Strain Typing Research Articles

Description and Genomic Characteristics of Weissella fermenti sp. nov., Isolated from Kimchi.

A Gram-positive, non-motile, and non-spore-forming lactic acid bacterium, designated as BK2T, was isolated from kimchi, a Korean traditional fermented vegetable food, and the taxonomic characteristics of strain BK2T, along with strain LMG 11983, were analyzed. Both strains optimally grew at 30°C, pH 7.0, and 1.0% NaCl. Cells of both strains were heterofermentative and facultatively anaerobic rods, demonstrating negative reactions for catalase and oxidase. Major fatty acids (>10%) identified in both strains were C18:1 ω9c, C16:0, and summed feature 7 (comprising C19:1 ω6c and/or C19:1 ω7c). The genomic DNA G+C contents of both strains were 44.7 mol%. The 16S rRNA gene sequence similarity (99.9%), average nucleotide identity (ANI; 99.9%), and digital DNA-DNA hybridization (dDDH; 99.7%) value between strains BK2T and LMG 11983 indicated that they are different strains of the same species. Strain BK2T was most closely related to Weissella confusa JCM 1093T and Weissella cibaria LMG 17699T, with 100% and 99.4% 16S rRNA gene sequence similarities, respectively. However, based on the ANI and dDDH values (92.3% and 48.1% with W. confusa, and 78.4% and 23.5% with W. cibaria), it was evident that strain BK2T represents a distinct species separate from W. confusa and W. cibaria. Based on phylogenetic, phenotypic, and chemotaxonomic features, strains BK2T and LMG 11983 represent a novel species of the genus Weissella, for which the name Weissella fermenti sp. nov. is proposed. The type of strain is BK2T (=KACC 22833T=JCM 35750T).

Myocardial strain-curve deformation patterns after Fontan operation.

Myocardial deformation analysis by cardiac MRI (CMR) yielding global circumferential and longitudinal strain (GCS and GLS) is an increasingly utilized method to accurately quantify systolic function and predict clinical events in patients with Fontan circulation. The purpose of this study was to use principal component analysis (PCA) to investigate myocardial temporal deformation patterns derived from strain-time curves to learn about latent strain features beyond peak values. We conducted the study with specific attention to dominant single left or right ventricle (SLV and SRV) morphologies. Methods and Results: Patients remote from Fontan operation who underwent follow-up CMR were analyzed for standard volumetric and function hemodynamics including myocardial deformation parameters including GCS and GLS. We applied PCA to investigate in an unbiased fashion the strain-time curve morphology and to calculate patient specific shape scores. All variables were subjected to single variable Cox regression analysis to detect composite clinical outcome including death, heart transplant, protein losing enteropathy and plastic bronchitis. A total of 122 patients, (SLV = 67, SRV = 55) with a mean age of 12.7years underwent comprehensive CMR analysis. The PCA revealed 3 primary modes of strain-curve variation regardless of single ventricle morphology and type of strain investigated. Principle components (PCs) described changes in (1) strain-time curve amplitude, (2) time-to-peak strain, and (3) post-systolic slope of the strain-time curve. Considering only SLV patients, GCS was only CMR variable predictive of clinical events (HR 1.46, p = 0.020). In the SRV group, significant CMR predictors of clinical events were derived indexed end-diastolic (HR 1.02, p = 0.023) and end-systolic (HR 1.03, p = 0.022) volumes, GCS (HR 1.91, p = 0.003) and its related first component score (HR 1.20, p = 0.005), GLS (HR 1.32, p = 0.029) and its third component score (HR 1.58, p = 0.017). CMR derived global strain measures are sensitive markers of clinical outcomes in patients with Fontan circulation, particularly in patients with the SRV morphology. Myocardial strain-time curve morphology specific to SLV and SRV patients inspired by unbiased PCA technique can further aid with predicting clinical outcomes.

Optimizing expression of Nanobody® molecules in Pichia pastoris through co-expression of auxiliary proteins under methanol and methanol-free conditions

BackgroundAblynx NV, a subsidiary of Sanofi, has a long-standing focus on the development of Nanobody® molecules as biopharmaceuticals (Nanobody® is a registered trademark of Ablynx NV). Nanobody molecules are single variable domains, and they have been met with great success part due to their favorable expression properties in several microbial systems. Nevertheless, the search for the host of the future is an ongoing and challenging process. Komagataella phaffi (Pichia pastoris) is one of the most suitable organisms to produce Nanobody molecules. In addition, genetic engineering of Pichia is easy and an effective approach to improve titers.ResultsHere we report that P. pastoris engineered to co-express genes encoding four auxiliary proteins (HAC1, KAR2, PDI and RPP0), leads to a marked improvement in the expression of Nanobody molecules using the AOX1 methanol induction system. Titer improvement is mainly attributed to HAC1, and its beneficial effect was also observed in a methanol-free expression system.ConclusionOur findings are based on over a thousand fed-batch fermentations and offer a valuable guide to produce Nanobody molecules in P. pastoris. The presented differences in expressability between types of Nanobody molecules will be helpful for researchers to select both the type of Nanobody molecule and Pichia strain and may stimulate further the development of a more ecological methanol-free expression platform.

Isolation of new Paraburkholderia strains for polyhydroxybutyrate production.

Polyhydroxyalkanoates (PHAs) are bioplastics that can serve as substitutes for petroleum-based plastics with the advantages of being biodegradable, biocompatible, and biobased. The microbial production of polyhydroxyalkanoates is generally conducted in the presence of sugar mixes rich in monosaccharides. In this study, molecular and cultural approaches based on forest soils enriched with hydrocarbon complexes led to the identification and isolation of microbial strains affiliated with Paraburkholderia sp. that dominated the microbial communities that are recognized among the top polyhydroxyalkanoates producers. The genome sequencing of those isolated affiliated strains showed that compared to the reference type strain of their species, they harbored more gene copies of the enzymes involved in PHB synthesis. The microbial conversion of sugar mixes for the newly isolated strains showed a higher PHB production (g/L) and content (%) than was exhibited by the reference strain type of that genus Paraburkholderia for PHB production (P. sacchari LMG 19450T).

Genetic and Functional Analyses of Cutibacterium Acnes Isolates Reveal the Association of a Linear Plasmid with Skin Inflammation

Cutibacterium acnes is a commensal bacterium on the skin that is generally well-tolerated, but different strain types have been hypothesized to contribute to the disease acne vulgaris. To understand how some strain types might contribute to skin inflammation, we generated a repository of C.acnes isolates from skin swabs of healthy subjects and subjects with acne and assessed their strain-level identity and capacity to stimulate cytokine release. Phylotype II K-type strains were more frequent on healthy and nonlesional skin of subjects with acne than those isolated from lesions. Phylotype IA-1 C-type strains were increased on lesional skin compared with those on healthy skin. The capacity to induce cytokines from cultured monocyte-derived dendritic cells was opposite to this action on sebocytes and keratinocytes and did not correlate with the strain types associated with the disease. Whole-genome sequencing revealed a linear plasmid in high-inflammatory isolates within similar strain types that had different proinflammatory responses. Single-cell RNA sequencing of mouse skin after intradermal injection showed that strains containing this plasmid induced a higher inflammatory response in dermal fibroblasts. These findings revealed that C.acnes strain type is insufficient to predict inflammation and that carriage of a plasmid could contribute to disease.

Respiratory Viral Infections Among Children Hospitalized in a Great Referral Hospital in Iran During the Coronavirus Pandemic

Background: Types of viral strains play an important role in developing respiratory infections in children, but what strains are prominent in each community and how they are distributed in both sexes and different ages is not yet fully understood in many countries, such as Iran. Objectives: We aimed to determine the prevalence of viral infections caused by different types of respiratory viruses in children with viral respiratory symptoms admitted to a referral children's hospital in Iran. Methods: In this cross-sectional study, 87 nasopharyngeal specimens were collected from 90 children hospitalized in our children's hospital with respiratory symptoms. Multiplex real-time PCR techniques detected 17 different viruses. Results: The most common respiratory symptom was cough revealed in 82.8%, followed by respiratory distress in 13.8% and tachypnea in 6.9%. Respiratory viruses were detected in 28.7% of patients, and co-infections were observed in one case. The most frequent viral strain detected was the Coronavirus (COVID-19) in 16.1%, followed by the Influenza A virus in 5.7%. The frequency of other viruses is Metapneumovirus 3.4%, respiratory syncytial virus (RSV) 2.3%, Bocavirus (1.1%), HCOV NL63 1.1%, and Parainfluenza 3.4%. The distribution of viral strains was independent of children's gender and age. Conclusions: Viruses were detected in 31% of children with respiratory symptoms, so other causative respiratory infections, except COVID-19, should be considered in children with respiratory symptoms even during the COVID-19 pandemic.

High carriage rate of extended-spectrum β-lactamase Enterobacterales and diarrheagenic Escherichia coli in healthy donor screening for fecal microbiota transplantation.

The safety of fecal microbiota transplantation (FMT) has been highlighted by extended-spectrum β-lactamase (ESBL)-producing Escherichia coli bacteremia transmitted from donors and acquisition of diarrheagenic E. coli (Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC)) via FMT. The use of donor screening criteria to lower the risk of pathogen transmission via FMT is essential. This study aimed to demonstrate the outcomes of our strict donor screening program. This study was conducted at our FMT center between January 2019 and June 2022. Our donor screening program included an initial questionnaire and subsequent blood and stool testing. We further used selective culture for third-generation cephalosporin-resistant (3GCR) Enterobacterales and multiplex PCR to detect diarrheagenic E. coli in stools. The resistance mechanisms and sequence type of 3GCR Enterobacterales were determined. A total of 742 individuals were assessed, and 583 participants (78.6%) were excluded after questionnaire. Of the remaining 159 participants undergoing stool and blood tests, 37 participants were finally qualified (5.0%, 37/742). A high fecal carriage rate of ESBL-producing Enterobacterales (35.2%, 56/159), including E. coli (n=53) and Klebsiella pneumoniae (n=5), and diarrheagenic E. coli (31.4%, 50/159), including EPEC (n=41), enteroaggregative E. coli (n=11), enterotoxigenic E. coli (n=4), and STEC (n=1), was noted. CTX-M-79 and CTX-M-15 were dominant in E. coli and K. pneumoniae, respectively. The sequence types of the ESBL-producing strains were diverse. The screening for 3GCR Enterobacterales and diarrheagenic E. coli in stool is necessary. Our findings also support the effectiveness of multiplex PCR panels in FMT donor screening programs.

Characteristics and outcomes of Clostridioides difficile infection after a change in the diagnostic testing algorithm.

Polymerase chain reaction (PCR) testing for the detection of C. difficile is a highly sensitive test. Some clinical laboratories have included a 2-step testing algorithm utilizing PCR plus toxin enzyme immunoassays (EIAs) to increase specificity. To determine the risk factors and outcomes of C. difficile PCR-positive/toxin-positive encounters compared to PCR-positive/toxin-negative encounters. Retrospective study. A Veterans' Affairs hospital. A retrospective case-control study of patient encounters with a positive C. difficile test by PCR and either a toxin EIA-positive assay (ie, cases) or toxin EIA-negative assay (ie, controls). Clinically relevant exposures and risk factors were determined to assess CDI recurrence at 30 days. Available encounter stool specimens were cultured for C. difficile and were subjected to restriction endonuclease analysis (REA) strain typing. Among 130 C. difficile PCR-positive patient encounters, 80 (61.5%) were toxin EIA negative and 50 (38.5%) were toxin EIA positive. Encounters that were toxin positive were more frequently treated (96.0%) compared to toxin-negative encounters (71.3%; P < .01). A multivariable logistic regression model revealed that toxin-negative encounters were less likely to suffer a recurrent CDI episode within 30 days (odds ratio [OR], 0.20, 95% confidence interval [CI], 0.05-0.83). Additionally, a higher C. difficile PCR cycle threshold predicted a lower risk of CDI recurrence at 30 days. (OR, 0.82; 95% CI, 0.68-0.98). During the study period, the REA group Y strain accounted for most toxin-negative encounters (32.5%; P = .05), whereas REA group BI strain accounted for most toxin-positive encounters (24.3%; P = .02). A testing strategy of PCR plus toxin EIA helped predict recurrent CDI.

Effect of the COVID-19 Pandemic on Rates and Epidemiology of Clostridioides difficile Infection in One VA Hospital.

The COVID-19 pandemic was associated with increases in some healthcare-associated infections. We investigated the impact of the pandemic on the rates and molecular epidemiology of Clostridioides difficile infection (CDI) within one VA hospital. We anticipated that the potential widespread use of antibiotics for pneumonia during the pandemic might increase CDI rates given that antibiotics are a major risk for CDI. Hospital data on patients with CDI and recurrent CDI (rCDI) were reviewed both prior to the COVID-19 pandemic (2015 to 2019) and during the pandemic (2020-2021). Restriction endonuclease analysis (REA) strain typing was performed on CD isolates recovered from stool samples collected from October 2019 to March 2022. CDI case numbers declined by 43.2% in 2020 to 2021 compared to the annual mean over the previous 5 years. The stool test positivity rate was also lower during the COVID-19 pandemic (14.3% vs. 17.2%; p = 0.013). Inpatient hospitalization rates declined, and rates of CDI among inpatients were reduced by 34.2% from 2020 to 2021. The mean monthly cases of rCDI also declined significantly after 2020 [3.38 (95% CI: 2.89-3.87) vs. 1.92 (95% CI: 1.27-2.56); p = <0.01]. Prior to the pandemic, REA group Y was the most prevalent CD strain among the major REA groups (27.3%). During the first wave of the pandemic, from 8 March 2020, to 30 June 2020, there was an increase in the relative incidence of REA group BI (26.7% vs. 9.1%); After adjusting for CDI risk factors, a multivariable logistic regression model revealed that the odds of developing an REA group BI CDI increased during the first pandemic wave (OR 6.41, 95% CI: 1.03-39.91) compared to the pre-pandemic period. In conclusion, the incidence of CDI and rCDI decreased significantly during the COVID-19 pandemic. In contrast, REA BI (Ribotype 027), a virulent, previously epidemic CD strain frequently associated with hospital transmission and outbreaks, reappeared as a prevalent strain during the first wave of the pandemic, but subsequently disappeared, and overall CDI rates declined.

Photoelectric properties of monolayer NbS2 and NbSe2 under three types of strain: A first-principles study

Based on the first principles, the crystal structure, photoelectric properties, and structural stability of two transition metal dichalcogenides (TMDCs) under different strain treatments are systematically calculated. The properties of NbX2 under tensile and compressive strains are discussed for the first time. Recently, a single-layer 1T structure with X atoms surrounding the transition metal atoms was synthesized in experiments. The 1T multi-forms were octahedral coordination structures, and some of the systems showed excellent semiconductor properties and stability. The results show that NbS2 has an indirect band gap under different strains and tends to transition to semiconductors which can effectively improve the material activity. NbSe2 has more excellent properties in the optical field and can be used to manufacture infrared lenses, anti-reflection coatings, and UV reflectors. This study provides a new understanding of the unconventional structure of NbX2 and provides theoretical guidance for the work in the field of TMDCs.

Evaluation of pathogen distribution and antimicrobial resistance in breast plastic surgery infection.

The demand for mammaplasty has increased in recent years, and infection remains one of the common and serious post-operative complications. In this study, we analyzed the pathogen distribution and antibiotic susceptibility of breast plastic surgery infections, and compared the differences in pathogenic species between surgical procedures. The number of each species was counted in the microbial samples of breast plastic surgery infections in Plastic Surgery Hospital of Chinese Academy of Medical Sciences from January 2011 to December 2021. The in vitro antibiotic sensitivity testing data were analyzed using WHONET 5.6 software. The surgical techniques, the period of infection, and other details were gathered in accordance with the clinical data. There were a total of 42 cases included, and 43 different types of pathogenic bacteria, mostly gram-positive bacteria, were found. CoNS (13/43) and Staphylococcus aureus (22/43) made up the majority. The most prevalent of the five Gram-negative bacteria was Pseudomonas aeruginosa. Results of drug sensitivity tests indicate that S. aureus is highly sensitive to vancomycin, cotrimoxazole, and linezolid, whereas CoNS is highly sensitive to vancomycin, linezolid, and chloramphenicol. Both of these bacteria show high resistance to erythromycin and penicillin. Breast augmentation, breast reconstruction, and breast reduction surgery were the most frequently associated breast surgery procedures in this study with infections, with the highest number of infections occurring following breast augmentation with fat grafting, breast reduction surgery, and breast reconstruction with autologous tissue. Various breast plastic surgery procedures have different common pathogens of infection, but the most prevalent are CoNS and S. aureus. Additionally, the majority of the infections in this study were in the early stages. Gram-positive bacteria were the predominant cause of breast plastic surgery infections, and the types of infection strains, the period of infection onset, and the antibiotic susceptibility of prevalent strains varied between breast plastic procedures.

In vitro activity of cefiderocol against MBL-producing Gram-negative bacteria collected in North America and Europe in five consecutive annual multinational SIDERO-WT surveillance studies (2014-2019).

To evaluate the in vitro antibacterial activity of cefiderocol, a siderophore cephalosporin against MBL-producing clinical isolates. MBL-producing strains were selected from clinical isolates of Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii complex collected in North America and Europe in five consecutive annual multinational SIDERO-WT surveillance studies from 2014 to 2019. MICs of cefiderocol and comparator agents were determined by the broth microdilution method according to the CLSI guideline. A total of 452 MBL-producing strains consisting of 200 Enterobacterales, 227 P. aeruginosa and 25 A. baumannii complex were identified. The highest number of MBL-producing Enterobacterales strains were detected in Greece. MBL-producing strains of both P. aeruginosa and A. baumannii complex were isolated most frequently in Russia. For Enterobacterales, 91.5% or 67.5% of MBL-producing strains had cefiderocol MIC values ≤4 mg/L (CLSI susceptibility breakpoint) or ≤2 mg/L (EUCAST susceptibility breakpoint), respectively. All MIC values of cefiderocol for MBL-producing P. aeruginosa strains were ≤4 mg/L (CLSI susceptibility breakpoint), and 97.4% of them had cefiderocol MIC values ≤2 mg/L (EUCAST susceptibility breakpoint). For A. baumannii complex, 60.0% or 44.0% of MBL-producing strains had cefiderocol MIC values ≤4 mg/L (CLSI susceptibility breakpoint) or ≤2 mg/L (EUCAST pharmacokinetic-pharmacodynamic susceptibility breakpoint), respectively. Against all types of MBL-producing strains, MIC distribution curves of cefiderocol were located in the lowest numerical values, compared with other β-lactams and β-lactam/β-lactamase inhibitor combinations tested and ciprofloxacin. Although the types of MBL-producing strains isolated by country varied, cefiderocol showed potent in vitro activity against all types of MBL-producing Gram-negative bacteria regardless of the bacterial species.

Piezo-gated flexible transistors: A path to energy-efficient multi-functional piezotronic devices

Developing smart sensors and advanced electronics for the need of the internet of things prevails yet under the constraint of energy efficiency. Therefore, the development of multi-functional devices toward low power is the research spotlight. Due to the low energy consumption, semiconductor-based piezo-gated devices hold great promises. Herein, a flexible multi-dimensional strain sensor by growing two ZnO thin film-based terminals on both sides of a mica substrate is developed. This Multidimensional Piezo-Gated Flexible Transistor (M-PGFT) device successfully detects the positive (upward) bending, negative (downward) bending, and compressive load. Where the gauge factor (GF) of the Top/Bottom terminals under positive and negative bending strains are −3.44/+9.78 and +5.44/−4.03, respectively, and +94.93/+74.70 for compressive load is obtained. Notably, the GF of both ZnO terminals is asymmetric under two bending types of loads, whereas symmetric under compressive load. This unique behavior made this smart multi-dimensional strain sensor to distinguish between different types of strains. Furthermore, the same device can be implemented as a piezo-gated three-valued logic device, demonstrating standard ternary inversion and standard ternary NAND operations. This study presents a unique and simple design to fabricate flexible multi-functional piezo-gated devices for multi-dimensional strain sensing and three-valued logic operation at low power.

Production Performance of Debu and Kelabu Sentul Chicken at Different Igy Concentrations

Sentul chicken is one of Indonesia’s native chickens that has high potential for meat and egg production performance. The production of Sentul chicken breeds with high body resistance is one of the solutions to fulfill this potential. This research aimed to ascertain the impact of various IgY concentrations (high, medium, and low) on the production of 90 Debu and 90 Kelabu Sentul chickens. Parameters observed included pre-laying and laying performance. Six replications of a completely randomized design were used to conduct the experiment. The Anova test was used to analyze the data that was collected. The findings revealed that different strains and sex of Debu and Kelabu Sentul chickens did not significantly affect the value of IgY concentration. In the entire population studied, only 12.09% of Debu Sentul chickens and 11. 32% of Kelabu Sentul chickens had low Igy concentration values. In pre-laying period, the difference in the types of chicken strains has no significant effect on the body weight gain and thefeed convertion. Chickens with high IgY concentrations performed worse during the laying phase than hens with low IgY concentrations. The research found that the population of Kelabu Sentul chickens with high IgY concentration value is more than Debu Sentul chickens, which makes them suitable to be used as breeders.

The effect of prolonged intubation on ventilator associated pneumonia: endotracheal tube cuff is really steril or not?

Aims: Endotracheal tube (ETT) design, size, cuff material, cuff pressure, and intubation duration are critical in preventing nosocomial pneumonia. We aimed to evaluate the possible infection focus potential of ETT cuff and pilot balloon, particularly in prolonged intubated patients. Methods: A total number of 66 patients who underwent orotracheal intubation and received conventional mechanical ventilation more than 48 hours in the intensive care unit (ICU), were included in this prospective cohort study. Results:The mean duration of intubation was 10.36±4.82 days. Bacteriologically confirmed positive tracheal aspirate culture was 18.2% (n=12). The most frequent positive culture was detected inside of ETT lumen with a percentage of 83.3% (n=55) and followed by cuff (27.3%, n=18), pilot balloon (13.6%, n=9), respectively. It was documented that rates of lung infections were significantly increased after 14 days (p = 0.017) and rates of cuff positive cultures were significantly increased after 10 and 14 days of incubation (p= 0.001, p=0.004). The same type of bacteriological strains was identified from both pilot balloon (n=9) and ETT cuff (n=9), simultaneously. In the remaining 9-cuff positive patients pilot balloons were sterile and ETT lumens were positive culture with the same strains as identified from the cuff. There was a statistically significant positive correlation between the intubation duration and the number of infected ETT parts (p&lt;0.001). Conclusion: ETT cuff was demonstrated to be a potential infection focus in the present study. In addition, it was observed that ETT cuff colonization increased in proportion to the intubation duration. We suggest changing ETT at appropriate time intervals in order to reduce ventilator-associated pneumonia in intubated patients.

ANTIMICROBIAL ACTIVITY OF BUTANOL COMPLEXES AND ISOLATED SECONDARY METABOLITES OF SOME SPECIES OF GENUS SILENE OF THE FAMILY CARYOPHYLLACEAE

Extracts of 9 species of the Caryophyllaceae family were screened for antimicrobial activity on the example of 17 types of strains of microorganisms. The inhibitory activity of 7 butanol extracts has been shown against a number of pathogenic strains belonging to the species Mycobacterium smegmatis, Phroteus mirabilis, Serratia marcescens, Staphylococcus aureus, Streptococcus faecalis. The extracts studied were most effective on the bacteria Bacillus cereus and Bacillus subtilis. It was found that all the studied plant species of the genera Lychnis and Silene contain ecdysteroids and flavonoids. The isolated individual compounds contained in all the studied species showed antimicrobial action on strains of a number of bacteria: the ecdysteroid 20-hydroxyecdysone suppressed the growth of the strain of R. mirabilis, while the flavonoid shaftoside inhibited strains of bacteria S. aureus, B. cereus, B. subtilis, R. mirabilis, M. smegmatis. Gram-negative bacteria Salmonella typhimurium, Klebsiella pneumoniae, Shigella sonnei, Pseudomonas aeruginosa are not susceptible to the antimicrobial action of extracts under experimental conditions.&#x0D; Determination of antimicrobial activity was carried out by methods – diffusion and joint incubation of plant extracts and cells of test strains of microorganisms in a liquid nutrient medium LB at the ratio sample : medium 1 : 2 and 1 : 1. Increase in the concentration of secondary metabolites of ecdysteroids and flavonoids in experiments with butanol complexes of substances with a sample ratio the 1 : 1 environment led to an increase in antimicrobial activity up to the complete inhibition of Gram-positive bacteria B. subtilis and B. cereus by extracts of plant species Silene graefferi, S. colpophylla, S. sendtneri, S. linicola, S. jenisseensis S. viridiflora (seeds) and individual shaftoside.

An efficient and safe strategy for germ cell-specific automatic excision of foreign DNA in F1 hybrid transgenic silkworms.

The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products. In sericulture, only the first filial generation (F1 ) hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms, but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs. To address this issue, we developed a safe and efficient strategy using the GAL4/Upstream activating sequence (UAS) system, the FLP/flippase recognition target (FRT) system, and the gonad-specific expression gene promoters (RSHP1p and Nanosp) for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms. We established 2 types of activator strains, R1p::GAL4-Gr and Nsp::GAL4-Gr, containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp, respectively, and 1 type of effector strain, UAS::FLP-Rg, containing the UAS-linked FLP gene expression cassette. The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%, whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73% to 80.3%. Additionally, we identified a gene, sw11114, that is expressed in both testis and ovary of Bombyx mori, and can be used to establish novel gonad-specific expression systems in transgenic silkworms. This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.

In vitro Assessment of Antibacterial Activity and Potential Genotoxic Effect of Fruit Extracts of Capparis spinosa L. Plant

This research was carried out to assess the minimum inhibitory concentration (MIC) and the genotoxic potential of ethanolic and aqueous (cold and hot) fruit extracts of Capparis spinosa L. (C. spinosa) plant against different types of bacterial strains. The antimicrobial effect of these extracts against the tested bacteria was investigated using broth microdilution method. The potential genotoxic effect was evaluated by ERIC-PCR technique. Results of the current study revealed that the MIC values of ethanolic fruit extract against the tested bacterial had a range of 12.5 mg/ml to 25 mg/ml. However, aqueous fruit extracts had an MIC with a range of 50 mg/ml to 100 mg/mL. The potential genotoxic activity of cold aqueous extract was determined according to the changes in ERIC-PCR profile of E. coli strain treated with extract in comparison to that untreated (negative control). Results of this study suggest the genotoxic effect of aqueous fruit extract on E. coli. Further research is required to assess and identify the biological molecules and their mechanisms in the context of the genotoxicity. In vivo genotoxicity assessment or with the presence of liver extract is recommended to evaluate the safety of using fruits for therapeutic purposes and a valuable nutrient source.

Competition among Flavescence Dorée Phytoplasma Strains in the Experimental Insect Vector Euscelidius variegatus.

Phytoplasmas are plant pathogenic wall-less bacteria transmitted in a persistent propagative manner by hemipteran insects, mainly belonging to the suborder Auchenorrhyncha (Fulgoromorpha and Cicadomorpha). Flavescence dorée (FD) is a quarantine disease of grapevine, causing great damage to European viticulture and associated with phytoplasmas belonging to 16SrV-C (FD-C) and -D (FD-D) subgroups. FD-C and FD-D strains share similar pathogenicity, but mixed infections are rare in nature. To investigate the competition among FDp strains, specimens of the laboratory vector Euscelidius variegatus (Hemiptera: Cicadellidae) were forced to acquire both phytoplasma haplotypes upon feeding on FD-C- and FD-D-infected plants or after the injection of both strains. The pathogen colonization of insect bodies and heads was monitored with multiplex qPCR, and the efficiencies of phytoplasma transmission were estimated. Single infection, irrespective of strain type, was more frequent than expected, indicating that competition among FD strains occurs. Hypotheses of competition for resources and/or host active sites or the direct antibiosis of one strain against the other are discussed, based on the genetic complexity of FDp populations and on the high genome variability of the FD-D strain. As FD management still mainly relies on insecticides against vectors, the characterization of FDp haplotypes and the description of their epidemiology also have practical implications.

Seizures regulate the cation-Cl- cotransporter NKCC1 in a hamster model of epilepsy: implications for GABA neurotransmission.

The balance between the activity of the Na+/K+/Cl- cotransporter (NKCC1) that introduces Cl- into the cell and the K+/Cl- cotransporter (KCC2) that transports Cl- outside the cell is critical in determining the inhibitory or excitatory outcome of GABA release. Mounting evidence suggests that the impairment of GABAergic inhibitory neurotransmission plays a crucial role in the pathophysiology of epilepsy, both in patients and animal models. Previous studies indicate that decreased KCC2 expression is linked to audiogenic seizures in GASH/Sal hamsters, highlighting that Cl- imbalance can cause neuronal hyperexcitability. In this study, we aimed to investigate whether the Na+/K+/Cl- cotransporter NKCC1 is also affected by audiogenic seizures and could, therefore, play a role in neuronal hyperexcitability within the GASH/Sal epilepsy model. NKCC1 protein expression in both the GASH/Sal strain and wild type hamsters was analyzed by immunohistochemistry and Western blotting techniques. Brain regions examined included cortex, hippocampus, hypothalamus, inferior colliculus and pons-medulla oblongata, which were evaluated both at rest and after sound-inducing seizures in GASH/Sal hamsters. A complementary analysis of NKCC1 gene slc12a2 expression was conducted by real-time PCR. Finally, protein and mRNA levels of glutamate decarboxylase GAD67 were measured as an indicator of GABA release. The induction of seizures caused significant changes in NKCC1 expression in epileptic GASH/Sal hamsters, despite the similar brain expression pattern of NKCC1 in GASH/Sal and wild type hamsters in the absence of seizures. Interestingly, the regulation of brain NKCC1 by seizures demonstrated regional specificity, as protein levels exclusively increased in the hippocampus and hypothalamus. Complementary real-time PCR analysis revealed that NKCC1 regulation was post-transcriptional only in the hypothalamus. In addition, seizures also modulated GAD67 mRNA levels in a brain region-specific manner. The increased GAD67 expression in the hippocampus and hypothalamus of the epileptic hamster brain suggests that NKCC1 upregulation overlaps with GABA release in these regions during seizures. Our results indicate that seizure induction causes dysregulation of NKCC1 expression in GASH/Sal animals, which overlaps with changes in GABA release. These observations provide evidence for the critical role of NKCC1 in how seizures affect neuronal excitability, and support NKCC1 contribution to the development of secondary foci of epileptogenic activity.