Culture Wars: Empirically Determining the Best Approach for Plasmid Library Amplification.

DNA libraries are critical components of many biological assays. These libraries are often kept in plasmids that are amplified in E. coli to generate sufficient material for an experiment. Library uniformity is critical for ensuring that every element in the library is tested similarly and is thought to be influenced by the culture approach used during library amplification. We tested five commonly used culturing methods for their ability to uniformly amplify plasmid libraries: liquid, semisolid agar, cell spreader-spread plates with high or low colony density, and bead-spread plates. Each approach was evaluated with two library types: a random 80-mer library, representing high complexity and low coverage of similar sequence lengths, and a human TF ORF library, representing low complexity and high coverage of diverse sequence lengths. We found that no method was better than liquid culture, which produced relatively uniform libraries regardless of library type. However, when libraries were transformed with high coverage, the culturing method had minimal impact on uniformity or amplification bias. Plating libraries was the worst approach by almost every measure for both library types and, counterintuitively, produced the strongest biases against long sequence representation. Semisolid agar amplified most elements of the library uniformly but also included outliers with orders of magnitude higher abundance. For amplifying DNA libraries, liquid culture, the simplest method, appears to be best.

Application of Slice Culture System for Successional Dental Lamina in Diphyodont Mammals.

Replacement teeth develop from the successional dental lamina (SDL). Understanding how SDL transitions from quiescence to initiation is crucial for preserving dental lamina stem cells in the jawbone microenvironment and for complete tooth regeneration. Miniature pigs are good models for studying human tooth replacement because of their similarities to humans. However, the molecular mechanisms and cellular composition that initiate SDL development remain unclear. One possible reason for this is the limitations of the current methods for culturing SDL in vitro, such as the inability to directly observe tooth morphological changes during culture and low tissue viability. This study aimed to improve the in vitro culture method for SDL. Using a McIlwain Tissue Chopper, we obtained mandibular slices containing deciduous canine and SDL of permanent canine. The slices were approximately 500 µm thick and were cultured on a Transwell membrane supported with metal grids over medium. The SDL developed into the bud stage on the second day and entered the cap stage on the fifth day in vitro. The expression of proliferation markers, cell death markers, and key odontogenetic genes in vitro was similar to that observed in vivo. In conclusion, we successfully applied a slice culture system to the SDL of miniature pigs. This slice culture method allowed us to directly visualize SDL initiation and further elucidate the molecular mechanisms underlying the initiation of permanent tooth development.

Human Sapovirus Replication in Human Intestinal Enteroids.

Human sapoviruses (HuSaVs), like human noroviruses (HuNoV), belong to the Caliciviridae family and cause acute gastroenteritis in humans. Since their discovery in 1976, numerous attempts to grow HuSaVs in vitro were unsuccessful until 2020, when these viruses were reported to replicate in a duodenal cancer cell-derived line. Physiological cellular models allowing viral replication are essential to investigate HuSaV biology and replication mechanisms such as genetic susceptibility, restriction factors, and immune responses to infection. In this study, we demonstrate replication of two HuSaV strains in human intestinal enteroids (HIEs) known to support the replication of HuNoV and other human enteric viruses. HuSaVs replicated in differentiated HIEs originating from jejunum, duodenum and ileum, but not from the colon, and bile acids were required. Between 2h and 3 to 6 days postinfection, viral RNA levels increased up from 0.5 to 1.8 log10-fold. Importantly, HuSaVs were able to replicate in HIEs independent of their secretor status and histo-blood group antigen expression. The HIE model supports HuSaV replication and allows a better understanding of host-pathogen mechanisms such as cellular tropism and mechanisms of viral replication. IMPORTANCE Human sapoviruses (HuSaVs) are a frequent but overlooked cause of acute gastroenteritis, especially in children. Little is known about this pathogen, whose successful in vitro cultivation was reported only recently, in a cancer cell-derived line. Here, we assessed the replication of HuSaV in human intestinal enteroids (HIEs), which are nontransformed cultures originally derived from human intestinal stem cells that can be grown in vitro and are known to allow the replication of other enteric viruses. Successful infection of HIEs with two strains belonging to different genotypes of the virus allowed discovery that the tropism of these HuSaVs is restricted to the small intestine, does not occur in the colon, and replication requires bile acid but is independent of the expression of histo-blood group antigens. Thus, HIEs represent a physiologically relevant model to further investigate HuSaV biology and a suitable platform for the future development of vaccines and antivirals.

Novel Antifungal and Antifeedant Metabolites from Penicillium chrysogenum Co-Cultured with Nemania primolutea and Aspergillus fumigatus.

The endophyte Nemania primolutea, inhibited the growth of Penicillium chrysogenum in the coculture system. Four new compounds, nemmolutines A-B (1-2), and penigenumin (3) from N. primolutea, penemin (4) from P. chrysogenum were isolated from the coculture. On the other hand, P. chrysogenum inhibited the Aspergillus fumigatus in the coculture. Induced metabolites (13-16) with monasone naphthoquinone scaffolds including a new one from P. chrysogenum were produced by the coculture of P. chrysogenum, and A. fumigatus. Interesting, cryptic metabolites penicichrins A-B isolated from wild P. chrysogenum induced by host Ziziphus jujuba medium were also found in induced P. chrysogenum cultured in PDB ordinary medium. So the induction of penicichrin production by supplementing with host extract occurred in the fungus P. chrysogenum not the host medium. The productions of penicichrins were the spontaneous metabolism, and the metabolites (13-16) were the culture driven. Compounds 4, 6, 8, 10, 11, 14, and 15 showed significant antifungal activities against the phytopathogen Alternaria alternata with MICS of 1-8 µg/mL, and compounds 7, 9, and 12 indicated significant antifeedant activities against silkworms with feeding deterrence indexes (FDIs) of 92 %, 66 %, and 64 %. The carboxy group in 4-(2-hydroxybutynoxy)benzoic acid derivatives, and xylabisboeins; the hydroxy group in mellein derivatives; and the quinoid in monasone naphthoquinone increased the antifungal activities.

Using plant growth-promoting microorganisms (PGPMs) to improve plant development under in vitro culture conditions.

MAIN CONCLUSION: The use of beneficial microorganisms improves the performance of in vitro - cultured plants through the improvement of plant nutrition, the biological control of microbial pathogens or the production of phytohormones that promote plant growth and development. Plant in vitro culture techniques are highly useful to obtain significant amounts of true-to-type and disease-free plant materials. One of these techniques is clonal micropropagation which consists on the establishment of shoot tip cultures, shoot multiplication, in vitro rooting and acclimatization to ex vitro conditions. However, in some cases, the existence of recalcitrant genotypes, with a compromised multiplication and rooting ability, or the difficulties to overcome the overgrowth of endophytic contaminations might seriously limit its efficiency. In this sense, the establishment of beneficial interactions between plants and plant growth-promoting microorganisms (PGPMs) under in vitro culture conditions might represent a valuable approach to efficiently solve those restrictions. During the last years, significant evidence reporting the use of beneficial microorganisms to improve the yield of in vitro multiplication or rooting as well as their acclimatization to greenhouse or soil conditions have been provided. Most of these positive effects are strongly linked to the ability of these microorganisms to provide in vitro plants with nutrients such as nitrogen or phosphorous, to produce plant growth regulators, to control the growth of pathogens or to mitigate stress conditions. The culture of A. thaliana under aseptic conditions has provided high-quality knowledge on the root development signaling pathways, involving hormones, triggered in the presence of PGPMs. Overall, the present article offers a brief overview of the use of microorganisms to improve in vitro plant performance during the in vitro micropropagation stages, as well as the main mechanisms of plant growth promotion associated with these microorganisms.

Optimised CO2-containing medium for in vitro culture and transportation of mouse preimplantation embryos without CO2 incubator.

Conventional in vitro culture and manipulation of mouse embryos require a CO2 incubator, which not only increases the cost of performing experiments but also hampers the transport of embryos to the other laboratories. In this study, we established and tested a new CO2 incubator-free embryo culture system and transported embryos using this system. Using an Anaero pouch, which is a CO2 gas-generating agent, to increase the CO2 partial pressure of CZB medium to 4%-5%, 2-cell embryos were cultured to the blastocyst stage in a sealed tube without a CO2 incubator at 37°C. Further, the developmental rate to blastocyst and full-term development after embryo transfer were comparable with those of usual culture method using a CO2 incubator (blastocyst rate: 97% versus 95%, respectively; offspring rate: 30% versus 35%, respectively). Furthermore, using a thermal bottle, embryos were reliably cultured using this system for up to 2 days at room temperature, and live offspring were obtained from embryos transported in this simple and very low-cost manner without reducing the offspring rate (thermal bottle: 26.2% versus CO2 incubator: 34.3%). This study demonstrates that CO2 incubators are not essential for embryo culture and transportation and that this system provides a useful, low-cost alternative for mouse embryo culture and manipulation.

Naturally-occurring and cultured bacteriophages in human therapy.

OBJECTIVE: The aim of the study was to show the importance of developing techniques that could exploit the potential of bacteriophages as therapeutics or food supplements. MATERIALS AND METHODS: PubMed database was searched using the following combination of keywords: (bacteriophage) AND (human therapy); (natural bacteriophage) AND (application). RESULTS: The increasing antibiotic resistance of many bacterial strains is making standard antibiotic treatments less effective. Phage therapy provides a non-antibiotic alternative with greater specificity and without harmful effects on the human microbiota. Phages target their specific bacteria, replicate, and then, destroy the host pathogen. Bacteriophages may be administered by several routes, including topical, oral and intravenous. They not only destroy the host pathogen but, in some cases, increase the sensitivity of host bacteria to antibiotics. Various studies have shown that combining phage therapy and antibiotic treatment can be effective against bacterial infections. Clinical trials of phage therapy have shown promising results for various human diseases and conditions. With advances in genetic engineering and molecular techniques, bacteriophages will be able to target a wide range of bacteria. CONCLUSIONS: In the future, phage therapy promises to become an effective therapeutic option for bacterial infections. Since many potentially beneficial bacteriophages can be found in food, supplements containing bacteriophages could be designed to remodel gut microbiota and eliminate pathogenic bacteria. Remodeling of gut microbiota could correct gut dysbiosis. The order of phages known to have these promising activities is Caudovirales, especially the families Siphoviridae and Myoviridae.

Clinical study on the diagnosis of porcine streptococcal meningitis with negative blood and cerebrospinal fluid culture by next-generation sequencing.

BACKGROUND: Streptococcus suis (Ss) is a Gram-positive and anaerobic zoonotic pathogen that is susceptible to all populations and can cause meningitis, septicemia, endocarditis and arthritis in humans. METHODS: In this study, patients with meningitis who were admitted to our hospital with negative blood and cerebrospinal fluid culture were divided into a next-generation sequencing group and a control group. In the next-generation sequencing group, we used the next-generation sequencing method to detect pathogenic bacteria in the patients' cerebrospinal fluid. In the control group, we used blood and cerebrospinal fluid bacterial culture method to detect pathogenic bacteria in the patients' cerebrospinal fluid. The detection rates of pathogenic bacteria in the cerebrospinal fluid of the two groups were compared and analyzed. RESULTS: A total of 18 patients were included in this study, including 8 patients in the next-generation sequencing group and 10 patients in the control group. The mean age (P = 0.613) and mean disease duration (P = 0.294) were similar in both groups. Patients in the next-generation sequencing group had a leukocyte count of 13.13 ± 4.79 × 109, a neutrophil percentage of 83.39 ± 10.36%, and a C-reactive protein level of 134.95 ± 107.69 mg/L. Patients in the control group had a temperature of 38.32 ± 1.07, a leukocyte count of 8.00 ± 2.99 × 109, and a neutrophil percentage of 74.61 ± 8.89%, and C-reactive protein level was 4.75 ± 6.8 mg/L. The statistical results showed that the leukocytes (P = 0.013) and C-reactive protein levels (P = 0.001) were significantly higher in the patients of the next-generation sequencing group than in the control group. No statistically significant differences were seen in body temperature and neutrophil percentage between the two groups (P > 0.05). The incidence of intracranial pressure and meningeal irritation signs were similar in the two groups (P > 0.05). The detection rate of Streptococcus suis in the cerebrospinal fluid of patients in the next-generation sequencing group was 100%, and the detection rate of Streptococcus suis in the cerebrospinal fluid of the control group was 0%. CONCLUSION: The detection rate of Streptococcus suis infection in cerebrospinal fluid by next-generation sequencing was significantly higher than that by blood and cerebrospinal fluid bacterial culture. Therefore, the diagnosis of porcine streptococcal meningitis by next-generation sequencing method is worthy of clinical promotion and application.

Scarlet Flax Linum grandiflorum (L.) In Vitro Cultures as a New Source of Antioxidant and Anti-Inflammatory Lignans.

In vitro cultures of scarlet flax (Linum grandiflorum L.), an important ornamental flax, have been established as a new possible valuable resource of lignans and neolignans for antioxidant and anti-inflammatory applications. The callogenic potential at different concentrations of α-naphthalene acetic acid (NAA) and thidiazuron (TDZ), alone or in combinations, was evaluated using both L. grandiflorum hypocotyl and cotyledon explants. A higher callus induction frequency was observed on NAA than TDZ, especially for hypocotyl explants, with a maximum frequency (i.e., 95.2%) on 1.0 mg/L of NAA. The presence of NAA (1.0 mg/L) in conjunction with TDZ tended to increase the frequency of callogenesis relative to TDZ alone, but never reached the values observed with NAA alone, thereby indicating the lack of synergy between these two plant growth regulators (PGRs). Similarly, in terms of biomass, NAA was more effective than TDZ, with a maximum accumulation of biomass registered for medium supplemented with 1.0 mg/L of NAA using hypocotyls as initial explants (DW: 13.1 g). However, for biomass, a synergy between the two PGRs was observed, particularly for cotyledon-derived explants and for the lowest concentrations of TDZ. The influence of these two PGRs on callogenesis and biomass is discussed. The HPLC analysis confirmed the presence of lignans (secoisolariciresinol (SECO) and lariciresinol (LARI) and neolignan (dehydrodiconiferyl alcohol [DCA]) naturally accumulated in their glycoside forms. Furthermore, the antioxidant activities performed for both hypocotyl- and cotyledon-derived cultures were also found maximal (DPPH: 89.5%, FRAP 866: µM TEAC, ABTS: 456 µM TEAC) in hypocotyl-derived callus cultures as compared with callus obtained from cotyledon explants. Moreover, the anti-inflammatory activities revealed high inhibition (COX-1: 47.4% and COX-2: 51.1%) for extract of hypocotyl-derived callus cultures at 2.5 mg/L TDZ. The anti-inflammatory action against COX-1 and COX-2 was supported by the IC50 values. This report provides a viable approach for enhanced biomass accumulation and efficient production of (neo)lignans in L. grandiflorum callus cultures.

Anther Culture in Cucurbita Species.

Production of homozygous pure parental lines is the first stage of hybrid vegetable breeding. Unfortunately, producing pure lines takes a long time by classical breeding methods, especially in open-pollinated vegetable species, and this period can be up to 8-10 years. Recently, doubled haploid (DH) technology, as a set of biotechnological methods, has emerged as an alternative to classical breeding methods and allows for the generation of 100% homozygous pure double haploid lines in 1 or 2 years. Although haploid plants were successfully produced via irradiated pollen technique and gynogenesis in some Cucurbita species, haploid plants have not been obtained from some lines due to genotype dependency, and haploidy frequency is still not sufficient for use in a breeding program. Thus, anther culture technique has emerged as an alternative technique in the DH process. The main objective of this chapter is to provide explanatory information on anther culture technique applied in the Cucurbita genus. For this purpose , key points and details of methods and protocols of the anther culture technique are described in summer squash (Cucurbita pepo L.), pumpkin (Cucurbita moschata Duch.), and winter squash (Cucurbita maxima Duch.).

Visualizing Blood Vessel Development in Cultured Mouse Embryos Using Lightsheet Microscopy.

Lightsheet microscopy is a form of fluorescence microscopy that can be used to visualize specimen with high resolution, a large depth-of-field, and minimal photodamage and photobleaching as compared to traditional confocal microscopy. As this technology becomes much more readily available, it will be useful in revealing new findings in the cardiovascular development field that may be hidden or difficult to image. In this manuscript, we describe an approach for mounting and culturing postimplantation mouse embryos to visualize blood vessel development with a lightsheet microscope.

A Comparison of Candida Detection in Sputum by the Conventional Culture and Fluorescent Polymerase Chain Reaction Methods.

BACKGROUND Candida is a pathogenic fungus. In recent years, the increase in immunosuppressive diseases has led to an increase in Candida infections, with the lungs being the most common site. Therefore, the aim of this study was to compare the positive detection rates of Candida in sputum samples by Candida culture and fluorescent polymerase chain reaction (PCR), and to explore a new method for rapid, accurate, and effective detection of Candida in sputum, providing swift evidence of clinical fungal infection. MATERIAL AND METHODS From October 2016 to March 2017, 300 sputum samples were collected and detected by the conventional culture method and fluorescent PCR method. The positive rate of Candida detection was compared between the 2 methods. RESULTS In the 300 sputum samples, the positive detection rate of Candida was 50% by the culture method and 65.67% by the fluorescent PCR method (P<0.001). Therefore, the positive detection rate of Candida was higher by the fluorescent PCR method. CONCLUSIONS The conventional culture method for Candida needs a longer duration (24 h to 48 h) and the positive detection rate is low. However, it takes only 3 h to detect Candida in sputum by the fluorescent PCR method, the positive detection rate is high, and can be used as a screening method for Candida in sputum samples. Additional large-scale clinical trials need to be completed to assess the correlation between fluorescent PCR and pulmonary Candida infection.

Sonication fluid culture of antibiotic-loaded bone cement spacer has high accuracy to confirm eradication of infection before reimplantation of new prostheses.

BACKGROUND: Sonication fluid culture of antibiotic-loaded bone cement spacer has been used to predict reinfection of two-stage revision, but its value remains disputable. This study aims to evaluate the association between the culture result of the sonicated spacer and the status of patients with periprosthetic joint infection receiving two-stage revision. MATERIALS AND METHODS: A comprehensive electronic literature search was performed through four databases including PubMed, Embase/Ovid, and EBSCO, and the Cochrane Library to retrieve studies in which sonication fluid culture of the antibiotic spacer was conducted before reimplantation. The pooled sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), and diagnostic odds ratio (DOR) were calculated to assess the association between the culture result of sonicated spacer and prognosis of the two-stage revision. RESULTS: Eleven eligible studies comprising 603 artificial joints with PJI (134 suffering a clinical failure of two-stage revision) were included in the quantitative analysis. The pooled incidences of positive culture of sonicated spacer and intraoperative tissue were 0.14 (95% confidence interval [CI] 0.08-0.21) and 0.14 (95% CI 0.08-0.20), respectively. A positive culture of sonicated antibiotic-loaded bone cement spacer illustrated moderate sensitivity (0.31, 95% CI 0.13-0.58) but high specificity (0.94, 95% CI 0.86-0.98) for the diagnosis of therapeutic failure of two-stage revision; the pooled DOR was 7.67 (95% CI, 3.63-16.22). Meanwhile, the pooled sensitivity, specificity, and DOR of intraoperative tissue culture during the two-stage revision to predict therapeutic failure were 0.32 (95% CI, 0.20-0.47), 0.96 (95% CI, 0.92-0.98), and 10.62 (95% CI, 4.90-23.01), respectively. CONCLUSIONS: Sonication fluid culture of antibiotic-loaded bone cement spacer revealed high accuracy for confirming eradication of infection before reimplantation of new prostheses and therefore could be used as a supplement for assessing therapeutic effect for PJI. However, both sonication fluid culture and intraoperative tissue culture from antibiotic-loaded bone cement spacer showed restricted yield for the prediction of a septic failure after the two-stage revision of PJI. Large-scale, prospective studies are still needed to testify current findings.

Optimized culture methods for isolating small extracellular vesicles derived from human induced pluripotent stem cells.

Extracellular vesicles that are derived from stem cells play an important role in the treatment of disease. To obtain high-quality small extracellular vesicles (sEVs), we optimized the culture conditions of human induced pluripotent stem cells (hiPSCs), the supernatant collection time, and sEVs extraction methods. Firstly, hiPSCs were cultured in extracellular vesicles-production medium (EVs-PM) containing different concentrations (0%, 0.25%, 0.5%, 2%, 5%, and 20%) of extracellular vesicle-depleted knockout serum replacement (ED-KSR), and the culture supernatants were collected continuously for 5 days. Then, the sEVs were isolated, followed by an evaluation of their characteristics. The survival rates of the hiPSCs lines that were cultured in EVs-PM containing 0.5% to 20% ED-KSR were not significantly different (P > 0.05). The survival rates of the hiPSCs in 0.5% ED-KSR after the culture supernatants were continuously collected for day 1, day 3, and day 5 were not statistically significant (P > 0.05). After 5 days of continuous collection of the supernatant, the hiPSCs expressed some pluripotent markers, while SSEA4 and TRA-1-60 expression changed gradually. The sEVs that were extracted by the two methods were all 50-200 nm, double-layered and oval or round cellular vesicles and expressed the marker proteins CD63, TSG101, and HSP70. The characteristics of sEVs extracted on day 1, day 3, and day 5 were almost identical on morphology, size and the relative quantity of annexin V-positive subpopulations. The PKH67 staining showed that the sEVs could be endocytosed by HepG2 cells and aggregated in the cytoplasm. The proliferation experiments showed that the sEVs can promote cell proliferation. In Conclusion, the 0.5% ED-KSR is the optimal concentration, and that the hiPSCs culture supernatant can be continuously collected for 5 days while maintaining high cell viability and some pluripotent characteristics. Both of the methods extraction can be used to obtain biologically active sEVs.

A Natural Product Chemist's Guide to Unlocking Silent Biosynthetic Gene Clusters.

Microbial natural products have provided an important source of therapeutic leads and motivated research and innovation in diverse scientific disciplines. In recent years, it has become evident that bacteria harbor a large, hidden reservoir of potential natural products in the form of silent or cryptic biosynthetic gene clusters (BGCs). These can be readily identified in microbial genome sequences but do not give rise to detectable levels of a natural product. Herein, we provide a useful organizational framework for the various methods that have been implemented for interrogating silent BGCs. We divide all available approaches into four categories. The first three are endogenous strategies that utilize the native host in conjunction with classical genetics, chemical genetics, or different culture modalities. The last category comprises expression of the entire BGC in a heterologous host. For each category, we describe the rationale, recent applications, and associated advantages and limitations.

Peat-based gnotobiotic plant growth systems for Arabidopsis microbiome research.

The complex structure and function of a plant microbiome are driven by many variables, including the environment, microbe-microbe interactions and host factors. Likewise, resident microbiota can influence many host phenotypes. Gnotobiotic growth systems and controlled environments empower researchers to isolate these variables, and standardized methods equip a global research community to harmonize protocols, replicate experiments and collaborate broadly. We developed two easily constructed peat-based gnotobiotic growth platforms: the FlowPot system and the GnotoPot system. Sterile peat is amenable to colonization by microbiota and supports growth of the model plant Arabidopsis thaliana in the presence or absence of microorganisms. The FlowPot system uniquely allows one to flush the substrate with water, nutrients and/or suspensions of microbiota via an irrigation port, and a mesh retainer allows for the inversion of plants for dip or vacuum infiltration protocols. The irrigation port also facilitates passive drainage, preventing root anoxia. In contrast, the GnotoPot system utilizes a compressed peat pellet, widely used in the horticultural industry. GnotoPot construction has fewer steps and requires less user handling, thereby reducing the risk of contamination. Both protocols take up to 4 d to complete with 4-5 h of hands-on time, including substrate and seed sterilization. In this protocol, we provide detailed assembly and inoculation procedures for the two systems. Both systems are modular, do not require a sterile growth chamber, and cost less than US$2 per vessel.

A study of the microbiological profile of filler-induced skin necrosis.

Skin necrosis is one of the most severe complications following filler injections, and can result in permanent aesthetic defects. Although an increasing number of studies have addressed the management of dermal filler complications, no study has described the spectrum of microbial pathogens. The aim of this study was to delineate the bacterial profile and prognostic factors of filler-related skin necrosis by reviewing the clinical and microbiological features of these patients. A retrospective medical record review of patients undergoing treatment for skin necrosis induced by fillers was conducted. In total, 10 cases were identified, with injection sites being the nasolabial fold (70%; n = 7), nasal dorsum (20%; n = 2) and nasal tip (10%; n = 1). Reviewing the culture results, the true culture-positive rate was found to be 50% after cases of contamination were excluded. To avoid permanent sequelae, all physicians should be aware of possible secondary infections when treating filler-induced skin necrosis.