Culture Time Research Articles

RecombiCraft library construction: A novel method for DNA library cloning and expansion using non-enzymatic single-step DNA recombination and liquid culture

In this study, we introduce RecombiCraft, an innovative, rapid, and cost-efficient method for constructing DNA libraries in E. coli. This method uses seamless ligation cloning extract (SLiCE) coupled with liquid culture amplification to effectively minimize sequence biases. The technique capitalizes on the natural homologous recombination capabilities of E. coli cell lysates, eliminating the need for multiple purified enzymes and reducing costs. We first synthesized the library backbone and inserts via PCR, employing high-fidelity polymerase to minimize sequence bias. The SLiCE technique was then used to assemble the DNA fragments introduced into E. coli through electroporation. To ensure the integrity of the library, we optimized culture times based on next-generation sequencing analysis which confirmed the minimal sequence bias. The RecombiCraft method demonstrates that this approach is economical and maintains the library’s uniformity. By using liquid culture, this method can complete DNA library generation in about 12 hours and final extraction is simple, making it a promising tool for genetic research and biotechnology applications.

Establishment and Validation of an Efficient Agrobacterium Tumefaciens-Mediated Transient Transformation System for Salix Psammophila

Salix psammophila, C. Wang & Chang Y. Yang, a desert-adapted shrub, is recognized for its exceptional drought tolerance and plays a vital role in ecosystem maintenance. However, research on S. psammophila has been limited due to the lack of an efficient and reliable genetic transformation method, including gene functional studies. The Agrobacterium-mediated transient overexpression assay is a rapid and powerful tool for analyzing gene function in plant vivo. In this study, tissue culture seedlings of S. psammophila were utilized as the recipient materials, and the plant expression vector pCAMBIA1301, containing the GUS reporter gene, was transferred into the seedlings via an Agrobacterium-mediated method. To enhance the efficiency of the system, the effects of secondary culture time, Agrobacterium concentration, infection time, and co-culture duration on the transient transformation efficiency of S. psammophila were explored. The optimal combination for the instantaneous transformation of S. psammophila tissue culture seedlings mediated by Agrobacterium was determined as follows: a secondary culture time of 30 d, a value of OD600 of 0.8, an infection time of 3 h, and a co-culture duration of 48 h. Subsequently, the effectiveness of the transformation system was validated using the S. psammophila drought response gene SpPP2C80. To further confirm the accuracy of the system, SpPP2C80-overexpressing Arabidopsis was constructed and drought resistance analysis was performed. The results were consistent with the transient overexpression of SpPP2C80 in S. psammophila tissue culture seedlings, indicating that this system can be effectively employed for studying gene function in S. psammophila. These findings provide essential information for investigating gene function in non-model plants and pave the way for advancements in molecular biology research in S. psammophila.

Development of a tumor organoid culture system with peptide-based hydrogels

Peptide-based hydrogel, the polymer materials with a special network structure, are widely used in various fields of biomedicine due to their stable properties and biocompatibility. Environment-responsive self-assembled peptide aqueous solutions can respond to environment changes by the self-assembly of peptides into nanofiber networks. Peptide-based hydrogels well simulate the extracellular matrix and cell growth microenvironment, being suitable for 3D cell culture and organoid culture. To establish a tumor organoid culture system with peptide-based hydrogels, we cultured Panc-1, U87, and H358 cells in a 3D spherical manner using CulX Ⅱ peptide-based hydrogels in 24-well plates for 15 days. The organoids showed a 3D spherical shape, and their sizes increased with the extension of the culture time, with a final diameter ranging from 150 to 300 μm. The organoids had a large number, varying sizes, good cell viability, clear edges, and a good shape, which indicated successful organoid construction. The tumor organoid culture system established in this study with CulX Ⅱ peptide-based hydrogels provides a model for studying tumor pathogenesis, drug development, and tumor suppression.

Diets supplemented with different solvents extracts of Lepista nuda alters longevity and fecundity, and the expression of related genes in Drosophila melanogaster (Diptera: Drosophilidae).

Exploring the effects of different solvent extracts from Lepista nuda [(Bull. ex Fr.) Cooke] on the lifespan and reproductive capacity of Drosophila melanogaster (Diptera: Drosophilidae), the extracts of the fruiting body and mycelium of L. nuda were prepared using water, 75% ethanol, and petroleum ether, and the active components were identified. These extracts were then incorporated into culture media and administered to D. melanogaster. The impact of the extracts from different solvents on the life span and fertility, and the contents of ecdysone (20E), juvenile hormone (JH), and vitellogenin (Vg), as well as the effects of autophagy gene, 20E synthesis gene Halloween, 20E receptor gene ECR, JH methyltransferase gene JHAMT and Vg gene Yolk1 transcripts were analyzed. The extracts from the fruiting body and mycelium of L. nuda can reduce lifespan, a phenomenon associated with the varied expression of 15 compounds across 6 distinct groups. The average survival time of female fruit flies was lower than that of the male fruit flies. Fertility had also been significantly reduced, indicating a positive correlation between lifespan and fertility. In addition, with the extension of cultivation time, the content of 20E, JH, and Vg, as well as the transcripts of Halloween, ECR, JHAMT, and Yolk1 in the L. nuda fruiting body extract treatment group, all significantly decreased. Among the 3 solvent extracts, phenylalanine, citric acid, quinic acid, and punicalagin in the L. nuda aqueous extract exhibit the most potent collective toxicity toward fruit flies. The insecticidal properties of these compounds function by modulating autophagy and the expression of insect hormones.

Myocarditis and pericarditis in young patients following Campylobacter jejuni enterocolitis infection: a systematic review of case studies

BackgroundCampylobacter is known to be the leading cause of foodborne illness. Campylobacter jejuni, specifically, most commonly causes self-limiting enterocolitis, but infection can lead to extraintestinal manifestations, including rare yet severe cardiac complications, such as myocarditis and/or pericarditis. This review aims to determine whether a relationship exists between the timing of a positive stool culture and the overall clinical course in patients with Campylobacter jejuni-associated myocarditis and/or pericarditis.MethodsA systematic search was conducted using PubMed, MEDLINE, CINAHL Ultimate, Academic Search Premier, and Nursing and Allied Health Premium databases. A gray literature search was also performed. Covidence.org was used to screen, select, and extract data by two independent reviewers. Following a full-text evaluation, the quality of each study was assessed using the Joanna Briggs Institute Critical Appraisal Checklist for Case Reports.ResultsThe search resulted in 434 records, of which 7 case reports met the inclusion criteria, each study including one patient. The average number of days from hospital presentation to obtaining a stool sample was 1.7. The average number of days from stool culture obtainment to clinical course improvement was 3.3, with four case studies reporting a range of 2–4 days.ConclusionThis systematic review raises awareness regarding the manifestations of Campylobacter jejuni, specifically the severe cardiac symptoms that may present in young individuals. Given the significant sequelae that can develop, providers must elicit a thorough history consisting of questions related to recent travel, food consumption, and gastrointestinal symptoms, especially among young individuals presenting with cardiac complaints.

Conglomeration, Digital Disruption, and COVID-19: Upheaval-Driven Changes to the Roles of U.S. Local Television News Directors

This mixed methods study analyzes how broadcast news directors renegotiate their roles under organizational and cultural times of change. Even before COVID-19, local television news directors, one of the two predominant groups of journalism managers in the United States, were experiencing shifts in roles related to their growing work responsibilities. These changes were the result of long-term workplace upheavals including conglomeration and digital disruption. When the COVID-19 pandemic hit, news directors found themselves managing through this additional, short-term upheaval. The authors of this study sought to discover how industry upheavals impact broadcast newsroom management roles.

Diagnosis of Chlamydia abortus by Isolation in Cell Culture and Real Time PCR in Aborted Sheep and Goats

Background: Ovine enzootic abortion (OEA) caused by Chlamyidia abortus is one of the most important abortive disease in small ruminants. Diagnosis of Ovine enzootic abortion depends on the isolation and detection of the agent or its nucleic acid. The aim of the present study was to detect Chlamydia abortus using both isolation method and real-time PCR in Brucella free flocks in Iran. Methods: Twenty-eight vaginal and conjunctival swab samples which were Chlamydia abortus seropositive, were selected from ewes and does with recent abortion. Then the samples were tested by real-time PCR and positive molecular samples were inoculated into McCoy cells. Results: Using real-time PCR, 18 samples (64.3%) were positive and 7 (25%) of them were isolated in cell culture. Conclusion: The present results indicate that Chlamydia can play a relatively significant role in the abortion in does and ewes in Iran. Although the isolation of Chlamydia abortus have 100% specificity, because of low sensitivity, time consuming, cost and high probability of contamination, it is not suitable for routine laboratory diagnosis. Therefore, applying real-time PCR which have high sensitivity and specificity is recommended.

A rotenone organotypic whole hemisphere slice model of mitochondrial abnormalities in the neonatal brain

Mitochondrial abnormalities underscore a variety of neurologic injuries and diseases and are well-studied in adult populations. Clinical studies identify critical roles of mitochondria in a wide range of developmental brain injuries, but models that capture mitochondrial abnormalities in systems representative of the neonatal brain environment are lacking. Here, we develop an organotypic whole-hemisphere (OWH) brain slice model of mitochondrial dysfunction in the neonatal brain. We extended the utility of complex I inhibitor rotenone (ROT), canonically used in models of adult neurodegenerative diseases, to inflict mitochondrial damage in OWH slices from term-equivalent rats. We quantified whole-slice health over 6 days of exposure for a range of doses represented in ROT literature. We identified 50 nM ROT as a suitable exposure level for OWH slices to inflict injury without compromising viability. At the selected exposure level, we confirmed exposure- and time-dependent mitochondrial responses showing differences in mitochondrial fluorescence and nuclear localization using MitoTracker imaging in live OWH slices and dysregulated mitochondrial markers via RT-qPCR screening. We leveraged the regional structures present in OWH slices to quantify cell density and cell death in the cortex and the midbrain regions, observing higher susceptibilities to damage in the midbrain as a function of exposure and culture time. We supplemented these findings with analysis of microglia and mature neurons showing time-, region-, and exposure-dependent differences in microglial responses. We demonstrated changes in tissue microstructure as a function of region, culture time, and exposure level using live-video epifluorescence microscopy of extracellularly diffusing nanoparticle probes in live OWH slices. Our results highlight severity-, time-, and region-dependent responses and establish a complimentary model system of mitochondrial abnormalities for high-throughput or live-tissue experimental needs.

Neutrophils Respond Selectively to Physical Cues: Roughness Modulates Its Granule Release, and NETosis.

Our study examined how different titanium alloy Ti6Al4V (Ti64) and zirconia (ZrO2) surfaces, ranging from rough to very smooth, affect the expression of elastase (NE), matrix metalloproteinase (MMP)-8, MMP-9, and extracellular traps (NETs) by neutrophils. Discs of Ti64 and ZrO2, 10 mm in diameter and 1.5 mm thick, were created using diamond-impregnated polishing burs and paste to produce rough (Ra > 3 µm), smooth (Ra ≥ 1 to 1.5 µm), and very smooth (Ra < 0.1 µm) surfaces. Neutrophils from Wistar rats were cultured on these surfaces, and the culture supernatants were then examined for NE, MMP-8, and MMP-9 using ELISA. At the same time, NET formation was demonstrated immunohistochemically by staining neutrophils with CD16b and DNA with DAPI. Overall, the expressions of NE and MMP-8 were significantly higher from neutrophil culture on Ti64 and ZrO2 rough surfaces compared to the very smooth surface (R > S > VS) after 2 h and 4 h of culture. The expression of MMP-9 also increased with culture time; however, no significant surface effects on expression were observed. Similarly, rough Ti64 and ZrO2 surfaces (R & S) also showed significantly larger NET formation compared to the very smooth surface (VS) after 4 h and 8 h cultures. Our findings suggest that increasing surface roughness on Ti64 and ZrO2 triggers higher NE, MMP-8, and NET formation secretion.

Unlocking marine microbial treasures: new PBP2a-targeted antibiotics elicited by metals and enhanced by RSM-driven transcriptomics and chemoinformatics

Elicitation through abiotic stress, including heavy metals, is a new natural product drug discovery technique. In this research, three compounds 1, 2, and 6, were achieved by triggering zinc and nickel on marine Sphingomonas sp. and Streptomyces sp., which were absent in normal culture. Compound 5 was obtained for the first time from marine bacteria. All compounds showed potent antibacterial activity against Staphylococcus aureus and bactericidal effect at 300 µm, but 6 was more active. The potent compound 6 production was further enhanced through response surface methodology by optimizing the condition consisting of nickel 1 mM ions, 20 mg/L sucrose, 30 mg/L salt and culture time 14 days. Under these conditions, the SM-6 production was enhanced with a yield of 6.3 mg/L, which was absent in the normal culture. Further transcriptome analysis of compound 6 unveiled its antibacterial activity on S. aureus by modulating heat shock protein genes, disrupting protein folding and synthesis, and perturbing cellular redox balance, leading to a comprehensive inhibition of normal bacterial growth. In addition, ADMET has shown that all compounds are safe for cardiac and hepatotoxicity. To determine the anti-bacterial mechanism, all compounds were docked with PBP2a and DNA gyrase enzyme, and TLR-4 protein for predicting vaccine construct, and the best docking score was achieved against PBP2a enzyme with the highest score of −10.2 for compound 6. In-silico cloning was carried out to ensure the expression of proteins generated and were cloned using S.aureus as a host. The simulation studies have shown that both SM-6-PBP2a and TLR-4-PBP2a complex are stable with the system. This study presents a new approach to anti-bacterial drug discovery from microorganisms through heavy metals triggering and enhancing the compound production through response surface methodology.

Establishment and Use of Primary Cultured Astrocytes from Alexander Disease Model Mice.

Alexander disease (AxD) is an intractable neurodegenerative disease caused by mutations in glial fibrillary acidic protein (GFAP), which is predominantly expressed in astrocytes. Thus, AxD is a primary astrocyte disease. However, it remains unclear how GFAP mutations affect astrocytes and cause AxD pathology. Three features are characteristic of AxD astrocytes in vivo: (1) Rosenthal fibers (RFs), the hallmark of AxD; (2) aberrant Ca2+ signals (AxCa); and (3) upregulation of disease-associated genes (AxGen). We established a primary culture system for astrocytes from an AxD transgenic mouse model, and used it to analyze the above features of AxD pathogenesis in astrocytes in vitro. We observed the formation of RFs in AxD primary cultures. The abundance of RFs was greater in AxD-transgene-homozygous compared with -hemizygous astrocytes, indicating a gene dosage effect, and this abundance increased with time in culture, indicating a developmental process effect. However, cultured AxD astrocytes did not exhibit changes in either AxCa or AxGen. We therefore conclude that RFs in astrocytes form via a cell-autonomous mechanism, whereas AxCa and AxGen are likely to occur via a non-cell-autonomous mechanism through interactions with other cells, such as neurons, microglia, and vascular cells. Although primary cultured AxD astrocytes are suitable for elucidating the mechanisms of RFs formation and for intervention studies, it should be noted that they cannot reflect the pathophysiology of non-cell-autonomous events in astrocytes.

Images and Motifs on Painted Ceramics Type C and D in Predynastic Egypt

The image of the Great Mother, whose cult can be traced back to the time of the Badari culture and the early phases of the Nagada culture, was carried by historical memory through the millennia of the existence of ancient Egyptian culture. Painted vessels of type C and D and the images on them represent the image of the Great Mother and her semantically identical symbols in compositions that reveal a number of motifs associated with the maternal aspect, sacred marriage with a social leader, and funeral rites. In the dynamics of the development of religious and mythological ideas, this image was embodied in the images of goddesses with a maternal aspect, primarily the heavenly goddess Nut, who preserved in the hieroglyphic spelling of her name the oldest image of a round vessel nw, which in predynastic times, along with water, served as a semantic identity of the image of the Great Mother in her functions of birth, protection, feeding, care. The iconography of the image of the cow goddess, who became the Heavenly Cow Nut, who raised the solar god Ra, has also been preserved. In Chapter 17 of the Book of the Dead, she appears as the Sacred Eye of Ra, which gives birth to the solar Ra every morning. The image of the Sacred Eye is involved in the water and fire elements of the cosmic top, like her distant ancestor the Great Mother, who created an integral, diverse world, all the elements of which, be it heavenly bodies, ground and underground objects, natural and social, are her children, her creations.

Comparison Between Simple Batch and Fed-Batch Bioreactor Cultivation of Recombinant BCG.

Background/Objectives: Tuberculosis continues to be a significant global health concern, causing 1.3 million deaths in 2022, particularly affecting children under 5 years old. The Bacillus Calmette-Guérin (BCG) vaccine, developed in 1921, remains the primary defense against tuberculosis but requires modernized production methods. The recombinant BCG-pertussis strain shows potential in providing dual protection against tuberculosis and whooping cough, especially for vulnerable newborns, and enhanced efficacy against bladder cancer. Implementing submerged cultivation techniques for rBCG-pertussis production can offer increased productivity and standardization. Methods: This study explores a fed-batch cultivation strategy with pH-stat control to feed L-glutamic acid through the acid pump into 1 L bioreactor. Three pH values were evaluated for fed-batch and a simple batch without pH control was done for comparison. The viable cell concentration was compared before and after freeze-drying samples harvested during the cultures. Results: L-glutamic acid was identified as the preferred substrate for rBCG-pertussis. While the fed-batch strategy did not enhance the maximum specific growth rate compared to simple batch cultivation, it did improve the specific growth rate after day 4 in the pH 7.4-controlled fed-batch cultures, thereby reducing the cultivation time. Fed-batch cultures controlled at three pH levels exhibited lower optical density than the simple batch, although the viable cell counts were similar. Notably, samples harvested after day 8 from the simple batch cultures showed a reduction in CFU/mL after freeze-drying, whereas all fed-batch samples exhibited high recovery of viable cell counts post lyophilization. Conclusions: The additional glutamate supplied to the fed-batch cultures may have protected the cells during the lyophilization process.

Passivation of Cadmium and Arsenic in Acidified Paddy Soil by Calcium Fertilizer with Biochar-ferromanganese Composites

Due to the aggravation of atmospheric nitrogen and sulfur deposition and the unreasonable application of fertilizer, soil acidification is becoming increasingly serious. In heavy metal-contaminated soils, acidification not only seriously affects fertility but also the effectiveness and sustainability of conventional passivation remediation materials such as biochar. The application of calcium fertilizer may improve soil acidification, alleviate the aging of biochar materials in soil, and improve its remediation ability to composite polluted soil. However, the mechanism of its effect is still unclear. Based on this, this study selected iron-manganese oxide （FM） and hickory cattails biochar （BC） to prepare biochar-ferromanganese composites （BFM） and conducted simulated acidification on it. Through characterization and an aqueous adsorption test, the changes in physicochemical properties and adsorption properties of the material after acidification were explored. Then, orthogonal tests were carried out to explore the effects of the combination of BFM and calcium fertilizer on soil pH and the availability of Cd and As under acidification conditions and to obtain the best combination scheme. The results showed that the optimal ratio of BC and FM in BFM was 7∶3 （quality ratio）, the removal rates of Cd（Ⅱ） and As（Ⅲ） were as high as 94.58% and 97.14%, respectively, and the adsorption capacities were 120.74 mg·g-1 and 129.29 mg·g-1 （solid-liquid ratio 1∶500）. After acidification treatment, the pore structure of BFM surface decreased, and the types and quantities of functional groups changed, resulting in the removal rates of Cd（Ⅱ） and As（Ⅲ） in aqueous solution decreasing by 73.97%-92.84% and 73.56%-93.61%, respectively. The combined application of calcium fertilizer and BFM could significantly increase soil pH, with an increase range of 3.06%-37.84%. The effect of increasing pH decreased with the increase in culture time and acidification degree. Compared with that in the blank control, the content of available Cd in soil was significantly reduced by 22.67%-97.78%. The content of available As in soil was generally stable. According to the effect curve analysis of the orthogonal test results, under the condition of weak acidification degree （pH=5.6）, the application of 2% supplemental amount with 2% silica-calcium fertilizer and 2% calcium-magnesium phosphate fertilizer had a good passivation effect on soil Cd. Under the condition of strong acidification degree （pH=4.0）, the application of 2% supplemental amount and 2% silica-calcium fertilizer had a good passivation effect on soil As. In summary, simulated acidification will affect the adsorption performance of BFM, and calcium fertilizer combined with it can increase soil pH, improve soil acidification, alleviate the aging of BFM in acidified soil, and improve its repair ability to heavy metal-polluted soil.

Graph-learning-based machine learning improves prediction and cultivation of commercial-grade marine microalgae Porphyridium

A graph learning [Binarized Attributed Network Embedding (BANE)] model enhances the single-target and multi-target prediction performances of random forest and eXtreme Gradient Boosting (XGBoost) by learning complex interrelationships between cultivation parameters of Porphyridium. The BANE-XGBoost has the best prediction performance (train R2 > 0.96 and test R2 > 0.87). Based on Shapley Additive Explanation (SHAP) model, illumination intensity, culture time, and KH2PO4 are the most critical factors for Porphyridium growth. The combined facilitating roles of cultivation parameters are found using the SHAP value-based heat map and group. To reach high biomass and daily production rate concurrently, one-way and two-way partial dependent plots models find the optimal conditions. The top 2 critical parameters (illumination intensity and KH2PO4) were selected to verify using the graphical user interface website based on the optimized model and lab experiments, respectively. This study shows thegraph-learning-based model can improve prediction performance and optimize intricate low-carbon microalgal cultivation.

Whole genome sequencing of two strains of Staphylococcus simulans and their effects on the nitrosomyoglobin formation based on nitric oxide synthase

In order to evaluate the safety and functional characteristics of two potential meat starter cultures, Staphylococcus simulans HZ01 (OM758216) and Staphylococcus simulans HZ02 (OM758217), we performed the whole genome sequences, and then investigated their effects on nitrosomyoglobin formation based on nitric oxide synthase (NOS) catalysis. The results revealed that the two strains did not possess any virulence genes. The gene annotation indicated the gene function is mainly due to translation, ribosomal structure and biogenesis, and amino acid transport and metabolism. Both strains possess nos gene. The phylogenetic tree based on nos gene indicated the two strains formed an independent evolutionary branch. Subsequently, NOS was found to be expressed in both HZ01 and HZ02. NOS activity was higher in HZ01 than in HZ02, whereas NO production in inoculated groups changed considerably with increasing culture time. In the model system, nitrosomyoglobin formed in S. simulans inoculated groups, and the derivatives had absorption peaks at 545 nm and 580 nm. The secondary structure of these derivatives exhibited differences in β-turn proportion and α-helix proportion. Physicochemical properties of fermented sausages inoculated groups indicated that S. simulans HZ01 and HZ02 can significantly improve the color of sausages with no addition of nitrite/nitrate. This result confirmed that the two S. simulans strains can improve the nitrosomyoglobin formation based on the NOS pathway, which contributes to the color of fermented sausages, without the requirement of nitrite or nitrate. Nevertheless, the contribution of the NOS pathway in fermented meat products should be further evaluated.

Protective immune-response of Aeromonas hydrophila phage lysate in crucian carp against direct virulent challenge with A. hydrophila-TPS

Aeromonas hydrophila was a common opportunistic pathogen that was widely found in various aquatic environment and could cause multiple infections in humans and animals. The haemorrhagic septicemia and bacterial enteritis in fish triggered by this pathogen led to significant losses in the aquaculture industry. In this study, we aimed to develop a phage lysate vaccine by lysing the A. hydrophila-TPS strain using phage PZY-Ah, which was previously isolated and preserved in the laboratory. First, we focused on optimizing phage lysis conditions, including different host bacteria culture time, phage lysis time, and phage to bacterial ratios. The optimal conditions were established as follows: culturing the A. hydrophila-TPS strain for 6 h, adding phage at a ratio of 1:10, and mixing for 4 h, which resulted in maximum lysis of the host bacteria. Subsequently, we assessed the immune responses of groups receiving formaldehyde-inactivated vaccines compared to those receiving various concentrations of the phage lysate vaccine. Both the TPS-phage lysate and formaldehyde-inactivated vaccine groups exhibited increased levels of specific immune enzymes (ACP, AKP, LZM, SOD, CAT) and cytokines (IL-1β, TNF-α and IFN-γ) in serum, as well as enhanced humoral immunity (IgM, C3 and C4) in crucian carp. Furthermore, challenge tests conducted post-immunization demonstrated that the high concentration of the TPS strain-lysate vaccine group (1 × 108 CFU/mL) achieved the highest immune protection rate at 88.89 %. Overall, the development of the TPS-phage lysate vaccine significantly enhanced the immunity of crucian carp, providing a higher level of protection and establishing a foundation for the research and development of phage-based aquatic vaccines.

Phenotypic diversity of Bordetella pertussis against Trimethoprim/Sulphamethoxazole in vitro tests

BackgroundTrimethoprim/sulfamethoxazole (TMP/SMZ), traditionally a second-line drug, is now proposed as the primary alternative treatment for macrolide-resistant Bordetella pertussis (B.pertussis). Thus, there is an urgent need to evaluate the phenotypic profile of B. pertussis regarding susceptibility to TMP/SMZ. MethodsKirby-Bauer (KB) disk diffusion methods and E-test strips were employed to assess the in vitro susceptibility profiles of 128 B. pertussis strains isolated in China between 2017 and 2022 against TMP/SMZ. Additionally, whole genome sequencing was performed on one strain exhibiting a heterogeneous phenotype. ResultsB. pertussis displayed three phenotypes to TMP/SMZ based on the first 10 days of culture: a predominant fuzzy-bordered phenotype (118/128, 92.2 %), a minority with a clear-bordered phenotype (8/128, 6.3 %), and a sporadic heterogeneous phenotype (2/128, 1.6 %) where TMP/SMZ heterozygous colonies emerged on the 9th day of incubation. The diameter of the inhibition zone around the paper disk will decrease as the culture time extends, while the MIC value will change in the opposite direction. Almost in all tests, the bacterial colony will cover the entire inhibitory zone after 10-14 days of incubation. Strains exhibiting heterogeneity were compared with the complete genomes of Tohama I and CS strains, and 191 and 179 point mutation sites were identified, respectively, with multiple variants associated with the expression of transcriptional regulators. ConclusionThis study highlights the diverse drug sensitivity phenotypes of B. pertussis to TMP/SMZ and the importance of uniform testing protocols. Continuous and dynamic monitoring of B. pertussis isolates for TMP/SMZ sensitivity, along with investigating its correlation with epidemiological changes, is essential for establishing a scientific foundation for the effective management and control of pertussis.

Overcoming problematic growth phenotypes in organoids from patients with monogenic GI disease.

Patient-derived organoids provide a unique model system to explore disease-causing mutations ex vivo. By using organoids from duodenal or colonic biopsies of pediatric patients with intestinal epithelial disorders, we can directly assay the patient cells to tailor treatment to their unique disease state. The advent of organoid technology from patients with severe intestinal disorders such as Congenital Diarrhea Enteropathies (CoDE) and Very-Early-Onset Inflammatory Bowel Disease (VEO-IBD) has allowed for rapid advances in the understanding of and the treatment of these monogenic disorders. Still, the expansion of these lines for scalable studies is not trivial, and success rates of expansion are variable between groups, and even lab members within the same group. These protocols have been validated on patients with CoDE or VEO-IBD and age-matched control patients. Here, we present our recommended protocols for the cultivation of organoids from pediatric patients with CoDE and VEO-IBD. These protocols have been validated on organoids generated from the duodenum (duodenoids), ileum (ileoids), colon (colonoids) and iPSC-derived intestinal colonoids from pediatric healthy donors or donors with CoDE or VEO-IBD (Gwilt et al., 2023). Using our modified culture media, extended culture times from biopsy preparation and thawing frozen lines, gentle passaging techniques with the incomplete removal of the organoids from the matrigel, and modified monolayer protocols (Maeda et al., 2023; Maeda et al., 2022), we have been able to successfully culture and expand several lines for more than 5 years. The conditions and protocols used here provide a basis for reproducible phenotypes, scaling for larger functional studies on patient lines, and for reproducibility of results between several investigators. We provide a useful starting point and troubleshooting guidelines for the optimization of culturing organoids from any patient with novel disease pathology.

Fiber-based biomaterial scaffolds for cell support towards the production of cultivated meat

The in vitro production of animal-derived foods via cellular agriculture is emerging as a key solution to global food security challenges. Here, the potential for fiber-based scaffolds, including silk and cotton, in the cultivation of muscle cells for tissue formation was pursued. Mechanical properties and cytocompatibility with the mouse myoblast cell line C2C12 and immortalized bovine muscle satellite cells (iBSCs) were assessed, as well as pre-digestion options for the materials due to their resilience within the human digestive track. The fibers supported cell adhesion, proliferation, and guided muscle cell orientation, facilitating myotube formation per differentiation. A progressive increase in biomass was also documented. Interestingly, iBSC proliferation was enhanced with coatings of recombinant proteins while C2C12 cells showed minimal response. Thus, both cotton and silk yarns were suitable as fiber-based scaffolds towards cell supportive goals, suggesting an alternative path toward structured protein-rich foods via this initial stage of textile engineering for food. Biomass prediction models were generated, enabling forecasts of cell growth and maturation across various scaffold conditions and cell types. This capability enhances the precision of the cultivation process towards an engineering approach, building on the inherent benefits of hierarchical muscle tissue structure, but here via textile engineering with these initial muscle-coated edible fibers. Further, the approach offers to reduce costs by optimizing cultivation time and media needs. These approaches are part of a foundation for future scalable and sustainable cultivated meat production. Statement of SignificanceThis research investigates the use of one-dimensional fiber-based scaffolds for cultivated meat production, contributing to advancements in cellular agriculture. It introduces a method to measure changes in biomass and scaffold degradation throughout the cultivation process. Additionally, our development of biomass prediction models improves the precision and predictability of cultivated meat production. This research not only aids in scaling up cultivated meats but also enhances the use of textile engineering techniques in tissue engineering, paving the way for producing complex, three-dimensional meat structures more sustainably.