Continuous Culture Research Articles

Two classes of DNA gyrase inhibitors elicit distinct evolutionary trajectories toward resistance in gram-negative pathogens

Comprehensive knowledge of mechanisms driving the acquisition of antimicrobial resistance is essential for the development of new drugs with minimized resistibility. To gain this knowledge, we combine experimental evolution in a continuous culturing device, the morbidostat, with whole genome sequencing of evolving cultures followed by characterization of drug-resistant isolates. Here, this approach was used to assess evolutionary dynamics of resistance acquisition against DNA gyrase/topoisomerase TriBE inhibitor GP6 in Escherichia coli and Acinetobacter baumannii. The evolution of GP6 resistance in both species was driven by a combination of two classes of mutational events: (i) amino acid substitutions near the ATP-binding site of the GyrB subunit of the DNA gyrase target; and (ii) various mutations and genomic rearrangements leading to upregulation of efflux pumps, species-specific (AcrAB/TolC in E. coli and AdeIJK in A. baumannii) and shared by both species (MdtK). A comparison with the experimental evolution of resistance to ciprofloxacin (CIP), previously performed using the same workflow and strains, revealed fundamental differences between these two distinct classes of compounds. Most notable were non-overlapping spectra of target mutations and distinct evolutionary trajectories that, in the case of GP6, were dominated by upregulation of efflux machinery prior to (or even in lieu) of target modification. Most of the efflux-driven GP6-resistant isolates of both species displayed a robust cross-resistance to CIP, while CIP-resistant clones showed no appreciable increase in GP6-resistance.

Precision matters: Repeatability and reproducibility of total PSA and homocysteine measurements in Alinity i-system.

The precision of test measurements is critical in clinical diagnostics, especially for biomarkers like total PSA and homocysteine, which are essential to disease assessment. Using the CMIA approach, this study investigates the repeatability and reproducibility of these biomarkers on the Abbott Alinity system. The present study was conducted in the clinical chemistry laboratory at Mohammed VI University Hospital of Oujda. The evaluation of the Alinity i-system's analytical performance for total PSA and homocysteine focused on assessing repeatability and intermediate precision. The assessment followed the protocols and guidelines established by the French Accreditation Committee (COFRAC). Our analysis yielded favorable findings regarding the performance of the Alinity assays. The coefficients of variation for both the within-run and between-run precision were less than 5.89% and 4.29%, respectively. These findings produce acceptable outcomes compared to the manufacturer's claims and the SFBC database. Our study underscores the tests' precision, affirming the CMIA method's reliability in measuring total PSA and homocysteine levels. The assessment of the Alinity i-system for total PSA and homocysteine showed significant analytical performance. Our findings have implications for laboratory personnel, researchers, and physicians supporting a continuous diagnostic accuracy improvement culture.

Timing of High-Energy Supplementation to Early-Season-Pasture-Forage Fed Continuous Culture

A continuous culture study was conducted duplicating the daily meal pattern of pasture-grazed cattle and evaluating the time of two supplements differing in their composition (one complex: SUPP A and one simplistic: SUPP B) on their effects on nutrient digestion and metabolism of ruminal microorganisms. SUPP A contained ingredients containing substantial amounts of both structural and non-structural carbohydrates (NSC), fat, and protein, all having fractions with differing rates of availability. SUPP B’s predominate organic matter ingredients were only corn grain and soybean meal and contained approximately 3% less crude protein than SUPP A. All treatments were fed forage at five distinct times, mimicking forage intake on pasture, with supplements being fed at either a morning (AM) or evening (PM) feeding. The treatments were a) SUPP A fed AM, b) SUPP B fed AM, c) SUPP A fed PM, and d) Supp B fed PM. The results showed that dry matter, organic matter, and NSC digestibility’s were unaffected by supplement type or time of feeding. Supplement A showed greater fiber digestion than SUPP B, regardless of time of supplementation. The PM feeding decreased the digestion of both neutral detergent fiber and acid detergent fiber in SUPP B with no effect on fiber digestion in SUPP A. Culture pH remained more stable over the 24-hour day with SUPP B feeding than it did with SUPP A feeding while average pH was not affected by supplement or time of feeding. Total volatile fatty acid flow was unaffected by treatment, as were flows of acetic and propionic acids. Butyric acid flow decreased significantly on both supplements with the PM feeding. As expected, there were fewer total branch chain volatile fatty acids generated with SUPP B. Microbial growth measured in g microbial N flow/day increased for both supplements at the PM feeding. Flow of by-pass feed nitrogen was unaffected by supplement but was lower with PM feeding. Microbial efficiency was increased as a result of the PM feedings. The timing of a large amount of supplementation relative to the timing of daily forage meals can alter the effects seen in continuous culture work and may translate to effects in-vivo systems as well.

Regional centromere configuration in the fungal pathogens of the Pneumocystis genus.

Centromeres are constricted chromosomal regions that are essential for cell division. In eukaryotes, centromeres display a remarkable architectural and genetic diversity. The basis of centromere-accelerated evolution remains elusive. Here, we focused on Pneumocystis species, a group of mammalian-specific fungal pathogens that form a sister taxon with that of the Schizosaccharomyces pombe, an important genetic model for centromere biology research. Methods allowing reliable continuous culture of Pneumocystis species do not currently exist, precluding genetic manipulation. CENP-A, a variant of histone H3, is the epigenetic marker that defines centromeres in most eukaryotes. Using heterologous complementation, we show that the Pneumocystis CENP-A ortholog is functionally equivalent to CENP-ACnp1 of S. pombe. Using organisms from a short-term in vitro culture or infected animal models and chromatin immunoprecipitation (ChIP)-Seq, we identified CENP-A bound regions in two Pneumocystis species that diverged ~35 million years ago. Each species has a unique short regional centromere (<10 kb) flanked by heterochromatin in 16-17 monocentric chromosomes. They span active genes and lack conserved DNA sequence motifs and repeats. These features suggest an epigenetic specification of centromere function. Analysis of centromeric DNA across multiple Pneumocystis species suggests a vertical transmission at least 100 million years ago. The common ancestry of Pneumocystis and S. pombe centromeres is untraceable at the DNA level, but the overall architectural similarity could be the result of functional constraint for successful chromosomal segregation.IMPORTANCEPneumocystis species offer a suitable genetic system to study centromere evolution in pathogens because of their phylogenetic proximity with the non-pathogenic yeast S. pombe, a popular model for cell biology. We used this system to explore how centromeres have evolved after the divergence of the two clades ~ 460 million years ago. To address this question, we established a protocol combining short-term culture and ChIP-Seq to characterize centromeres in multiple Pneumocystis species. We show that Pneumocystis have short epigenetic centromeres that function differently from those in S. pombe.

Effect of increased pCO2 and temperature on the phytoplankton community in the coastal of Yellow Sea

In order to study the dynamics of marine phytoplankton communities in response to anticipated in temperature and CO2, a shipboard continuous culture experiment (Ecostat) was conducted. The experiment involved simulations under current atmospheric CO2 concentrations (400 ppm) and projected year-2100 CO2 levels (1000 ppm), as well as varying temperature under present (22 °C) versus increased temperature (26 °C) in the Yellow Sea during the summer of 2020. The results showed that both the increased pCO2 and temperature had significant effects on microphytoplankton and picophytoplankton, with the warming effect proving to be more significant. The different responses of various species to acidification and warming and their coupling effect led to the changes in microphytoplankton and picophytoplankton community structure. Elevated temperature and greenhouse treatments promoted the growth of dominant diatoms and Synechococcus, such as Guinardia flaccida and Pseudo-nitzschia delicatissima. This phenomenons widened the ecological niche, and the changes in the growth patterns of dominant species consequently influenced the content of cellular elements. Mantel's analysis further demonstrated that both warming and greenhouse promoted the growth of diatoms and Synechococcus. Projections of marine phytoplankton community trends by the end of the century based on Growth Rate Ratio (GRR), indicated that not only would species with GRR < 1 decrease, but also numerous species with growth rates >1 at elevated pCO2 levels would be ousted from competition. This experiment demonstrates the need to investigate whether extended exposure to increased pCO2 and temperature over more extended time scales would similarly induce shifts in the biological and biogeochemical dynamics of the Yellow Sea.

Culture and expansion of murine proximal airway basal stem cells

BackgroundThe stem cell characteristic makes basal cells desirable for ex vivo modeling of airway diseases. However, to date, approaches allowing them extensively in vitro serial expansion and maintaining bona fide stem cell property are still awaiting to be established. This study aims to develop a feeder-free culture system of mouse airway basal stem cells (ABSCs) that sustain their stem cell potential in vitro, providing an experimental basis for further in-depth research and mechanism exploration.MethodsWe used ROCK inhibitor Y-27632-containing 3T3-CM, MEF-CM, and RbEF-CM to determine the proper feeder-free culture system that could maintain in vitro stem cell morphology of mouse ABSCs. Immunocytofluorescence was used to identify the basal cell markers of obtained cells. Serial propagation was carried out to observe whether the stem cell morphology and basal cell markers could be preserved in this cultivation system. Next, we examined the in vitro expansion and self-renewal ability by evaluating population doubling time and colony-forming efficiency. Moreover, the differentiation potential was detected by an in vitro differentiation culture and a 3D tracheosphere assay.ResultsWhen the mouse ABSCs were cultured using 3T3-CM containing ROCK inhibitor Y-27632 in combination with Matrigel-coated culture dishes, they could stably expand and maintain stem cell-like clones. We confirmed that the obtained clones comprised p63/Krt5 double-positive ABSCs. In continuous passage and maintenance culture, we found that it could be subculture to at least 15 passages in vitro, stably maintaining its stem cell morphology, basal cell markers, and in vitro expansion and self-renewal capabilities. Meanwhile, through in vitro differentiation culture and 3D tracheosphere culture, we found that in addition to maintaining self-renewal, mouse ABSCs could differentiate into other airway epithelial cells such as acetylated tubulin (Act-Tub) + ciliated and MUC5AC + mucus-secreting cells. However, they failed to differentiate into alveoli epithelial cells, including alveolar type I and alveolar type II.ConclusionWe established an in vitro feeder-free culture system that allows mouse ABSCs to maintain their stem cell characteristics, including self-renewal and airway epithelium differentiation potential, while keeping up in vitro expansion stability.

Core microbiota for nutrient digestion remained and ammonia utilization increased after continuous batch culture of rumen microbiota in vitro.

This study aimed to investigate the digestive function, urea utilization ability, and bacterial composition changes in rumen microbiota under high urea (5% urea in diet) over 23 days of continuous batch culture in vitro. The gas production, dry matter digestibility, and bacterial counts were determined for the continuously batch-cultured rumen fluid (CRF). The changes in fermentation parameters, NH3-N utilization efficiency, and microbial taxa were analyzed in CRF and were compared with that of fresh rumen fluid (RF), frozen rumen fluid (FRF, frozen rumen fluid at -80°C for 1 month), and the mixed rumen fluid (MRF, 3/4 RF mixed with 1/4 CRF) with in vitro rumen fermentation. The results showed that the dry matter digestibility remained stable while both the microbial counts and diversity significantly decreased over the 23 days of continuous batch culture. However, the NH3-N utilization efficiency of the CRF group was significantly higher than that of RF, FRF, and MRF groups (p < 0.05), while five core genera including Succinivibrio, Prevotella, Streptococcus, F082, and Megasphaera were retained after 23 days of continuous batch culture. The NH3-N utilization efficiency was effectively improved after continuous batch culture in vitro, and Streptococcus, Succinivibrio, Clostridium_sensu_stricto_1, p.251.o5, Oxalobacter, Bacteroidales_UCG.001, and p.1088.a5_gut_group were identified to explain 75.72% of the variation in NH3-N utilization efficiency with the RandomForest model. Thus, core bacterial composition and function retained under high urea (5% urea in diet) over 23 days of continuous batch culture in vitro, and bacterial biomarkers for ammonia utilization were illustrated in this study. These findings might provide potential applications in improving the efficiency and safety of non-protein nitrogen utilization in ruminants.

Effect of ammonium/nitrate ratio on microalgae continuous cultures: Species-specificity of nutrient uptake and modelling perspectives

Microalgae-based wastewater treatment is promising in view of circularity as they can uptake most of the nitrogen sources usually present in waste streams: ammonium, nitrite and nitrate as well as some organic species. However, regulation pathways are not fully understood yet. This complexity is a challenge for accurate modelling of these processes, necessary for the design and optimization of wastewater treatment systems. It is generally observed in microalgae that the presence of ammonium inhibits the assimilation of other nitrogen compounds. This was confirmed in this work for Synechocystis sp. PCC6803, even in acclimated continuous cultures. A different uptake regulation was instead observed for the species Auxenochlorella protothecoides, able to uptake nitrate even when high concentrations of ammonium are present. From a modelling perspective, the Droop model was implemented to account for the exploitation of both ammonium and nitrate: a switch function was used to describe the ammonium inhibition, but this was applicable for Synechocystis only. For A. protothecoides, a changing nutrient uptake rate is needed, which was found to be function of the dissolved nitrogen concentration. A bioinformatic analysis was performed to infer putative players involved in nitrogen uptake, accounting for the different behavior of the two taxonomic groups.

Review Paper: “Quality Control and Assurance Strategy for Improved Operational Logistics”

Abstract - his project explores the implementation of a robust Quality Control and Assurance (QA/QC) strategy within operational logistics to enhance efficiency and elevate the overall quality of services. Through a comprehensive analysis of end-to-end logistics processes, we identified key areas for improvement, including bottlenecks in workflows, gaps in standardized procedures, and opportunities for technology integration. The strategy involves the adoption of real-time monitoring technologies, the development of standardized operating procedures, and the establishment of a continuous improvement culture. Training programs for employees and closer collaboration with suppliers are integral components, ensuring that the workforce is equipped to implement and adhere to quality standards. The project emphasizes the importance of data analytics for informed decision-making, regulatory compliance, and risk management. By adopting a phased roadmap for technology integration, the strategy aims to optimize logistics operations while minimizing disruptions. Regular audits, customer feedback mechanisms, and the definition of key performance indicators (KPIs) ensure ongoing evaluation and adherence to quality standards. This comprehensive QA/QC strategy, tailored to the organization's needs, is poised to drive efficiency, reduce errors, and enhance overall customer satisfaction in the operational logistics landscape.

Key elements and contextual factors that influence successful implementation of large-system transformation initiatives in the New Zealand health system: a realist evaluation

BackgroundDespite three decades of policy initiatives to improve integration of health care, delivery of health care in New Zealand remains fragmented, and health inequities persist for Māori and other high priority populations. An evidence base is needed to increase the chances of success with implementation of large-system transformation (LST) initiatives in a complex adaptive system.MethodsThis research aimed to identify key elements that support implementation of LST initiatives, and to investigate contextual factors that influence these initiatives. The realist logic of enquiry, nested within the macro framing of complex adaptive systems, formed the overall methodology for this research and involved five phases: theory gleaning from a local LST initiative, literature review, interviews, workshop, and online survey. NVivo software programme was used for thematic analysis of the interview, workshop, and the survey data. We identified key elements and explained variations in success (outcomes) by identifying mechanisms triggered by various contexts in which LST initiatives are implemented.ResultsThe research found that a set of 10 key elements need to be present in the New Zealand health system to increase chances of success with implementation of LST initiatives. These are: (i) an alliancing way of working; (ii) a commitment to te Tiriti o Waitangi; (iii) an understanding of equity; (iv) clinical leadership and involvement; (v) involved people, whānau, and community; (vi) intelligent commissioning; (vii) continuous improvement; (viii) integrated health information; (ix) analytic capability; and (x) dedicated resources and time. The research identified five contextual factors that influenced implementation of LST initiatives: a history of working together, distributed leadership from funders, the maturity of Alliances, capacity and capability for improvement, and a continuous improvement culture. The research found that the key mechanism of trust is built and nurtured over time through sharing of power by senior health leaders by practising distributed leadership, which then creates a positive history of working together and increases the maturity of Alliances.DiscussionTwo authors (KMS and PBJ) led the development and implementation of the local LST initiative. This prior knowledge and experience provided a unique perspective to the research but also created a conflict of interest and introduced potential bias, these were managed through a wide range of data collection methods and informed consent from participants. The evidence-base for successful implementation of LST initiatives produced in this research contains knowledge and experience of senior system leaders who are often in charge of leading these initiatives. This evidence base enables decision makers to make sense of complex processes involved in the successful implementation of LST initiatives.ConclusionsUse of informal trust-based networks provided a critical platform for successful implementation of LST initiatives in the New Zealand health system. Maturity of these networks relies on building and sustaining high-trust relationships among the network members. The role of local and central agencies and the government is to provide the policy settings and conditions in which trust-based networks can flourish.OtherThis study was approved by the Victoria University of Wellington Human Ethics Committee (Ethics Approval Number 27,356). The research was supported by the Victoria University of Wellington research grant (222,809) and from the University of Auckland Department of Medicine research fund (H10779).

A membrane photobioreactor with flashing light for enhancing biomass and lipid production by a benthic diatom mutant Nitzschia sp. L8

In this work, a membrane photobioreactor integrating with a flashing light (MFLPBR) has been developed for dense culture of a fast growing and oil-rich benthic diatom mutant Nitzschia sp. L8. Under 45 μmol/m2/s light intensity, 0.5 Hz flashing light (FL) could obviously improve the photosynthetic activity, specific growth rate and biomass yield of L8, with the increments by 18.5 %, 32.2 % and 137.6 % comparing to the continuous illumination culture. The total lipid content of L8 reached up to 53.5 % of dry weight, indicating a diversion of carbon flow from carbohydrates to lipids, resulting in increased biomass and lipid accumulation under FL. When L8 was subject to attached growth on the membrane surface in MFLPBR, cell growth was further improved, reaching the biomass yield and lipid productivity of 0.423 g/L and 30.35 mg/L/d in MFLPBR. The MFLPBR was proved to be a feasible strategy to promote the growth of benthic diatom for biomass and lipid production.

Transcriptomic analyses of host-virus interactions during in vitro infection with wild-type and glycoprotein g-deficient (ΔgG) strains of ILTV in primary and continuous cell cultures.

Infectious laryngotracheitis (ILT) remains a significant concern for the poultry industry worldwide due to its impact on animal welfare and its substantial economic consequences. The disease is caused by the alphaherpesvirus, infectious laryngotracheitis virus (ILTV). This study investigated in vitro host-virus interactions of a glycoprotein G (gG) deletion mutant vaccine strain of ILTV (ΔgG ILTV), and its parent wild-type strain (CSW-1 ILTV). Inoculations were performed separately for the two strains of ILTV using both a primary (chicken embryonic kidney, CEK) and a continuous culture (leghorn male hepatoma, LMH) of chicken cells. Transcriptome analysis was performed at 12 hours post infection. Each cell-type displayed distinct effects on host and viral gene transcription, with a greater number of viral and host genes differentially transcribed in CEK cells and LMH cells, respectively. Both cell-types infected with either strain demonstrated enrichment of pathways related to signalling, and gene ontologies (GO) associated with chemotaxis. Infection with either strain upregulated both SOCS proteins and certain proto-oncogenes, which may contribute to prolonged viral persistence by promoting immunosuppression and preventing apoptosis, respectively. Patterns of gene transcription related to cytokines, chemokines, endosomal TLRs, and interferon responses, as well as pathways associated with histone acetylation, transport, and extracellular matrix organization were similar within each cell type, regardless of the viral strain. In CEK cells, GO terms and pathways were downregulated uniquely after CSW-1 ILTV infection, indicating a viral-strain specific effect in this cell-type. Overall, this study highlights that the observed differences in host and ILTV gene transcription in vitro were more strongly influenced by the cell-types used rather than the presence or absence of gG. This underscores the importance of cell-line selection in studying host-virus interactions and interpreting experimental results.

Adaptation of the technology for culturing bovine embryos on commercial media for human embryos

We have studied the efficiency of using commercial media developed for human-assisted reproductive technologies (ART) for bovine embryos. The dynamics of bovine cells and embryos at the stages of maturation, fertilization, and preimplantation development were analyzed on three different media types, including ones particularly designed for bovine ART. Cumulus-oocyte complexes were divided into three groups: group 1 – maturation, fertilization, and culture were performed on media IVF-Bioscience (BO-IVM, BO-IVF, BO-IVC) developed for bovine animals; group 2 – maturation, fertilization, and culture were done on Vitrolife human ART media (G-IVF, G-TL); group 3 – maturation, fertilization, and culture were done on Irvine Scientific (Continuous Single Culture Complete) ART media. Significant distinctions in maturation, cleavage and blastulation indicators were found between embryos cultured on Vitrolife G-series human ART media and embryos processed on IVF-Bioscience bovine cattle media and Irvine Scientific Continuous Single Culture Complete human ART media. The difference in performance between the last two media was less pronounced. The obtained results make it possible to simplify the maturation of oocytes and the cultivation of bovine embryos to achieve reproducible outcomes without variability from batch to batch.

Novel electrochemical strategies for the microbial conversion of CO2 into biomass and volatile fatty acids using a fluid-like bed electrode in a three-phase reactor.

Microbial electrosynthesis (MES) constitutes a bioelectrochemical process where bacteria uptake electrons extracellularly from a polarized electrode to incorporate them into their anabolic metabolism. However, the efficiency of current MES reactor designs can be lower than expected due to limitations regarding electron transfer and mass transport. One of the most promising bioreactor configurations to overcome these bottlenecks is the Microbial Electrochemical Fluidized Bed Reactor (ME-FBR). In this study, microbial CO2 fixation is investigated for the first time in a ME-FBR operated as a 3-phase reactor (solid-liquid-gas). An electroconductive carbon bed, acting as a working electrode, was fluidized with gas and polarized at different potentials (-0.6, -0.8 and -1 V vs. Ag/AgCl) so it could act as an electron donor (biocathode). Under these potentials, CO2 fixation and electron transfer were evaluated. Autotrophic electroactive microorganisms from anaerobic wastewater were enriched in a ME-FBR in the presence of 2-bromoethanosulfonic acid (BES) to inhibit the growth of methanogens. Cyclic voltammetry analysis revealed interaction between the microorganisms and the cathode. Furthermore, volatile fatty acids like propionate, formate and acetate were detected in the culture supernatant. Acetate production had a maximum rate of ca. 1 g L-1 day-1 . Planktonic cell biomass was produced under continuous culture at values as high as ca. 0.7 g L-1 dry weight. Overall, this study demonstrates the feasibility of employing a fluidized electrode with gaseous substrates and electricity as the energy source for generating biomass and carboxylic acids.

Decision-Making Under Uncertainty: How Organizations Adapt to Environmental Changes?

In an increasingly volatile global business environment, organizations must navigate complex and unpredictable changes. This study aims to explore the adaptive strategies organizations employ to manage decision-making under uncertainty, focusing on how these strategies enhance organizational resilience and effectiveness in the face of environmental changes. This qualitative study collected data through semi-structured interviews with 26 participants from various industries including technology, healthcare, manufacturing, financial services, and retail. Participants ranged from senior management to operational staff, ensuring a comprehensive view of organizational strategies across different levels. The interviews continued until theoretical saturation was achieved, ensuring depth and breadth in the collected data. Thematic analysis was employed to identify and analyze patterns within the data. Three main themes were identified as central to organizational adaptation: Strategic Adaptation, Operational Responses, and Organizational Learning. Strategic Adaptation included subthemes such as Scenario Planning, Resource Allocation, Communication Strategies, and Leadership and Governance. Operational Responses covered Change Management, Innovation and Creativity, Process Optimization, and Risk Management. Organizational Learning focused on Knowledge Management and Employee Engagement. Each theme encapsulated several strategies and practices crucial for navigating uncertainty effectively. The study concludes that effective adaptation to environmental changes relies heavily on a cohesive strategy that integrates strategic foresight, operational agility, and a continuous learning culture. These elements are crucial for building resilience and ensuring sustained organizational performance in uncertain environments. The findings suggest that a proactive approach to strategic planning, coupled with robust operational responses and an emphasis on organizational learning, forms the backbone of successful adaptive strategies.

Is it possible to detect surface antigen CD133 on patient-derived glioblastoma continuous cell cultures using fluorescent aptamers?

To detect CD133 in patient-derived glioblastoma continuous cell cultures using fluorescence microscopy and modified aptamers (molecular recognition elements) anti-CD133. To detect CD133, we used mousey fluorescence monoclonal antibodies anti-CD133 MA1-219, FAM-modified DNA aptamers anti-CD133 AP-1-M and Cs5. Non-aptamer DNA oligonucleotide NADO was used as a negative control. Detection was performed for three samples of patient-derived glioblastoma continuous cell cultures coded as 1548, 1721 and 1793. MA1-219 antibodies brightly stained cell culture 1548, to a lesser extent - 1721. There was diffuse staining of cell culture 1793. Cs5-FAM aptamer stained cells in a similar way, but much weaker. AP-1-M-FAM aptamer interacted with cells even weaker and diffusely stained only cell culture 1793. Non-aptamer NADO did not stain cell culture 1548 and very weakly diffusely stained cell culture 1793. For both molecular recognition elements (MA1-219 antibody and Cs5 aptamer), 3 cell culture samples can be arranged in the following order possibly reflecting CD133 status decrease: strong signal for cell culture 1548, much weaker for 1721, even weaker for 1793. Only cell culture 1548 can be considered CD133 positive with combination of Cs5+ and NADO signals. Cell culture 1793 is CD133 false positive with combination of Cs5+ and NADO+ signals.

Optimization of conditions for cultivation of pathogens of infectious rhinotracheitis and viral diarrhea

This article reflects the results of research on optimizing the conditions of cultivation of strain “ТК-A(VIEV)-В2” of infectious bovine rhinotracheitis (IBR) and strain “ВК-1” of bovine viral diarrhea (BVDV). It was found that the roller cultivation method is most easily applicable and provides the production of viral material with high infectious activity. A comparative analysis of the results of studying the sensitivity of continuous cell cultures to the pathogens of IBR and BVDV of cattle of the latter made it possible to select the MDBK cell line to obtain the maximum titer of viruses. The optimal dose for infecting cell culture with both pathogens was the content of viral particles from 0.2 to 0.5 TCD50/cm3 per cell. The maximum reproduction of viruses was observed using a culture concentration of cells from 110 to 120 thousand cells/cm3 and the duration of their cultivation for 2-3 days. The results obtained can be applied for the further development and creation of preventive and diagnostic tools for IBR and BVDV of cattle.

Long-term culture of patient-derived mammary organoids in non-biogenic electrospun scaffolds for identifying metalloprotein and motor protein activities in aging and senescence.

We recently identified TMEM230 as a master regulator of the endomembrane system of cells. TMEM230 expression is necessary for promoting motor protein dependent intracellular trafficking of metalloproteins for cellular energy production in mitochondria. TMEM230 is also required for transport and secretion of metalloproteinases for autophagy and phagosome dependent clearance of misfolded proteins, defective RNAs and damaged cells, activities that decline with aging. This suggests that aberrant levels of TMEM230 may contribute to aging and regain of proper levels may have therapeutic applications. The components of the endomembrane system include the Golgi complex, other membrane bound organelles, and secreted vesicles and factors. Secreted cellular components modulate immune response and tissue regeneration in aging. Upregulation of intracellular packaging, trafficking and secretion of endosome components while necessary for tissue homeostasis and normal wound healing, also promote secretion of pro-inflammatory and pro-senescence factors. We recently determined that TMEM230 is co-regulated with trafficked cargo of the endomembrane system, including lysosome factors such as RNASET2. Normal tissue regeneration (in aging), repair (following injury) and aberrant destructive tissue remodeling (in cancer or autoimmunity) likely are regulated by TMEM230 activities of the endomembrane system, mitochondria and autophagosomes. The role of TMEM230 in aging is supported by its ability to regulate the pro-inflammatory secretome and senescence-associated secretory phenotypein tissue cells of patients with advanced age and chronic disease. Identifying secreted factors regulated by TMEM230 in young patients and patients of advanced age will facilitate identification of aging associated targets that aberrantly promote, inhibit or reverse aging. Ex situ culture of patient derived cells for identifying secreted factors in tissue regeneration and aging provides opportunities in developing therapeutic and personalized medicine strategies. Identification and validation of human secreted factors in tissue regeneration requires long-term stabile scaffold culture conditions that are different from those previously reported for cell lines used as cell models for aging. We describe a 3 dimensional (3D) platform utilizing non-biogenic and non-labile poly ε-caprolactonescaffolds that supports maintenance of long-term continuous cultures of human stem cells, in vitro generated 3D organoids andpatient derived tissue. Combined with animal component free culture media, non-biogenic scaffolds are suitable for proteomic and glycobiological analyses to identify human factors in aging. Applications of electrospun nanofiber technologies in 3D cell culture allow for ex situ screening and the development of patient personalized therapeutic strategies and predicting their effectiveness in mitigating or promoting aging.

Streamlining the biodesulfurization process: development of an integrated continuous system prototype using Gordonia alkanivorans strain 1B.

Biodesulfurization is a biotechnological process that uses microorganisms as biocatalysts to actively remove sulfur from fuels. It has the potential to be cleaner and more efficient than the current industrial process, however several bottlenecks have prevented its implementation. Additionally, most works propose models based on direct cultivation on fuel, or batch production of biocatalysts followed by a processing step before application to batch biodesulfurization, which are difficult to replicate at a larger scale. Thus, there is a need for a model that can be adapted to a refining process, where fuel is being continuously produced to meet consumer needs. The main goal of this work was to develop the first bench-scale continuous biodesulfurization system that integrates biocatalyst production, biodesulfurization and fuel separation, into a single continuous process, taking advantage of the method for the continuous production of the biodesulfurization biocatalysts previously established. This system eliminates the need to process the biocatalysts and facilitates fuel separation, while mitigating some of the process bottlenecks. First, using the bacterium Gordonia alkanivorans strain 1B, continuous culture conditions were optimized to double biocatalyst production, and the produced biocatalysts were applied in batch biphasic biodesulfurization assays for a better understanding of the influence of different factors. Then, the novel integrated system was developed and evaluated using a model fuel (n-heptane + dibenzothiophene) in continuous biodesulfurization assays. With this system strain 1B surpassed its highest biodesulfurization rate, reaching 21 μmol h-1 g-1. Furthermore, by testing a recalcitrant model fuel, composed of n-heptane with dibenzothiophene and three alkylated derivatives (with 109 ppm of sulfur), 72% biodesulfurization was achieved by repeatedly passing the same fuel through the system, maintaining a constant response throughout sequential biodesulfurization cycles. Lastly, the system was also tested with real fuels (used tire/plastic pyrolysis oil; sweet and sour crude oils), revealing increased desulfurization activity. These results highlight the potential of the continuous biodesulfurization system to accelerate the transition from bench to commercial scale, contributing to the development of biodesulfurization biorefineries, centered on the valorization of sulfur-rich residues/biomasses for energy production.

Generation of Skeletal Muscle Organoids from Human Pluripotent Stem Cells.

Various protocols have been proven effective in the directed differentiation of mouse and human pluripotent stem cells into skeletal muscles and used to study myogenesis. Current 2D myogenic differentiation protocols can mimic muscle development and its alteration under pathological conditions such as muscular dystrophies. 3D skeletal muscle differentiation approaches can, in addition, model the interaction between the various cell types within the developing organoid. Our protocol ensures the differentiation of human embryonic/induced pluripotent stem cells (hESC/hiPSC) into skeletal muscle organoids (SMO) via cells with paraxial mesoderm and neuromesodermal progenitors' identity and further production of organized structures of the neural plate margin and the dermomyotome. Continuous culturing omits neural lineage differentiation and promotes fetal myogenesis, including the maturation of fibroadipogenic progenitors and PAX7-positive myogenic progenitors. The PAX7 progenitors resemble the late fetal stages of human development and, based on single-cell transcriptomic profiling, cluster close to adult satellite cells of primary muscles. To overcome the limited availability of muscle biopsies from patients with muscular dystrophy during disease progression, we propose to use the SMO system, which delivers a stable population of skeletal muscle progenitors from patient-specific iPSCs to investigate human myogenesis in healthy and diseased conditions. Key features • Development of skeletal muscle organoid differentiation from human pluripotent stem cells, which recapitulates myogenesis. • Analysis of early embryonic and fetal myogenesis. • Provision of skeletal muscle progenitors for in vitro and in vivo analysis for up to 14 weeks of organoid culture. • In vitro myogenesis from patient-specific iPSCs allows to overcome the bottleneck of muscle biopsies of patients with pathological conditions.