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.

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.

Accuracy of Using a Patient-Derived Tumor Organoid Culture Model to Predict the Response to Chemotherapy Regimens In Stage IV Colorectal Cancer: A Blinded Study.

BACKGROUND: Patient-derived tumor organoid culture has emerged as a preclinical model that has the potential to predict individual drug response. However, the predictive accuracy of patient-derived tumor organoid culture models for responses to chemotherapy regimens in stage IV colorectal cancer remains unknown. OBJECTIVE: The purpose of this study was to evaluate the predictive accuracy of the patient-derived tumor organoid culture model for responses to chemotherapy regimens in stage IV colorectal cancer. DESIGN: A pilot study was performed to define the half-maximal inhibitory concentration of the response to chemotherapy regimens in the patient-derived tumor organoid culture model. Then, a blinded study was performed to evaluate the predictive accuracy of the patient-derived tumor organoid culture model for responses to chemotherapy regimens. SETTINGS: Cancer samples were collected from patients with stage IV colorectal cancer at Nanfang Hospital of Southern Medical University in China. PATIENTS: In the pilot study, 30 patients were enrolled, and 43 samples were collected. In the blinded study, 71 patients were enrolled, and 96 samples were collected. INTERVENTION: Patient-derived tumor organoid culture and chemotherapy regimens were tested. MAIN OUTCOME MEASURES: The predictive accuracy of the patient-derived tumor organoid model for responses to chemotherapy regimens was measured. RESULTS: The median (range) time of organoid culture and drug testing was 9 days (range, 7-14 d). In the pilot study, 30 samples (69.77% [30/43]) were successfully cultured. The half-maximal inhibitory concentration of the chemotherapy response was 10 µmol/L according to clinical chemotherapy outcomes. In the blinded study, 77 samples (80.21% [77/96]) from 57 patients were successfully cultured. The sensitivity, specificity, and accuracy of the patient-derived tumor organoid model for predicting responses to chemotherapy regimens were 63.33%, 94.12%, and 79.69%. LIMITATIONS: This was a blinded study rather than a prospective randomized controlled study. CONCLUSIONS: The patient-derived tumor organoid culture model effectively predicts responses to existing chemotherapy regimens for individual patients. Video Abstract at http://links.lww.com/DCR/B511. PRECISIN EN EL USO DE MODELOS DE CULTIVO DE ORGANOIDES TUMORALES DERIVADOS DE PACIENTES PARA PREDECIR LA RESPUESTA DEL RGIMEN DE QUIMIOTERAPIA EN CNCER COLORRECTAL ESTADIO IV ESTUDIO CIEGO: ANTECEDENTES:El cultivo de organoides tumorales derivado del paciente ha surgido como un modelo preclínico que tiene el potencial de predecir la respuesta a un fármaco individual. Sin embargo, la exactitud predictiva en los modelos de cultivo de organoides tumorales derivados de pacientes para las respuestas a los regímenes de quimioterapia en el cáncer colorrectal en estadio IV sigue siendo desconocida.OBJETIVO:Evaluar la exactitud predictiva del modelo de cultivo organoide tumoral derivado de pacientes para las respuestas a los regímenes de quimioterapia en el cáncer colorrectal en estadio IV.DISEÑO:Se realizó un estudio piloto para definir la concentración inhibitoria media máxima de la respuesta a los regímenes de quimioterapia en el modelo de cultivo organoide tumoral derivado de pacientes. Luego, se realizó un estudio ciego para evaluar la exactitud predictiva del modelo de cultivo organoide tumoral derivado de pacientes para las respuestas a los regímenes de quimioterapia.AJUSTE:Se recolectaron muestras de cáncer de pacientes con cáncer colorrectal en estadio IV en el Hospital Nanfang de la Universidad Médica del Sur en China.PACIENTES:En el estudio piloto, se inscribieron 30 pacientes y se recolectaron 43 muestras. En el estudio ciego, se inscribieron 71 pacientes y se recolectaron 96 muestras.INTERVENCIÓN:Se probaron cultivos de organoides de tumores derivados del paciente y regímenes de quimioterapia.PRINCIPALES MEDIDAS DE RESULTADO:La precisión predictiva del modelo organoide tumoral derivado del paciente para las respuestas a los regímenes de quimioterapia.RESULTADOS:La mediana (rango) de tiempo de cultivo organoide y prueba de drogas fue de 9 (7-14) días. En el estudio piloto, se cultivaron con éxito 30 (69,77% [30/43]) muestras. La concentración inhibidora media máxima de la respuesta a la quimioterapia fue de 10 µmol / L según los resultados de la quimioterapia clínica. En el estudio ciego, se cultivaron con éxito 77 muestras (80,21% [77/96]) de 57 pacientes. La sensibilidad, especificidad y precisión del modelo organoide tumoral derivado del paciente para predecir las respuestas a los regímenes de quimioterapia fueron 63,33%, 94,12% y 79,69%, respectivamente.LIMITACIONES:Este fue un estudio ciego en lugar de un estudio prospectivo, aleatorizado y controlado.CONCLUSIONES:El modelo de cultivo organoide tumoral derivado de pacientes predice eficazmente las respuestas a los regímenes de quimioterapia existentes para pacientes individuales. Consulte Video Resumen en http://links.lww.com/DCR/B511.

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.

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.

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.

A synopsis of prostate organoid methodologies, applications, and limitations.

BACKGROUND: Current in vitro modeling systems do not fully reflect the biologic and clinical diversity of prostate cancer (PCa). Organoids are 3D in vitro cell cultures that recapitulate disease heterogeneity, retain prostate gland architecture, and mirror parental tumor characteristics. METHODS: To make better use of organoid models in the PCa research field, we provide a review of cutting-edge prostate organoid methodologies, applications, and limitations. RESULTS: We summarize methodologies for the establishment of benign prostate and PCa organoids and describe some of the model's practical applications and challenges. We highlight the patient-derived xenograft (PDX)-organoid interface model, which may allow for the generation of organoids from primary and rare PCa subtypes. Finally, we discuss potential future utilizations of PCa organoids in the realms of drug development and precision oncology. CONCLUSIONS AND FUTURE DIRECTIONS: Organoids represent a quasi in vivo modeling system that can be easily amenable to genetic modification and functional studies. As such, organoids may serve as an intermediate preclinical model for studying PCa. Future directions may include the refinement of culturing conditions to increase drug response fidelity in PCa organoids. The PDX-organoid interface model may enable the future establishment of primary and rare subtype PCa organoid lines.

Midtrimester amniotic fluid from healthy pregnancies has no microorganisms using multiple methods of microbiologic inquiry.

BACKGROUND: There is controversy about whether the amniotic fluid contains bacteria. With the use of sequencing-based methods, recent studies report that the amniotic fluid is colonized by microorganisms. However, background-contaminating DNA might lead to false-positive findings when such a low microbial biomass sample is examined. OBJECTIVE: The purpose of this study was to determine whether the midtrimester amniotic fluid of patients who subsequently had normal pregnancy outcomes contains a microbial signature. STUDY DESIGN: In this prospective cohort study, 42 amniotic fluid samples were collected from 37 pregnancies (5 twin and 32 singletons) during genetic amniocentesis in the midtrimester. The subsequent pregnancy outcomes of all the participants were followed. Multiple methods were used to detect the presence of microorganisms in this study, which included cultivation, quantitative real-time polymerase chain reaction, and 16S ribosomal RNA gene sequencing. Multiple positive control samples (n=16) served as quality control samples and included 3 adult fecal samples, 4 vaginal swabs, and 9 artificial bacterial communities that were run in parallel with negative control samples (n=12) that included 4 samples from the hospital operating room and 8 samples from the laboratory, to account for background-contaminating DNA during each step of the experiments. RESULTS: No bacteria under anaerobic or aerobic conditions or genital mycoplasmas were cultured from any of the amniotic fluid samples. Quantitative polymerase chain reaction did not reveal greater copy numbers of 16S ribosomal RNA gene in amniotic fluid samples than in negative control samples. 16S Ribosomal RNA gene sequencing did not indicate a significant difference in the microbial richness or community structures between amniotic fluid and negative control samples. CONCLUSION: With multiple methods of microbiologic inquiry, no microorganisms were identified in the midtrimester amniotic fluid of healthy pregnancies with a normal pregnancy outcome.

SCREENED: A Multistage Model of Thyroid Gland Function for Screening Endocrine-Disrupting Chemicals in a Biologically Sex-Specific Manner.

Endocrine disruptors (EDs) are chemicals that contribute to health problems by interfering with the physiological production and target effects of hormones, with proven impacts on a number of endocrine systems including the thyroid gland. Exposure to EDs has also been associated with impairment of the reproductive system and incidence in occurrence of obesity, type 2 diabetes, and cardiovascular diseases during ageing. SCREENED aims at developing in vitro assays based on rodent and human thyroid cells organized in three different three-dimensional (3D) constructs. Due to different levels of anatomical complexity, each of these constructs has the potential to increasingly mimic the structure and function of the native thyroid gland, ultimately achieving relevant features of its 3D organization including: 1) a 3D organoid based on stem cell-derived thyrocytes, 2) a 3D organoid based on a decellularized thyroid lobe stromal matrix repopulated with stem cell-derived thyrocytes, and 3) a bioprinted organoid based on stem cell-derived thyrocytes able to mimic the spatial and geometrical features of a native thyroid gland. These 3D constructs will be hosted in a modular microbioreactor equipped with innovative sensing technology and enabling precise control of cell culture conditions. New superparamagnetic biocompatible and biomimetic particles will be used to produce "magnetic cells" to support precise spatiotemporal homing of the cells in the 3D decellularized and bioprinted constructs. Finally, these 3D constructs will be used to screen the effect of EDs on the thyroid function in a unique biological sex-specific manner. Their performance will be assessed individually, in comparison with each other, and against in vivo studies. The resulting 3D assays are expected to yield responses to low doses of different EDs, with sensitivity and specificity higher than that of classical 2D in vitro assays and animal models. Supporting the "Adverse Outcome Pathway" concept, proteogenomic analysis and biological computational modelling of the underlying mode of action of the tested EDs will be pursued to gain a mechanistic understanding of the chain of events from exposure to adverse toxic effects on thyroid function. For future uptake, SCREENED will engage discussion with relevant stakeholder groups, including regulatory bodies and industry, to ensure that the assays will fit with purposes of ED safety assessment. In this project review, we will briefly discuss the current state of the art in cellular assays of EDs and how our project aims at further advancing the field of cellular assays for EDs interfering with the thyroid gland.

Microbiota-Derived Metabolic Factors Reduce Campylobacteriosis in Mice.

BACKGROUND & AIMS: Campylobacter jejuni, a prevalent foodborne bacterial pathogen, exploits the host innate response to induce colitis. Little is known about the roles of microbiota in C jejuni-induced intestinal inflammation. We investigated interactions between microbiota and intestinal cells during C jejuni infection of mice. METHODS: Germ-free C57BL/6 Il10-/- mice were colonized with conventional microbiota and infected with a single dose of C jejuni (109 colony-forming units/mouse) via gavage. Conventional microbiota were cultured under aerobic, microaerobic, or anaerobic conditions and orally transplanted into germ-free Il10-/- mice. Colon tissues were collected from mice and analyzed by histology, real-time polymerase chain reaction, and immunoblotting. Fecal microbiota and bile acids were analyzed with 16S sequencing and high-performance liquid chromatography with mass spectrometry, respectively. RESULTS: Introduction of conventional microbiota reduced C jejuni-induced colitis in previously germ-free Il10-/- mice, independent of fecal load of C jejuni, accompanied by reduced activation of mammalian target of rapamycin. Microbiota transplantation and 16S ribosomal DNA sequencing experiments showed that Clostridium XI, Bifidobacterium, and Lactobacillus were enriched in fecal samples from mice colonized with microbiota cultured in anaerobic conditions (which reduce colitis) compared with mice fed microbiota cultured under aerobic conditions (susceptible to colitis). Oral administration to mice of microbiota-derived secondary bile acid sodium deoxycholate, but not ursodeoxycholic acid or lithocholic acid, reduced C jejuni-induced colitis. Depletion of secondary bile acid-producing bacteria with antibiotics that kill anaerobic bacteria (clindamycin) promoted C jejuni-induced colitis in specific pathogen-free Il10-/- mice compared with the nonspecific antibiotic nalidixic acid; colitis induction by antibiotics was associated with reduced level of luminal deoxycholate. CONCLUSIONS: We identified a mechanism by which the microbiota controls susceptibility to C jejuni infection in mice, via bacteria-derived secondary bile acids.

Potential efficiency of conventional and advanced approaches used to detect Mycobacterium bovis in cattle.

The present study was aimed to assess the prevalence and efficiency of techniques for the diagnosis of bovine tuberculosis (bTB) including enzyme-linked immunosorbent assay (ELISA), Gamma interferon assay (IFN-γ) and polymerase chain reaction (PCR) in comparison to skin tuberculin test and culture technique. A total of 2600 cross-breed dairy cattle in Menoufia and Daqahlia governorates were tested by the single intradermal tuberculin test where the disease prevalence was 1.8%. Serum and whole blood samples were collected from positive tuberculin reactors for ELISA and IFN-γ assay, respectively. After slaughtering of positive tuberculin reactors, the post-mortem examination was carried out and tissue samples were collected for the bacteriological examination and PCR. The percentage of visible lesions of tuberculin reactors was 78.7%, while non-visible lesions were 21.27%. Culture technique revealed that the percentage of bTB was 63.8%. The ELISA and IFN-γ assay using short-term culture filtrate (ST-CF) prepared antigen revealed higher sensitivity (72.3% and 82.9%) than the bovine purified protein derivative (PPD-B) antigen. Although prepared ST-CF antigen has great efficiency and eligibility for the diagnosis of bTB, PCR appeared to have a higher sensitivity (85.1%) than other diagnostic methods when dealing with post-mortem samples. Gamma interferon assay using ST-CF antigen is recommended for antemortem diagnosis of bTB in cattle.

Activation of transposable elements and genetic instability during long-term culture of the human fungal pathogen Candida albicans.

It has been repeatedly reported that transposable elements (TE) become active and/or mobile in the genomes of replicatively and stress-induced senescent mammalian cells. However, the biological role of senescence-associated transposon activation and its occurrence and relevance in other eukaryotic cells remain to be elucidated. In the present study, Candida albicans, a prevalent opportunistic fungal pathogen in humans, was used to analyze changes in gene copy number of selected TE, namely Cirt2, Moa and Cmut1 during long-term culture (up to 90 days). The effects of stress stimuli (fluconazole, hydrogen peroxide, hypochlorite) and ploidy state (haploid, diploid, tetraploid cells) were also considered. An increase in copy number of Cirt2 and Moa was the most accented in tetraploid cells after 90 days of culture that was accompanied by changes in karyotype patterns and slightly more limited growth rate compared to haploid and diploid cells. Stress stimuli did not potentiate TE activity. Elevation in chromosomal DNA breaks was also observed during long-term culture of cells of different ploidy, however this was not correlated with increased TE activity. Our results suggest that increased TE activity may promote genomic diversity and plasticity, and cellular heterogeneity during long-term culture of C. albicans cells.

Culturing periprosthetic tissue in blood culture bottles results in isolation of additional microorganisms.

Despite low sensitivity, culture of periprosthetic tissue (PPT) specimens on agars and in broths has traditionally been used for the detection of causative microorganisms in patients suspected for prosthetic joint infection (PJI). The aim of this study was to evaluate the added diagnostic value of culturing PPT in blood culture bottles (BCB) over the conventional combination of standard agar and broth alone. This prospective cohort study was conducted over a 12-month period and included consecutive patients undergoing revision arthroplasty. Overall, 113 episodes from 90 subjects were studied; 45 subjects (50.0%) met the Infectious Diseases Society of America (IDSA) criteria for PJI, of whom the majority (75.6%) had an acute infection. Sensitivity and specificity of culture were assessed using IDSA criteria for PJI as gold standard. Although the increase in sensitivity from 84.44 (CI 70.54; 93.51) to 93.33% (81.73; 98.60) was not significant, added diagnostic value of culturing PPT in BCBs was demonstrated by the significantly higher number of detected pathogens in culture sets with BCBs compared to culture without BCBs (61 pathogens in conventional set versus 89 when BCBs were included for 57 PJI episodes, P = <0.0001). In 17 (29.8%) episodes, microorganisms were cultured from BCBs only, and in 9 (52.9%) of these episodes, virulent pathogens were found. This study demonstrates that PPT culture in BCBs leads to isolation of additional microorganisms, both virulent and low-virulent, which were not cultured with use of agars and broths alone. Isolation of additional causative microorganisms has serious consequences for the treatment strategy in PJI.

Storage and in vitro germination of orchids (Orchidaceae) seeds from Atlantic Forest - Brazil.

The orchid seed banks of Atlantic Forest may be considered a key strategy for the conservation of species threatened with extinction by indiscriminate collection or habitat destruction. The aim of this study was to evaluate the seed viability, to choose the best culture medium for the asymbiotic germination and evaluate germination, after storage for different periods and temperatures for the Brazilian native orchids: Gomesa praetexta (Rchb.f.) M.W.Chase & N.H.Williams, Gomesa forbesii (Hook.) M.W.Chase & N.H.Williams, Gomesa recurva R.Br. and Grandiphyllum divaricatum (Lindl.) Docha Neto. Knudson C (KC), Murashige & Skoog (MS), half-strength MS (1/2 MS macro- and micro-nutrients) and Woody Plant Medium (WPM) culture media were tested for germination. The WPM culture medium was the best for asymbiotic germination of all species evaluated, with high germination percentages and improved seedling development. Seeds of G. divaricatum, G. praetexta, G. recurva and G. forbesii indicated orthodox behavior, with high viability rates after 12 months of storage, being recommended the storage temperature of -80°C for the first three species and -20°C for G. forbesii. The protocol developed in the present study was efficient for seed bank storage, in vitro germination and seedling production of G. divaricatum and G. praetexta, contributing to conservation strategies of these species.

The Impact of Different Cultivation Systems on the Content of Selected Secondary Metabolites and Antioxidant Activity of Carlina acaulis Plant Material.

Roots and leaves of Carlina acaulis L. are still used in ethnomedicine in many European countries; however, the limited occurrence of the plants and protection of this species necessitate a search for alternative ways for obtaining this plant material. In this study, in vitro cultures, hydroponic cultures, and field cultivation were applied to obtain the C. acaulis plant material. Its quality was evaluated using antioxidant activity tests and high performance liquid chromatography analysis. Our study showed that the antioxidant activity and the content of chlorogenic and 3,5-di-caffeoylquinic acid in roots of plants cultivated in hydroponics and field conditions were comparable. However, the amount of carlina oxide was significantly higher in plants from the field. The flavonoid content in leaves obtained from both cultivation systems was at the same level; however, the antioxidant activity and the content of the investigated metabolites were higher in the soil cultivation system. The callus line exhibited high differentiation in phytochemical compositions depending on the treatments and medium compositions.

Implementation of a systematic culturing program to monitor the efficacy of endoscope reprocessing: outcomes and costs.

BACKGROUND AND AIMS: In 2015, the U.S. Food and Drug Administration and Centers for Disease Control and Prevention (CDC) issued guidance for duodenoscope culturing and reprocessing in response to outbreaks of carbapenem-resistant Enterobacteriaceae (CRE) duodenoscope-related infections. Based on this guidance, we implemented best practices for reprocessing and developed a systematic process for culturing endoscopes with elevator levers. The aim of this study is to report the outcomes and direct costs of this program. METHODS: First, clinical microbiology data from 2011 to 2014 were reviewed retrospectively to assess for possible elevator lever-equipped endoscope-related CRE infections. Second, a program to systematically culture elevator lever-equipped endoscopes was implemented. Each week, about 25% of the inventory of elevator lever-equipped endoscopes is cultured based on the CDC guidelines. If any cultures return bacterial growth, the endoscope is quarantined pending repeat culturing. The costs of the program, including staff time and supplies, have been calculated. RESULTS: From 2011 to 2014, none of 17 patients with documented CRE infection had undergone ERCP or endoscopic ultrasound in the previous 36 months. From June 2015 to September 2016, 285 cultures were performed. Three (1.1%) had bacterial growth, 2 with skin contaminants and 1 with an oral contaminant. The associated endoscopes were quarantined and reprocessed, and repeat cultures were negative. The total estimated cost of our program for an inventory of 20 elevator lever-equipped endoscopes was $30,429.60 per year ($1521.48 per endoscope). CONCLUSIONS: This 16-month evaluation of a systematic endoscope culturing program identified a low rate of positive cultures after elevator lever endoscope reprocessing. All positive cultures were with non-enteric microorganisms. The program was of modest cost and identified reprocessing procedures that may have led to a low rate of positive cultures.

A novel immortalized hepatocyte-like cell line (imHC) supports in vitro liver stage development of the human malarial parasite Plasmodium vivax.

BACKGROUND: Eradication of malaria is difficult because of the ability of hypnozoite, the dormant liver-stage form of Plasmodium vivax, to cause relapse in patients. Research efforts to better understand the biology of P. vivax hypnozoite and design relapse prevention strategies have been hampered by the lack of a robust and reliable model for in vitro culture of liver-stage parasites. Although the HC-04 hepatoma cell line is used for culturing liver-stage forms of Plasmodium, these cells proliferate unrestrictedly and detach from the culture dish after several days, which limits their usefulness in a long-term hypnozoite assay. METHODS: A novel immortalized hepatocyte-like cell line (imHC) was evaluated for the capability to support P. vivax sporozoite infection. First, expression of basic hepatocyte markers and all major malaria sporozoite-associated host receptors in imHC was investigated. Next, in vitro hepatocyte infectivity and intracellular development of sporozoites in imHC were determined using an indirect immunofluorescence assay. Cytochrome P450 isotype activity was also measured to determine the ability of imHC to metabolize drugs. Finally, the anti-liver-stage agent primaquine was used to test this model for a drug sensitivity assay. RESULTS: imHCs maintained major hepatic functions and expressed the essential factors CD81, SR-BI and EphA2, which are required for host entry and development of the parasite in the liver. imHCs could be maintained long-term in a monolayer without overgrowth and thus served as a good, supportive substrate for the invasion and growth of P. vivax liver stages, including hypnozoites. The observed high drug metabolism activity and potent responses in liver-stage parasites to primaquine highlight the potential use of this imHC model for antimalarial drug screening. CONCLUSIONS: imHCs, which maintain a hepatocyte phenotype and drug-metabolizing enzyme expression, constitute an alternative host for in vitro Plasmodium liver-stage studies, particularly those addressing the biology of P. vivax hypnozoite. They potentially offer a novel, robust model for screening drugs against liver-stage parasites.

Accessing the Hidden Microbial Diversity of Aphids: an Illustration of How Culture-Dependent Methods Can Be Used to Decipher the Insect Microbiota.

Microorganism communities that live inside insects can play critical roles in host development, nutrition, immunity, physiology, and behavior. Over the past decade, high-throughput sequencing reveals the extraordinary microbial diversity associated with various insect species and provides information independent of our ability to culture these microbes. However, their cultivation in the laboratory remains crucial for a deep understanding of their physiology and the roles they play in host insects. Aphids are insects that received specific attention because of their ability to form symbiotic associations with a wide range of endosymbionts that are considered as the core microbiome of these sap-feeding insects. But, if the functional diversity of obligate and facultative endosymbionts has been extensively studied in aphids, the diversity of gut symbionts and other associated microorganisms received limited consideration. Herein, we present a culture-dependent method that allowed us to successfully isolate microorganisms from several aphid species. The isolated microorganisms were assigned to 24 bacterial genera from the Actinobacteria, Firmicutes, and Proteobacteria phyla and three fungal genera from the Ascomycota and Basidiomycota phyla. In our study, we succeeded in isolating already described bacteria found associated to aphids (e.g., the facultative symbiont Serratia symbiotica), as well as microorganisms that have never been described in aphids before. By unraveling a microbial community that so far has been ignored, our study expands our current knowledge on the microbial diversity associated with aphids and illustrates how fast and simple culture-dependent approaches can be applied to insects in order to capture their diverse microbiota members.