Continuous Culture Research Articles

Comparative study on the morphological characteristics, nutritional quality, and tastes of large yellow croaker from five cage culture areas: Relay farming improved fish quality

Relay culture of large yellow croaker from Fujian to Zhejiang is a good model for improved fish quality. However, there is a scarcity of information regarding the comprehensive evaluation of quality alterations following relay culture. In the present study, the large yellow croaker from Ningde (ND) was transferred to net cages in Dongtou (DT), Nanji (NJ), Dongji (DJ) and Dacheng (DC) for continued culture. The 15-month-old fish from ND, DJ, DC, DT, and NJ were sampled on-site for a subsequent comparative study on the morphological characteristics, nutritional quality, and tastes. Results showed that the fish from DJ, DC, DT, and NJ had a lower condition factor (CF) and a higher ratio of body length to height (L/H) than those from ND. The fish from DJ, DC, DT, and NJ, at rates of 77.8%, 91.3%, 95.7%, and 100%, exhibited different geometric morphometrics from those from ND using 10 typical landmarks and Morpho J software. Ralay culture also significantly affected the histology and ultrastructure of muscle fibers, as reflected by an increase in the muscle fiber density and decreases in the ratio of fiber area to gap, I band width, and sarcomere length in the relay culture fish compared to the ND fish. The relay culture fish showed low lipid content and high moisture content. Relay culture also significantly affected the levels of some nutritional composition, including deceases in the levels of hydrolyzed glycine, C16:0, C18:1n9c, C18:2n6c, Σn-6 PUFA (polyunsaturated fatty acids), Fe (iron), Mn (manganese), and Co (cobalt), and increases in the levels of C20:5n3 (EPA, eicosapentaenoic acid), C22:6n3 (DHA, docosahexaenoic acid), C18:0, Σn-3/Σn-6 PUFA, Σn-3 PUFA, Al (aluminum), Se (selenium), Na (sodium), and Ca (calcium). The levels of taste substances such as guanosine monophosphate (GMP) and most flavor amino acids sharply declined after relay culture. However, the ratio of taste activity value (TAV) of ΣDAA to ΣBAA (delicious amino acids/bitter amino acids) in the DT and NJ fish was higher than that in the ND fish. Significant negative relationships between market price and quality parameters were found, including CF, crude lipid, C18:1n9c, C18:2n6c, Σn-6 PUFA, Fe, Mn, and TAV of GMP, whereas there were positive relationships between market price and 4 quality parameters such as L/H, moisture, Σn-3/Σn-6 PUFA, and the ratio of TAC of ΣDAA to ΣBAA. In conclusion, relay culture improved large yellow croaker quality by changes in morphological characteristics, nutritional quality, and tastes, which provides a theoretical basis for quality improvement.

Generation of stable suspension producer cell lines for serum-free lentivirus production.

The production of lentiviral vectors (LVs) pseudotyped with the vesicular stomatitis virus envelope glycoprotein (VSV-G) is limited by the associated cytotoxicity of the envelope and by the production methods used, such as transient transfection of adherent cell lines. In this study, we established stable suspension producer cell lines for scalable and serum-free LV production derived from two stable, inducible packaging cell lines, named GPRG and GPRTG. The established polyclonal producer cell lines produce self-inactivating (SIN) LVs carrying a WAS-T2A-GFP construct at an average infectious titer of up to4.64×107TU mL-1in a semi-perfusion process in a shake flask and can be generated in less than two months. The derived monoclonal cell lines are functionally stable in continuous culture and produce an average infectious titer of up to 9.38×107TU mL-1in a semi-perfusion shake flask process. The producer clones are able to maintain a productivity of>1×107TUmL-1day-1for up to 29 consecutive days in a non-optimized 5L stirred-tank bioreactor perfusion process, representing a major milestone in the field of LV manufacturing. As the producer cell lines are based on an inducible Tet-off expression system, the established process allows LV production in the absence of inducers such as antibiotics. The purified LVs efficiently transduce human CD34+ cells, reducing the LV quantities required for gene and cell therapy applications.

Continuous microalgal culture module and method of culturing microalgae containing macular pigment

This study established and investigated continuous macular pigment (MP) production with a lutein (L):zeaxanthin (Z) ratio of 4–5:1 by an MP-rich Chlorella sp. CN6 mutant strain in a continuous microalgal culture module. Chlorella sp. CN6 was cultured in a four-stage module for 10 days. The microalgal culture volume increased to 200 L in the first stage (6 days). Biomass productivity increased to 0.931 g/L/day with continuous indoor white light irradiation during the second stage (3 days). MP content effectively increased to 8.29 mg/g upon continuous, indoor white light and blue light-emitting diode irradiation in the third stage (1 day), and the microalgal biomass and MP concentrations were 8.88 g/L and 73.6 mg/L in the fourth stage, respectively. Using a two-step MP extraction process, 80 % of the MP was recovered with a high purity of 93 %, and its L:Z ratio was 4–5:1.

The massive 340 megabase genome of Anisogramma anomala, a biotrophic ascomycete that causes eastern filbert blight of hazelnut

BackgroundThe ascomycete fungus Anisogramma anomala causes Eastern Filbert Blight (EFB) on hazelnut (Corylus spp.) trees. It is a minor disease on its native host, the American hazelnut (C. americana), but is highly destructive on the commercially important European hazelnut (C. avellana). In North America, EFB has historically limited commercial production of hazelnut to west of the Rocky Mountains. A. anomala is an obligately biotrophic fungus that has not been grown in continuous culture, rendering its study challenging. There is a 15-month latency before symptoms appear on infected hazelnut trees, and only a sexual reproductive stage has been observed. Here we report the sequencing, annotation, and characterization of its genome.ResultsThe genome of A. anomala was assembled into 108 scaffolds totaling 342,498,352 nt with a GC content of 34.46%. Scaffold N50 was 33.3 Mb and L50 was 5. Nineteen scaffolds with lengths over 1 Mb constituted 99% of the assembly. Telomere sequences were identified on both ends of two scaffolds and on one end of another 10 scaffolds. Flow cytometry estimated the genome size of A. anomala at 370 Mb. The genome exhibits two-speed evolution, with 93% of the assembly as AT-rich regions (32.9% GC) and the other 7% as GC-rich (57.1% GC). The AT-rich regions consist predominantly of repeats with low gene content, while 90% of predicted protein coding genes were identified in GC-rich regions. Copia-like retrotransposons accounted for more than half of the genome. Evidence of repeat-induced point mutation (RIP) was identified throughout the AT-rich regions, and two copies of the rid gene and one of dim-2, the key genes in the RIP mutation pathway, were identified in the genome. Consistent with its homothallic sexual reproduction cycle, both MAT1-1 and MAT1-2 idiomorphs were found. We identified a large suite of genes likely involved in pathogenicity, including 614 carbohydrate active enzymes, 762 secreted proteins and 165 effectors.ConclusionsThis study reveals the genomic structure, composition, and putative gene function of the important pathogen A. anomala. It provides insight into the molecular basis of the pathogen’s life cycle and a solid foundation for studying EFB.

Development of reliable in vitro long-term culture systems for hematopoietic cells of crayfish Cherax quadricarinatus and virus susceptibility assay

Long-term in vitro cultured crayfish cells can serve as a valuable tool for investigating various aspects of crayfish biology, encompassing cell physiology and viral mechanisms. Regrettably, they are difficult to maintain a highly active state and survive during long-term and continuous culture in vitro. In order to generate highly viable crayfish cell cultures, it is crucial to optimize dependable culture conditions and utilize a suitable cell medium. In this study, we have successfully developed reliable in vitro long-term culture systems for hematopoietic (HPT) cells of crayfish Cherax quadricarinatus by formulating an optimal crayfish cell growth medium (OCCM), including under two-dimensional (2D) and three-dimensional (3D) conditions. The novel medium was optimized by a mix of exogenous nutrients, crayfish plasma (CP) and shrimp serum-derived amino acids and saccharides within the Leibovitz's L-15 (L-15) medium. Our newly developed crayfish cell growth medium (CCM-28) enabled the culturing of HPT cells for over 142 days under 2D and over 60 days under 3D conditions, with consistent active cell proliferation observed over a 30-day incubation period. This proliferation was comparable to in vivo conditions, with a cell division rate exceeding 20%, suggesting that a high mitotic rate has been successfully achieved during long-term in vitro culture. Flow cytometry analysis further elucidated the mitosis-arrest of HPT cells cultured in vitro was predominantly observed in the G0/G1 phase. Furthermore, the cell subculture techniques have been established using mechanical dissociation method (in 2D) and substrate exchange method (in 3D). Additionally, the susceptibility of these cell cultures to covert mortality nodavirus (CMNV) and white spot syndrome virus (WSSV) was confirmed by capturing cytopathic effects (CPE) and analyzing viral copy numbers. Species identification using the cytochrome oxidase subunit I (COI) gene established the origin of the cell cultures from C. quadricarinatus. In conclusion, the successful development of highly viable crayfish cells in long-term in vitro culture systems, as demonstrated in this study, will significantly advance the research on immortalization in crayfish cells and the study of mechanisms related to viruses.

Association of persistent positive blood cultures and infective endocarditis: A cohort study among patients with suspected infective endocarditis

ObjectivesTo ascertain whether infective endocarditis (IE) was associated with persistent bacteraemia/candidaemia among patients with suspected IE. MethodsThis study included bacteraemic/candidaemic adult patients with echocardiography and follow-up blood cultures. Persistent bacteraemia/candidaemia was defined as continued positive blood cultures with the same microorganism for 48 h or more after antibiotic treatment initiation. Each case was classified for IE by the Endocarditis Team. ResultsAmong 1962 episodes of suspected IE, IE (605; 31%) was the most prevalent infection type. Persistent bacteraemia/candidaemia was observed in 426 (22%) episodes. Persistent bacteraemia was more common among episodes with Staphylococcus aureus bacteraemia compared to episodes with positive blood cultures for other pathogens (32%, 298/933 vs 12%, 128/1029; P < 0.001). Multivariable analysis demonstrated that cardiac predisposing factors (aOR 1.84, 95% CI 1.31-2.60), community or non-nosocomial healthcare-associated (2.85, 2.10-3.88), bacteraemia by high-risk bacteria, such as S. aureus, streptococci, enterococci or HACEK (1.84, 1.31-2.60), two or more positive sets of index blood cultures (6.99, 4.60-10.63), persistent bacteraemia/candidaemia for 48 h from antimicrobial treatment initiation (1.43, 1.05-1.93), embolic events within 48h from antimicrobial treatment initiation (12.81, 9.43-17.41), and immunological phenomena (3.87, 1.09-1.78) were associated with infective endocarditis. ConclusionsIE was associated with persistent bacteraemia/candidaemia, along with other commonly associated factors.

The role of silver nanoparticles alone and combined with imipenem on carbapenem-resistant Klebsiella pneumoniae.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infections poses a significant threat to human health, necessitating urgent development of new antimicrobial agents. Silver nanoparticles (AgNPs), which are among the most widely used engineered nanomaterials, have been extensively studied. However, the impact of AgNPs on CRKP and the potential for drug resistance development remain inadequately explored. In this study, broth dilution method was used to determine the minimum inhibitory concentration (MIC) was determined using the broth dilution method. Results indicated MIC values of 93.1±193.3µg ml-1 for AgNPs, 2.3±5.1µg ml-1 for AgNO3, and 25.1±48.3µg ml-1 for imipenem (IMI). The combined inhibitory effect of AgNPs and IMI on CRKP was assessed using the checkerboard method. Moreover, after 6-20 generations of continuous culture, the MIC value of AgNPs increased 2-fold. Compared to IMI, resistance of Kl. pneumoniae to AgNPs developed more slowly, with a higher fold increase in MIC observed after 20 generations. Whole-genome sequencing revealed four nonsynonymous single nucleotide polymorphism mutations in CRKP after 20 generations of AgNP treatment. We have demonstrated that AgNPs significantly inhibit CRKP isolates and enhance the antibacterial activity of imipenem against Kl. pneumoniae. Although the development of AgNP resistance is gradual, continued efforts are necessary for monitoring and studying the mechanisms of AgNP resistance.

Uncovering the role of oxygen on organic carbon cycling: insights from a continuous culture study with a facultative anaerobic bacterioplankton species (Shewanella baltica)

Deoxygenation is tied to organic carbon (Corg) supply and utilization in marine systems. Under oxygen-depletion, bacteria maintain Corg respiration using alternative electron acceptors such as nitrate. Since anaerobic respiration’s energy yield is lower, Corg remineralization may be reduced and its residence time increased. We investigated the influence of oxygen and alternative electron acceptors’ availability on Corg cycling by heterotrophic bacteria during a continuous culture experiment with Shewanella baltica, a facultative anaerobic γ-Proteobacteria in the Baltic Sea. We tested six different oxygen levels, from suboxic (&amp;lt;5 µmol L-1) to fully oxic conditions, using a brackish (salinity=14 g L-1) media supplied with high (HighN) or low (LowN) inorganic nitrogen concentrations relative to glucose as labile Corg source. Our results show that suboxia limited DOC (glucose) uptake and cell growth only under LowN, while higher availability of alternative electron acceptors seemingly compensated oxygen limitation under HighN. N-loss was observed under suboxia in both nitrogen treatments. Under HighN, N-loss was highest and a C:N loss ratio of ~2.0 indicated that Corg was remineralized via denitrification. Under LowN, the C:N loss ratio under suboxia was higher (~5.5), suggesting the dominance of other anaerobic respiration pathways, such as dissimilatory nitrate reduction to ammonium (DNRA). Bacterial growth efficiency was independent of oxygen concentration but higher under LowN (34 ± 3.0%) than HighN (26 ± 1.6%). Oxygen concentration also affected dissolved organic matter (DOM) cycling. Under oxic conditions, the release of dissolved combined carbohydrates was enhanced, and the amino acid-based degradation index (DI) pointed to more diagenetically altered DOM. Our results suggest bacterial Corg uptake in low-oxygen systems dominated by S. baltica can be limited by oxygen but compensated by high nitrate availability. Hence, suboxia diminishes Corg remineralisation only when alternative electron acceptors are lacking. Under high nitrate:Corg supply, denitrification leads to a higher N:C loss ratio, potentially counteracting eutrophication in the long run. Low nitrate:Corg supply may favour other anaerobic respiration pathways like DNRA, which sustains labile nitrogen in the system, potentially intensifying the cycle of eutrophication. Going forward, it will be crucial to establish the validity of our findings for S. baltica in natural systems with diverse organic substrates and microbial consortia.

Matrix-free human pluripotent stem cell manufacturing by seed train approach and intermediate cryopreservation

BackgroundHuman pluripotent stem cells (hPSCs) have an enormous therapeutic potential, but large quantities of cells will need to be supplied by reliable, economically viable production processes. The suspension culture (three-dimensional; 3D) of hPSCs in stirred tank bioreactors (STBRs) has enormous potential for fuelling these cell demands. In this study, the efficient long-term matrix-free suspension culture of hPSC aggregates is shown.Methods and resultsSTBR-controlled, chemical aggregate dissociation and optimized passage duration of 3 or 4 days promotes exponential hPSC proliferation, process efficiency and upscaling by a seed train approach. Intermediate high-density cryopreservation of suspension-derived hPSCs followed by direct STBR inoculation enabled complete omission of matrix-dependent 2D (two-dimensional) culture. Optimized 3D cultivation over 8 passages (32 days) cumulatively yielded ≈4.7 × 1015 cells, while maintaining hPSCs’ pluripotency, differentiation potential and karyotype stability. Gene expression profiling reveals novel insights into the adaption of hPSCs to continuous 3D culture compared to conventional 2D controls.ConclusionsTogether, an entirely matrix-free, highly efficient, flexible and automation-friendly hPSC expansion strategy is demonstrated, facilitating the development of good manufacturing practice-compliant closed-system manufacturing in large scale.

Progress with the Learning Health System 2.0: a rapid review of Learning Health Systems’ responses to pandemics and climate change

BackgroundPandemics and climate change each challenge health systems through increasing numbers and new types of patients. To adapt to these challenges, leading health systems have embraced a Learning Health System (LHS) approach, aiming to increase the efficiency with which data is translated into actionable knowledge. This rapid review sought to determine how these health systems have used LHS frameworks to both address the challenges posed by the COVID-19 pandemic and climate change, and to prepare for future disturbances, and thus transition towards the LHS2.0.MethodsThree databases (Embase, Scopus, and PubMed) were searched for peer-reviewed literature published in English in the five years to March 2023. Publications were included if they described a real-world LHS’s response to one or more of the following: the COVID-19 pandemic, future pandemics, current climate events, future climate change events. Data were extracted and thematically analyzed using the five dimensions of the Institute of Medicine/Zurynski-Braithwaite’s LHS framework: Science and Informatics, Patient-Clinician Partnerships, Continuous Learning Culture, Incentives, and Structure and Governance.ResultsThe search yielded 182 unique publications, four of which reported on LHSs and climate change. Backward citation tracking yielded 13 additional pandemic-related publications. None of the climate change-related papers met the inclusion criteria. Thirty-two publications were included after full-text review. Most were case studies (n = 12, 38%), narrative descriptions (n = 9, 28%) or empirical studies (n = 9, 28%). Science and Informatics (n = 31, 97%), Continuous Learning Culture (n = 26, 81%), Structure and Governance (n = 23, 72%) were the most frequently discussed LHS dimensions. Incentives (n = 21, 66%) and Patient-Clinician Partnerships (n = 18, 56%) received less attention. Twenty-nine papers (91%) discussed benefits or opportunities created by pandemics to furthering the development of an LHS, compared to 22 papers (69%) that discussed challenges.ConclusionsAn LHS 2.0 approach appears well-suited to responding to the rapidly changing and uncertain conditions of a pandemic, and, by extension, to preparing health systems for the effects of climate change. LHSs that embrace a continuous learning culture can inform patient care, public policy, and public messaging, and those that wisely use IT systems for decision-making can more readily enact surveillance systems for future pandemics and climate change-related events.Trial registrationPROSPERO pre-registration: CRD42023408896.

Opportunities and Perspectives for Three Dimensional Culture of Mesenchymal Stem Cell-Derived Exosomes.

Exosomes are nanosized extracellular vesicles (EVs) that participate in intercellular communication through surface proteins and the delivery of internal cargo. The exosomes have gained attention for their potential as disease biomarkers and therapeutic agents. The therapeutic ability of exosomes has been verified by copious previous studies. Effective methods for extensive clinical applications are being researched for exosome-based regenerative therapies, including the application of 3D cultures to enhance exosome production and secretion, which can resolve limited exosome secretion from the parent cells. Cell culture has emerged as a crucial approach for biomedical research because of its many benefits. Both well-established continuous cell lines and primary cell cultures continue to be invaluable for basic research and clinical application. Previous studies have shown that three-dimensional cultured exosomes (3D-Exo) improve therapeutic properties and yields compared with traditional culture systems. Since the majority of studies have focused on exosomes derived from mesenchymal stem cells (MSC-Exo), this review will also concentrate on MSC-Exo. In this review, we will summarize the advantages of 3D-Exo and introduce the 3D culture system and methods of exosome isolation, providing scientific strategies for the diagnosis, treatment, and prognosis of a wide variety of diseases.

Investigating the association between nitrate dosing and nitrite generation by the human oral microbiota in continuous culture.

The generation of nitrite by the oral microbiota is believed to contribute to healthy cardiovascular function, with oral nitrate reduction to nitrite associated with systemic blood pressure regulation. There is the potential to manipulate the composition or activities of the oral microbiota to a higher nitrate-reducing state through nitrate supplementation. The current study examined microbial community composition and enzymatic responses to nitrate supplementation in sessile oral microbiota grown in continuous culture. Nitrate reductase (NaR) activity and nitrite concentrations were not significantly different to tongue-derived inocula in model biofilms. These were generally dominated by Streptococcus spp., initially, and a single nitrate supplementation resulted in the increased relative abundance of the nitrate-reducing genera Veillonella, Neisseria, and Proteus spp. Nitrite concentrations increased concomitantly and continued to increase throughout oral microbiota development. Continuous nitrate supplementation, over a 7-day period, was similarly associated with an elevated abundance of nitrate-reducing taxa and increased nitrite concentration in the perfusate. In experiments in which the models were established in continuous low or high nitrate environments, there was an initial elevation in nitrate reductase, and nitrite concentrations reached a relatively constant concentration over time similar to the acute nitrate challenge with a similar expansion of Veillonella and Neisseria. In summary, we have investigated nitrate metabolism in continuous culture oral biofilms, showing that nitrate addition increases nitrate reductase activity and nitrite concentrations in oral microbiota with the expansion of putatively NaR-producing taxa.IMPORTANCEClinical evidence suggests that blood pressure regulation can be promoted by nitrite generated through the reduction of supplemental dietary nitrate by the oral microbiota. We have utilized oral microbiota models to investigate the mechanisms responsible, demonstrating that nitrate addition increases nitrate reductase activity and nitrite concentrations in oral microbiota with the expansion of nitrate-reducing taxa.

Impacts of priming on distinct immunosuppressive mechanisms of mesenchymal stromal cells under translationally relevant conditions

BackgroundThe multimodal properties of mesenchymal stromal cells (MSCs), particularly their ability to modulate immune responses is of high interest in translational research. Pro-inflammatory, hypoxic, and 3D culture priming are promising and often used strategies to improve the immunosuppressive potency of MSCs, but the underlying mechanisms are not well understood. Therefore, the aims of this study were (i) to compare the effects of pro-inflammatory, hypoxic, and 3D culture priming on the in vitro immunosuppressive potential of MSCs, (ii) to assess if immunosuppressive priming effects are temporally preserved under standard and translationally relevant culture conditions, and (iii) to investigate if the three priming strategies engage the same immunosuppressive mechanisms.MethodsFunctional in vitro T cell suppressive potency measurements were conducted to assess the impact of pro-inflammatory, hypoxic, and 3D culture priming on the immunosuppressive potential of human bone marrow-derived MSCs. Primed MSCs were either cultured under standard cell culture conditions or translationally relevant culture conditions, and their transcriptomic adaptations were monitored over time. Next-generation sequencing was performed to assess if different priming strategies activate distinct immunosuppressive mechanisms.Results(i) Pro-inflammatory, hypoxic, and 3D culture priming induced profound transcriptomic changes in MSCs resulting in a significantly enhanced T cell suppressive potential of pro-inflammatory and 3D culture primed MSCs. (ii) Priming effects rapidly faded under standard cell culture conditions but were partially preserved under translationally relevant conditions. Interestingly, continuous 3D culture priming of MSCs maintained the immunosuppressive potency of MSCs. (iii) Next-generation sequencing revealed that priming strategy-specific differentially expressed genes are involved in the T cell suppressive capacity of MSCs, indicating that different priming strategies engage distinct immunosuppressive mechanisms.ConclusionPriming can be a useful approach to improve the immunosuppressive potency of MSCs. However, future studies involving primed MSCs should carefully consider the significant impact of translationally relevant conditions on the preservation of priming effects. Continuous 3D culture could act as a functionalized formulation, supporting the administration of MSC spheroids for a sustainably improved immunosuppressive potency.

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.

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.