultures: A Study with Conventional and Probiotic Fermented Milks

Impact of High-Intensity Ultrasound on Fermentation, Viability and Predictive Growth of Lactic Acid Cultures: A Study with Conventional and Probiotic Fermented Milks

Влияние микрокапсулированных стартовых культур на образование биологически активных пептидов в готовых мясных продуктах

Of particular importance in the production of fermented meat products are starter cultures with a proteolytic system that promotes the proteolysis of the protein components of meat raw materials. Numerous compounds, which may have biological activity, were formed as a result of the proteolysis, for example, biologically active peptides or bacteriocins. Nevertheless, there are some technological difficulties in the use of bacteria that contribute to the formation of biologically active peptides in raw meat. The solution to this problem can be microencapsulation. In this study, industrially valuable strains of starter cultures were selected, and proteolytic activity genes were identified. Thus, the proteolytic genes of the PRTB10/PRTB20 and P15C/P06C families were found in the P. acidilactici 38 and P. pentosaceus 28 strains, while the genes of the Jp23/Jp25 and prti2/IP6Xba families were found in the L. acidophilus AT-45 strain. The prti2/IP6Xba genes were also found in the S. carnosus 111-2 strain. A technology is proposed for incorporating microorganisms into alginate capsules with an alginate gel concentration of 5%, which makes it possible to form strong capsules capable of maintaining the viability of starter cultures in the production of boiled sausage: when incubating heat-treated capsules to 72 °С on chalk agar, the survival rate of microorganisms from 30 to 90% was recorded. The protein profile of the samples of boiled sausages produced using microencapsulated starter cultures was studied. A significant interaction of some strains with proteins of the actomyosin complex was shown, and the enzymes of energy metabolism from muscle tissue were subjected to destructive changes - muscle creatine phosphate kinase, enolase, and adenylate kinase 1. The paper contains 4 Figures, 4 Tables, and 27 References. The Authors declare no conflict of interest.

Viability of selected strains of probiotic Lactobacillus spp. and sensory evaluation of concentrated yogurt (labneh) made from cow, camel, and cashew milk

The aim of this study is to investigate the effects of concentrated yogurt (labneh; L) made from cow (Co), camel (Ca), and cashew (Ew) milk (M) and their mixture (100% and 50%) using three strains of probiotic Lactobacillus spp. as starter culture (S) i.e. Lactobacillus plantarum (Lp; S1), L. casei (Lc; S2), L. rhamnosus (Lr; S3) or Lp, Lc, and Lr (S4) in co-cultures with Streptococcus thermophilus and L. delbrueckii subsp. Lactis on the changes of post-acidification and the viability of lactic acid bacteria (LAB) during 0, 7, 14, and 21 days of storage. In addition, sensory evaluation of all yogurt samples was run during the first day of storage. The lowest pH among all samples was seen in Ew-/Ca-ML50%S3 and Ew-/Co-ML50%S3 during the 21 days of storage. S. thermophilus increased to the highest numbers in Co-/Ca-ML50%S3 during 7th and 14th day as compared to other treated samples. All EwML100% were the most accepted by the panels compared to other samples. In conclusion, the combination of EwM with Co-/Ca-M during labneh preparation might enhance the viability of all different starter cultures especially S3 during the storage.

Abstract P045: Preclinical transcriptome-based evaluation of the translatable potential of new treatments in Triple-Negative Breast Cancer

Abstract Objective: This study will evaluate the potential of four recently proposed TNBC treatments—which all successfully reduced tumor viability in vitro and/or in vivo—to inhibit genes involved in CSC survival, metastatic metabolomic signature, and tumor immunosuppression. Methods: TNBC cell lines and/or patient-derived xenografts were treated with five different treatments: DCC-2036, 9Gy proton irradiation, miR302b+cisplatin combination, and DFX+doxorubicin combination. Genome-wide mRNA profiling (via either RNA-seq or microarray) was performed on control and treated groups. Data was obtained from NCBI GEO datasets. We assessed the differential expression of genes associated with CSC growth and metastatic metabolomic signature in TNBC tumors. Limma statistical analysis was performed. GSEA was also used to complement results from individual gene expression analysis. Results: DCC-2036 treatment significantly induced the expression of CSC TNBC biomarkers—such as ALDH2, CD44, CCR5, and SNAI1—and genes associated with TNBC metastatic metabolomic signature—such as PPARGC1A. DCC-2036 showed inconsistent effects on the expression of immunosuppressive markers. Gene expression profiles of the remaining treatment groups are currently being analyzed. 9Gy proton irradiation has mixed effects on the expression of our candidate genes, yet mostly induced the expression of stemness, metastatic, and immunosuppressive markers. miR302b+cisplatin and DFX+doxorubicin both failed to inhibit the candidate genes, yet without significantly inducing their expression. GSEA analysis confirmed the results obtained for all four treatments. Conclusions: Observing cancer rebound in TNBC patients after treatment with traditional cancer drugs is common and often happens when treatments fail to inhibit CSC growth, metabolic pathways associated with metastasis, and oncogenic immunosuppressive pathways. Our analysis shows that all four treatments failed to significantly impact the expression of protein pathways associated with increased metastasis and immunosuppression. It is worth noting that the researchers did report a decrease in tumor viability due to treatment of their experimental models with all four treatments. However, these findings correspond to the viability of the whole cell culture or tumor, not the viability of specifically the CSCs; in TNBC, CSCs make up only a small proportion of the total mass or the tumor, so the reported antiproliferative effects of the treatments do not necessarily suggest the treatment has effectively targeted the CSC population. Therefore, we hypothesize that these treatments will likely not show positive effects in clinical studies. Furthermore, none of the researchers performed any assays evaluating CSC growth—such as CSC-labelled flow cytometry—or metastasis—such as secondary tumor transplantation. Therefore, we encourage the researchers to perform more rigorous assays to evaluate the translatable potential of their treatments. Finally, the outline of this study provides a useful rationale for to evaluate emerging TNBC therapies. Citation Format: Ammar Salkini. Preclinical transcriptome-based evaluation of the translatable potential of new treatments in Triple-Negative Breast Cancer [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P045.