Antimicrobial efficacy of chitosan versus sodium hypochlorite: A systematic review and meta-analysis.

AIM: This meta-analysis aimed to compare the antibacterial efficacy of chitosan/chitosan nanoparticles (Ch/Ch-NPs) versus sodium hypochlorite/chlorhexidine (NaOCl/CHX). MATERIALS AND METHODS: A search was performed in four electronic databases until December 08, 2023. Studies with missing, unclear, and insufficient data sets were excluded. The included studies were assessed by two independent reviewers using the Joanna Briggs Institute Critical Appraisal Checklist for Quasi-Experimental Studies. The meta-analysis of standardized mean difference was performed using a random effects model. Additionally, funnel plots as well as Egger's regression intercept test were used to evaluate potential publication bias. RESULTS: A total of 426 samples were used in nine included studies. There was no difference in antibacterial efficacy between Ch/Ch-NPs-NaOCl (SMD: 0.005; 95% CI: -0.844-0.854; p = 0.990). However, the antibacterial efficacy of NaOCl was statistically more effective than Ch/Ch-NPs (SMD: 0.807; 95% CI: 0.015-1.599; p = 0.046) using the bacterial culture method, and Ch/Ch-NPs was statistically higher than NaOCl (SMD: -1.827; 95% CI: -2.720, -0.934; p < 0.000) using confocal laser scanning microscopy. CONCLUSIONS: Ch/Ch-NPs may be an alternative to NaOCl against Enterococcus faecalis. The methods used in the in vitro studies evaluating the antibacterial efficacy of irrigation solutions against E. faecalis may have had an impact on the results.

A liquid culture method assisted by ATP analysis for accelerating laboratory experiments on ultraviolet disinfection of airborne bacteria.

The solid culture method for measuring the efficiency of ultraviolet (UV) disinfection of airborne bacteria is time-consuming, typically taking 12-48 h. To expedite such experiments, this study proposed a liquid culture method assisted by adenosine triphosphate (ATP) analysis, as a liquid culture is faster than a solid culture, and measurement of ATP does not require waiting for visible colonies to form. Escherichia coli (E. coli) was used as the experimental bacterium. This study first compared the log reduction of bacteria in liquid as measured by the proposed method and by the traditional solid culture method. The minimum liquid culture time was determined for different bacterial concentration ranges. Finally, the feasibility of the proposed method was validated by UV disinfection experiments on airborne bacteria. The results indicated that the proposed method measured a similar log reduction to that of the solid culture method in liquid experiments. The minimum liquid culture time for E. coli in 105-106 colony forming units (CFU)/mL was 2 h. The validation experiments demonstrated that the proposed method is capable of measuring the UV disinfection efficiency of airborne bacteria. The proposed method can accelerate laboratory experiments on UV disinfection of airborne bacteria, which in turn can support the effective design and utilization of UV disinfection in real life.

Comparative seropositivity of Listeria monocytogenes in the serum of pregnant women with and without a history of abortion by serological and culture methods.

Objective: Listeria monocytogenes is a major cause of miscarriage and postpartum infections in infant. Determining antibody levels against listeriolysin O can be valuable for diagnosing both invasive listeriosis and febrile gastroenteritis. However, serological methods that detect antibodies against incomplete forms of listeriolysin O can be more specific. The objective of this study was to identify (Listeria monocytogenes) in the serum of pregnant women using serological and culture methods. Methods: Clinical samples (120 cases) were collected from pregnant women with a gestational age of less than 20 weeks. Diagnosis of Listeria monocytogenes was conducted using culture methods to identify anti-Listeria antibodies. Statistical analysis of the results was conducted using IBM SPSS Statistics version 23.0 (New York, USA), Pearson's Chi Square and fisher tests. Results: The number of positive samples by culture and ELISA was 24.16% (29) and 28.3% (34), respectively. Out of the 29 positive sample by the culture method, 10 individuals had no abortion history, 16 and 3 individuals had 1 and 2 abortions and no sample had 3 abortions. Maybe, the more abortions a person has had, the less likely they are to be infected. In the Enzyme Linked Immuno-Sorbent Assay (ELISA) method, 13 individuals tested positive for both IgG and IgM antibodies and 38 individuals tested negative. Additionally, among the positive individuals with 1, 2, and 3 miscarriages, 0, 17, and 3 people were positive for the IgG antibody and 0, 18, and 3 individuals were positive for the IgM antibody. The analysis results indicated that there was no significant relationship between culture and abortion history (p = 0.316), IgG ELISA and history of miscarriage (p = 0.672) and IgM ELISA and history of miscarriage (p = 0.552). Conclusion: There was no significant relationship between infection with Listeria monocytogenes and abortion (p ⩾ 0.05) in our samples. These results should be interpreted with caution due to the limitation of our small sample size.

[Methods for assessing the microbiological diversity of the ocular surface]. / Metody otsenki mikrobiologicheskogo raznoobraziya glaznoi poverkhnosti.

This review compares data from scientific studies on the microbial community of the ocular surface (OS) in conditionally healthy individuals using cultural methods (including culture-dependent diagnostic tests), microscopic and molecular genetic methods, and assesses the influence of research methods and sample preparation on the results. Concordance and discordance of the sets of identified microorganisms were analyzed using overlapping and non-overlapping methods of studying the microbial community of a healthy OS. The article presents tables showing the names of microorganisms that were identified in different sources. Cross-verification in taxa of different ranks helped confirm the following most frequently found microorganisms on healthy OS: coccomorphic microorganisms of the genera Staphylococcus, Micrococcus, Kocuria, Streptococcus, Enterococcus; gram-positive spore-forming bacilli of the genera Bacillus and Paenibacillus; gram-positive non-spore-forming rod-shaped bacteria, including Corynebacterium, but excluding Propionibacterium and Microbacterium; gram-negative non-spore-forming rod-shaped microorganisms of the genera Moraxella and Serratia. The study also assessed the effect of wearing soft contact lenses on the composition of the microbial community of the OS.

Community structure of endophytic bacteria of Sargassum thubergii in the intertidal zone of Qingdao in China.

Endophytic bacteria are one of the symbiotic microbial groups closely related to host algae. However, less research on the endophytic bacteria of marine algae. In this study, the endophytic bacterial community of Sargassum thunbergii was investigated using the culture method and high-throughput sequencing. Thirty-nine endophytic bacterial strains, belonging to two phyla, five genera and sixteen species, were isolated, and Firmicutes, Bacillus and Metabacillus indicus were the dominant taxa at the phylum, genus and species level, respectively. High-throughput sequencing revealed 39 phyla and 574 genera of endophytic bacteria, and the dominant phylum was Proteobacteria, while the dominant genus was Ralstonia. The results also indicated that the endophytic bacteria of S. thunbergii included various groups with nitrogen fixation, salt tolerance, pollutant degradation, and antibacterial properties but also contained some pathogenic bacteria. Additionally, the endophytic bacterial community shared a large number of groups with the epiphytic bacteria and bacteria in the surrounding seawater, but the three groups of samples could be clustered separately. In conclusion, there are a variety of functional endophytic bacteria living in S. thunbergii, and the internal condition of algae is a selective factor for the formation of endophytic bacterial communities. This study enriched the database of endophytic bacteria in marine macroalgae, paving the way for further understanding of the interrelationships between endophytic bacteria, macroalgae, and the environment.

Evaluation of culture methods and chemical reagent combinations on CRISPR/Cas9 gene editing systems by lipofection in pig zygotes.

The delivery of CRISPR/Cas ribonucleoprotein (RNP) complexes is gaining attention owing to its high cleavage efficiency and reduced off-target effects. Although RNPs can be delivered into porcine zygotes via electroporation with relatively high efficiency, lipofection-mediated transfection appears to be versatile because of its ease of use, low cost, and adaptation to high-throughput systems. However, this system requires improvements in terms of embryo development and mutation rates. Therefore, this study elucidated the effects of culture methods and reagent combinations on the CRISPR/Cas9 gene editing systems by using three lipofection reagents: Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (CM), Lipofectamine™ 2000 Transfection Reagent (LP), and jetCRISPR™ RNP Transfection Reagent (Jet). Porcine zona pellucida-free zygotes were incubated for 5 h with Cas9, a guide RNA targeting CD163, and the above lipofection reagents. When examining the effect of culture methods using 4-well (multiple embryo culture) and 25-well plates (single embryo culture) on the efficiency of CM-mediated zygote transfection, the culture of embryos in 25-well plates significantly increased the blastocyst formation rate; however, there was no difference in mutation rates between the 4-well and 25-well plates. When assessing the effects of individual or combined reagents on the efficiency of zygote transfection, the mutation rate was significantly lower for individual LP compared to individual CM- and Jet-mediated transfections. Moreover, combinations of lipofection transfection reagents did not significantly increase the mutation rate or mutation efficiency.

Principles of Indirect Co-culture Method Using Transwell.

The co-culture method is a simple type of cell culture method used to evaluate the effects of communication between various types of cells in an in vitro setting. In the co-culture method, two or more eukaryotic cell types, or eukaryotic and prokaryotic cells, are cultured together. The co-culture method reflects in vivo cell behaviors and thereby emerges as a pivotal technique with diverse applications in cancer research and cell biology. Two categories of co-culture methods (indirect methods and direct methods) are well known. Direct co-culture methods allow physical contact between the various cell types (juxtacrine signaling). In indirect methods, cells are physically separated into two different populations (for example, using a Transwell) that allow communication only via secretory factors (paracrine signaling). Herein, we focus on the principles of the indirect co-culture method. Nowadays, this method is used to explore the effects of mesenchymal stem cell (MSC) secretome on cancer cells. These studies have unveiled intricate cell behavior dynamics, demonstrating how the MSC secretome influences cancer cell proliferation, invasion, apoptosis, and polarity.

Enhanced bacterial cellulose production in Gluconacetobacter xylinus by overexpression of two genes (bscC and bcsD) and a modified static culture.

Bacterial cellulose (BC), a nanostructured material, is renowned for its excellent properties. However, its production by bacteria is costly due to low medium utilization and conversion rates. To enhance the yield of BC, this study aimed to increase BC yield through genetic modification, specifically by overexpressing bcsC and bcsD in Gluconacetobacter xylinus, and by developing a modified culture method to reduce medium viscosity by adding water during fermentation. As a result, BC yields of 5.4, 6.2, and 6.8 g/L were achieved from strains overexpressing genes bcsC, bcsD, and bcsCD, significantly surpassing the yield of 2.2 g/L from wild-type (WT) strains. In the modified culture, the BC yields of all four strains increased by >1 g/L with the addition of 20 mL of water during fermentation. Upon comparing the properties of BC, minimal differences were observed between the WT and pbcsC strains, as well as between the static and modified cultures. In contrast, BC produced by strains overexpressing bcsD had a denser microstructural network and exhibited demonstrated higher tensile strength and elongation-to-break. Compared to WT, BC from bcsD overexpressed strains also displayed enhanced crystallinity, higher degree of polymerization and improved thermal stability.

Improvement and application of culture method for hemorrhagic amniotic fluid cells / 临床检验杂志

Objective To compare the success rates of different culture methods for hemorrhagic amniotic fluid.Methods Thirty-one hemorrhagic amniotic fluid samples from pregnant women who were subjected to chromosomal examination at Anhui Maternal and Child Health Hospital were collected from January 2021 to December 2022.Two culture methods,the slide in situ culture box method(re-ferred to as the slide method)and the plastic bottle culture combined with slide in situ culture box method(referred to as the combined culture method),were used for cell cultivation.All the cells were harvested and stained with Giemsa staining,then the number of eligi-ble karyotype was counted and the success rates were compared between the two methods.Results Among the 31 cases of the slide method,21 were successfully cultured with a success rate of 67.7％.For the combined culture method,all the 31 cases were success-fully cultured with a success rate of 100％.The success rate of the combined culture method was significantly higher than that of the slide method(P＜0.05).Of 31 bloody amniotic fluid samples,three cases were fresh bleeding,and an average number of eligible kary-otype was 8 in the slide method and 32 in the combined culture method.Twenty-eight cases were old bleeding,and an average number of eligible karyotype was 13 in the slide method and 53 in the combined culture method.The number of eligible karyotype in the com-bined culture method was significantly higher than that of the slide method(P＜0.05).Conclusion The combined culture method is suitable for the cultivation of hemorrhagic amniotic fluid samples,and should be worthy of promotion in the clinics.

Liver organoid culture methods.

Organoids, three-dimensional structures cultured in vitro, can recapitulate the microenvironment, complex architecture, and cellular functions of in vivo organs or tissues. In recent decades, liver organoids have been developed rapidly, and their applications in biomedicine, such as drug screening, disease modeling, and regenerative medicine, have been widely recognized. However, the lack of repeatability and consistency, including the lack of standardized culture conditions, has been a major obstacle to the development and clinical application of liver organoids. It is time-consuming for researchers to identify an appropriate medium component scheme, and the usage of some ingredients remains controversial. In this review, we summarized and compared different methods for liver organoid cultivation that have been published in recent years, focusing on controversial medium components and discussing their advantages and drawbacks. We aimed to provide an effective reference for the development and standardization of liver organoid cultivation.

Thermal Inactivation of Hepatitis E Virus in Pork Products Estimated with a Semiquantitative Infectivity Assay.

Hepatitis E virus genotype 3 (HEV-3) is a food-borne pathogen causative of hepatitis E infections in humans. In Europe, HEV-3 is mainly transmitted through the consumption of raw or undercooked pork. In order to determine the effectiveness of control measures that can be taken in the industry or by the consumer, it is pivotal to determine the infectivity of HEV present in pork products after thermal food-processing steps. First, we implemented a method for the detection of infectious HEV-3c and HEV-3e in a cell culture medium and in extracts from inoculated pork products. Next, we investigated the effect of the thermal inactivation of HEV by mimicking food-processing steps specific for dried sausage and liver homogenate matrices. After four weeks, HEV-inoculated dried sausage subjected to 21 °C or lower temperatures was still infectious. For the liver homogenate, the highest HEV-3c/e inactivation of the conditions tested was observed at 71 °C for five min or longer. Finally, our method was able to successfully detect and estimate viral loads of infectious HEV in naturally infected pig livers. Our data provide a basis for the future use of the quantitative microbial risk assessment of infectious HEV in pork products that are subjected to thermal food processing steps.

Composition of pathogenic microorganism in chronic osteomyelitis based on metagenomic sequencing and its application value in etiological diagnosis.

BACKGROUND: Traditionally, conventional microbiological culture methods have been used to detect pathogenic microorganisms in chronic osteomyelitis. However, these methods have been found to have a low detection rate, complicating the precise guidance of infection treatment. This study employed metagenomic next-generation sequencing (mNGS) to detect these microorganisms in chronic osteomyelitis with three main objectives: 1). Gain a deeper understanding of the composition of pathogenic microorganisms in chronic osteomyelitis. 2). Compare the microbial detection rates between mNGS and the standard culture methods used in laboratories to enhance the effectiveness of the traditional culture methods. 3). Explore the potential of mNGS in etiological diagnosis. METHODS: Fifty clinically confirmed intraoperative bone tissue samples of chronic osteomyelitis from January 2021 to December 2021 were collected and subjected to mNGS and microbiological testing, respectively. The orthopaedic surgeon combined clinical manifestations and related examinations to determine the causative pathogens. RESULTS: The culture method obtained 29 aerobic and parthenogenic anaerobic bacteria, 3 specific anaerobic bacteria, and 1 yeast-like fungus. Thirty-six aerobic and parthenogenic anaerobic bacteria, 11 specific anaerobic bacteria, and 1 yeast-like fungus were obtained by mNGS, and 2 Mycobacterium tuberculosis(MTB) strains were detected. However, there was no significant difference in the overall positive detection rate between mNGS and the culture method (P = 0.07), and the two were not statistically significant in detecting aerobic and partly anaerobic bacteria (P = 0.625). But, mNGS was significantly superior to culture in detecting anaerobic bacteria and Mycobacterium tuberculosis (P<0.05). CONCLUSIONS: The mNGS method has enhanced our understanding of the distribution of pathogenic microorganisms in chronic osteomyelitis. Traditional culture methods help isolate and cultivate aerobic and facultative anaerobic bacteria, and fungi, and are also utilized for antibacterial drug sensitivity tests. However, mNGS has shown superior capabilities in detecting anaerobic bacteria, MTB, and mixed infection bacteria. This finding offers invaluable guidance for improving laboratory microbial culture and detection conditions. Hence, mNGS should be judiciously used for chronic osteomyelitis, and PCR can be implemented for certain difficult-to-culture microorganisms, such as MTB.

Analgesic Effect of the Lysine-Containing Short Peptide Is Due to Modulation of the NaV1.8 Channel Activation Gating System.

The present work continues our recent series of articles that aim to elucidate the ligand-receptor binding mechanism of short cationic peptides to the NaV1.8 channel in the nociceptive neuron. The applied methodological approach has involved several methods: the patch-clamp experimental evaluation of the effective charge of the NaV1.8 channel activation gating system, the organotypic tissue culture method, the formalin test, and theoretical conformational analysis. The lysine-containing short peptide Ac-KEKK-NH2 has been shown to effectively modulate the NaV1.8 channel activation gating system. As demonstrated by the organotypic tissue culture method, the studied short peptide does not trigger the downstream signaling cascades controlling neurite outgrowth and should not be expected to evoke adverse side effects. Conformational analysis of the Ac-KEKK-NH2 molecule has revealed that the distances between the positively charged amino groups of the lysine side chains are equal to 11-12 Å. According to the previously suggested mechanism of ligand-receptor binding of short peptides to the NaV1.8 channel molecule, Ac-KEKK-NH2 should exhibit an analgesic effect, which has been confirmed by the formalin test. The data obtained unequivocally indicate that the studied lysine-containing short peptide is a promising candidate for the role of a novel analgesic medicinal substance.

Development and diagnostic evaluation of indirect enzyme linked immunosorbent assay for Epidermophyton floccosum infection in humans.

BACKGROUND: Epidermophyton floccosum is an anthrophophilic dermatophyte widely distributed in the tropics and subtropics. It can invade keratinized tissues of humans and cause superficial mycoses called dermatophytosis (ringworm). OBJECTIVE: The main objective of this study was to develop an in-house indirect enzyme-linked immunosorbent assay (ELISA) and to evaluate its performance for the immunological diagnosis of E. floccosum infection in humans. METHODS: An in-house indirect ELISA was developed using partially purified E. floccosum antigens, pre immunized rabbit serum as negative control, immunized rabbit polyclonal antibodies as positive control, enzyme labeled goat anti rabbit antibodies and goat anti human antibodies. A total of 50 serum samples from E. floccosum infected patients as confirmed by direct microscopy and culture and 30 samples from humans without history of dermatophyte infection that served as controls were used to evaluate the performance of an in-house indirect ELISA developed in this study. Analytical and diagnostic performance characteristics were determined to evaluate its diagnostic value. RESULTS: The diagnostic sensitivity, specificity, positive and negative predictive values of E. floccosum indirect ELISA were 90.00 %, 83.33 %, 90.83 %, 83.83 % respectively. The performance of indirect ELISA assay was compared with gold standard diagnostic tests such as KOH hydrolysis test and fungal culture. The correlation coefficients of E. floccosum indirect ELISA with KOH hydrolysis and fungal culture method were 0.612** and 0.826** (P < 0.01) respectively indicating significant correlation between these tests. CONCLUSION: This revealed the great potentiality of E. floccosum indirect ELISA in early, specific and precise detection of E. floccosum infection in humans.

Continued Organic Fertigation after Basal Manure Application Does Not Impact Soil Fungal Communities, Tomato Yield or Soil Fertility.

There is currently a limited understanding of the complex response of fungal microbiota diversity to organic fertigation. In this work, a 2-year field trial with organic tomato crops in a soil previously amended with fresh sheep manure was conducted. Two hypotheses were compared: (i) fertigation with organic liquid fertilizers versus (ii) irrigation with water. At the end of both years, soils were analyzed for physical-chemical parameters and mycobiome variables. Plate culture and DNA metabarcoding methods were performed in order to obtain a detailed understanding of soil fungal communities. Fertigation did not increase any of the physical-chemical parameters. Concerning soil fungal communities, differences were only found regarding the identification of biomarkers. The class Leotiomycetes and the family Myxotrichaceae were identified as biomarkers in the soil fungal community analyzed by means of DNA metabarcoding of the "fertigation" treatment at the end of Year 1. The Mortierella genus was detected as a biomarker in the "water" treatment, and Mucor was identified in the "fertigation" treatment in the cultivable soil fungi at the end of Year 2. In both years, tomato yield and fruit quality did not consistently differ between treatments, despite the high cost of the fertilizers added through fertigation.

Research Note: Detection of Campylobacter spp. in chicken meat using culture methods and quantitative PCR with propidium monoazide.

Globally, Campylobacter spp. are prominent causative agents of food-borne gastroenteritis. These pathogens are commonly detected using conventional culture methods; however, culture methods are unable to detect viable but nonculturable (VBNC) bacteria. Currently, the detection rate of Campylobacter spp. on chicken meat does not correlate with the seasonal peak of human campylobacteriosis. We hypothesized that this may be due to the presence of undetectable VBNC Campylobacter spp. Therefore, we previously established a quantitative PCR assay using propidium monoazide (PMA-qPCR), which can detect viable Campylobacter cells. In this study, PMA-qPCR was conducted to detect viable Campylobacter spp. in chicken meat, and the detection rates of PMA-qPCR and the culture method throughout all 4 seasons were compared. A total of 105 chicken meat samples (whole legs, breast fillets, and livers) were screened for the presence of Campylobacter spp. using both PMA-qPCR and the conventional culture method. The detection rates of the 2 methods did not differ significantly; however, the positive and negative samples were not always consistent. Detection rates in March were significantly lower compared to months with the highest detection rates. These results suggest that, to increase the detection rate of Campylobacter spp., the 2 methods should be used in parallel. In this study, PMA-qPCR could not detect VBNC Campylobacter spp. effectively in C. jejuni-spiked chicken meat. Further studies using improved viability-qPCR should be performed to describe the impact of the VBNC state of Campylobacter spp. on the detection of this bacterium in chicken meat.

Role of the Rhamnosyl Residue of Ouabain in the Activation of the Na,K-ATPase Signaling Function.

The signaling or non-pumping Na,K-ATPase function was first observed by us in the nociceptive neuron; Na,K-ATPase transduced the signals from the opioid-like receptors to NaV1.8 channels. This study elucidates the role of the rhamnosyl residue of ouabain in the activation of the Na,K-ATPase signaling function. The effects resulting from activation of Na,K-ATPase signaling by the Ca2+ chelate complex of ouabain (EO) are not manifested upon removal of the rhamnosyl residue, as demonstrated in viable cells by the highly sensitive patch-clamp and organotypic cell culture methods. Docking calculations show that the rhamnosyl residue is involved in five intermolecular hydrogen bonds with the Na,K-ATPase α1-subunit, which are fundamentally important for activation of the Na,K-ATPase signaling function upon EO binding. The main contribution to the energy of EO binding is provided by its steroid core, which forms a number of hydrogen bonds and hydrophobic interactions with Na,K-ATPase that stabilize the ligand-receptor complex. Another critically important role in EO binding is expected to be played by the chelated Ca2+ cation, which should switch on strong intermolecular ionic interactions between the EO molecule and two α1-Na,K-ATPase amino acid residues, Glu116 and Glu117.

Various Three-Dimensional Culture Methods and Cell Types for Exosome Production.

Cell-based therapies have been used as promising treatments for several untreatable diseases. However, cell-based therapies have side effects such as tumorigenesis and immune responses. To overcome these side effects, therapeutic effects of exosomes have been researched as replacements for cell-based therapies. In addition, exosomes reduced the risk that can be induced by cell-based therapies. Exosomes contain biomolecules such as proteins, lipids, and nucleic acids that play an essential role in cell-cell and cell-matrix interactions during biological processes. Since the introduction of exosomes, those have been proven perpetually as one of the most effective and therapeutic methods for incurable diseases. Much research has been conducted to enhance the properties of exosomes, including immune regulation, tissue repair, and regeneration. However, yield rate of exosomes is the critical obstacle that should be overcome for practical cell-free therapy. Three-dimensional (3D) culture methods are introduced as a breakthrough to get higher production yields of exosomes. For example, hanging drop and microwell were well known 3D culture methods and easy to use without invasiveness. However, these methods have limitation in mass production of exosomes. Therefore, a scaffold, spinner flask, and fiber bioreactor were introduced for mass production of exosomes isolated from various cell types. Furthermore, exosomes treatments derived from 3D cultured cells showed enhanced cell proliferation, angiogenesis, and immunosuppressive properties. This review provides therapeutic applications of exosomes using 3D culture methods.

Establishing patient-derived organoids from human endometrial cancer and normal endometrium.

Endometrial cancer is the most common gynecologic malignancy in the United States and is one of the few malignancies that had an increasing incidence and mortality rate over the last 10 years. Current research models fail to recapitulate actual characteristics of the tumor that are necessary for the proper understanding and treatment of this heterogenous disease. Patient-derived organoids provide a durable and versatile culture system that can capture patient-specific characteristics such as the mutational profile and response to therapy of the primary tumor. Here we describe the methods for establishing, expansion and banking of endometrial cancer organoids to develop a living biobank. Samples of both endometrial tumor tissue and matched normal endometrium were collected from 10 patients. The tissue was digested into single cells and then cultured in optimized media to establish matched patient endometrial cancer and normal endometrial tissue organoids. Organoids were created from all major endometrial cancer histologic subtypes. These organoids are passaged long term, banked and can be utilized for downstream histological and genomic characterization as well as functional assays such as assessing the response to therapeutic drugs.

The "comfort timing" strategy: a potential pathway for the cultivation of uncultured microorganisms and a possible adaptation for environmental colonisation.

Efforts to isolate uncultured microorganisms over the last century and a half, as well as the advanced 'omics' technologies developed over the last three decades, have greatly increased the knowledge and resources of microbiology. However, many cellular functions such as growth remain unknown in most of the microbial diversity identified through genomic sequences from environmental samples, as evidenced by the increasingly precise observations of the phenomenon known as the 'great plate count anomaly'. Faced with the many microbial cells recalcitrant to cultivation present in environmental samples, Epstein proposed the 'scout' model, characterised by a dominance of dormant cells whose awakening would be strictly stochastic. Unfortunately, this hypothesis leaves few exploitable possibilities for microbial cultivation. This review proposes that many microorganisms follow the 'comfort timing' strategy, characterised by an exit from dormancy responding to a set of environmental conditions close to optimal for growth. This 'comfort timing' strategy offers the possibility of designing culture processes that could isolate a larger proportion of uncultured microorganisms. Two methods are briefly proposed in this article. In addition, the advantages of dormancy, of the 'scout' model and of the 'comfort timing' strategy for survival under difficult conditions, but also for colonisation of environments, are discussed.