Polymerase Chain Reaction Method Research Articles

Occurrence of Extended-spectrum β-lactamase (ESBL) and Carbapenemase-producing Escherichia coli isolated from Childhood Diarrhoea in Yaoundé, Cameroon

IntroductionExtended-spectrum β-lactamase (ESBL)-producing pathogenic E. coli is a global public health issue, especially in sub-Saharan African countries such as Cameroon. It contributes to increase significantly hospital length of stay, morbidity, mortality and economic costs because of treatment failures. This study aims at determining the resistance background and virulence profiles of ESBL-E. coli isolates among childhood diarrhoea during the cholera outbreak occuring in Yaoundé, Cameroon.Materials and methodsDuring a four-month periods, from March 1st to June 30th, 2023, a total of 84 stool samples were collected from 90 under five children presenting clinical signs of gastroenteritis and attending four hospitals in Yaoundé, Cameroon. Bacterial identification was done using API20E and antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method. After extraction, genomic DNA was subjected to conventional and multiplex polymerase chain reaction methods (PCRs) for detection of resistance and virulence genes. Statistical analysis was performed using Epi info™ (7.2.5.0). Statistical significance was considered at a p-value < 0.05.ResultsOut of 150 patients contacted, 90 patients were enrolled, 84 samples were collected, 52.38%(44/84) and 3.57%(03/84) were confirmed as extended-spectrum β-lactamase and carbapenemase-producing E. coli respectively. The risk factors were analyzed, and children who drank natural fruit juice (OR: 0.4, p-value: 0.03) were found to be significantly associated with ESBL-producing E. coli. The ESBL-producing E. coli isolates showed a high level of resistance to amoxicillin-clavulanic acid, cefotaxime, ceftazidime, cefepime, colistin, and tetracycline. The blaCTX−M was more prevalent ß-lactamase resistance gene. The tetracycline resistance genes tet(A) and tet(B) were also detected. The most important virulence genes detected were FimH (81.81%) and papA (79.54%).ConclusionThese findings suggest implementing routine surveillance and screening for antimicrobial resistance among children under five. Antimicrobial stewardship strategies (ASP) need to be implemented to curb the emergence and dissemination of ESBL-producing E. coli. In addition, a national surveillance program for antimicrobial resistance needs to be implemented at local and regional levels in order to reduce morbidity in Cameroon.

A multi-pathogen behavioral exposure model for young children playing in public spaces in developing communities.

Diarrheal disease is the second leading cause of death among children under five in developing communities, despite active interventions to improve access to water, sanitation, and hygiene resources. Even interventions with high fidelity and compliance saw minimal improvements in health outcomes, necessitating a need for looking beyond traditionally targeted exposure pathways. This study focuses on the pathogen exposure a young child may receive while playing in the public spaces of informal settlements, where animal feces, human feces, and garbage are frequently observed. The study utilized 79 soil samples previously collected across ten cluster sites in Corail, Haiti and processed using DelAgua cultural assays and quantitative Polymerase Chain Reaction methods. Molecular assays screened for Aeromonas, Vibrio cholera, and several pathogenic Escherichia coli species. Behavioral observations of young children (<5 years old) were also conducted in overlapping areas with the environmental sampling to quantify frequency of risky and mitigating behaviors. Environmental and behavioral data were combined to obtain the posterior distribution of children's pathogen exposure from playing in a public space for one hour. The model estimated that children have a likelihood of approximately 70% of being exposed to a pathogen during one hour of play and a greater than 30% chance of being exposed to multiple pathogens in the same period. While children and toddlers practice fewer risky behaviors compared to infants, they were shown to have higher likelihood of exposure and slightly higher pathogen dose per exposure. As anticipated, a high correlation between genes from the same E. coli species was observed in the model output. These findings demonstrate the need to consider public spaces as an important exposure pathway for young children for both future research and interventions.

Analysis of the species composition and infection of ticks with pathogens of Lyme disease in the territory of the Republic of Tatarstan for the period 2012–2022

BACKGROUND: The role of ixodid ticks in the transmission of pathogens of a number of natural focal infections can hardly be overestimated. Analysis of the manifestations of the epizootic process is important for planning preventive and anti-epidemic measures and is an integral component of epidemiological surveillance of natural focal infections. AIM: To analyze the species composition of ticks and the infection of ticks with borreliosis pathogens in the Republic of Tatarstan for the period 2012–2022. MATERIALS AND METHODS: An analysis of the species composition of ticks collected by the zoological group of the Center for Hygiene and Epidemiology in the Republic of Tatarstan from environmental objects during the period 2012–2022 was carried out. Testing of the ticks for Borrelia burgdorferi and Borrelia miyamotoi was carried out using the polymerase chain reaction method with hybridization-fluorescent detection. An analysis of statistical observation form No. 2 “Information on infectious and parasitic diseases” (the section “Tick bites”) for the Republic of Tatarstan from 2012 to 2022 was carried out. Statistical processing was performed using descriptive statistics methods in Microsoft Excel. RESULTS: The total number of people who applied for tick bites in the Republic of Tatarstan in 2012–2022 was 77,703, of which 59,168 (76.2%) were people over 18 years of age. During the study period, 5536 ticks were collected for research on the territory of the Republic of Tatarstan from environmental objects. The species composition of ticks was distributed as follows: Dermacentor reticulatus — 75.4%, Ixodes ricinus — 14.1%, Ixodes persulcatus — 10.5%. Infection of different types of ticks with Borrelia was not the same and was 20.4% for I. persulcatus, 14% for I. ricinus, 4.4% for D. reticulatus. In 4 regions of the republic, from ticks of the species I. ricinus pathogenic genomospecies B. miyamotoi were found (the infestation of this type of tick with B. miyamotoi was 0.4%). Indicators of morbidity of the population with ixodid tick-borne borreliosis and indicators of infection rates for ticks vary in different regions of the republic. CONCLUSIONS: The dominant species of ixodid ticks in natural epitopes of the Republic of Tatarstan in 2012–2022 were D. reticulatus (75.4% of the total number of collected ticks). For ticks of this species, the lowest level of infection with Borrelia was demonstrated (4.4% of ticks). This may be one of the factors responsible for the low, in comparison with other regions, incidence rates of tick-borreliosis in the Republic of Tatarstan. The identification of ticks of the species I. ricinus infected with B. miyamotoi confirms the need for targeted molecular genetic and serological examination for recurrent fever in patients with non-erythematous forms of febrile illness after an episode of tick bite.

Animal and plant protein intake association with mental health, tryptophan metabolites pathways, and gut microbiota in healthy women: a cross-sectional study

Mental health is affected by tryptophane (TRP) metabolism regulation. Diet-influenced gut microbiome regulates TRP metabolism. Thus, the present study aimed to explore the relationship between type of dietary protein intake, gut microbiota, TRP metabolites homeostasis, and mental well-being in healthy women. 91 healthy females aged 18–50 were recruited based on the study protocol. Validate and reliable questionnaires assessed dietary intake and mental health. Biochemical tests and gut microbiota composition were analyzed following the manufacturer’s instructions for each enzyme-linked immune sorbent assay (ELISA) kit and Real-time quantitative polymerase chain reaction (qPCR) methods respectively. Regression methods were used to estimate the considered associations. The results show that in the fully adjusted model, plant protein consumption was partially inversely associated with depression risk (OR = 0.27; 95% CI: 0.06, 1.09; P = 0.06). Higher dietary animal protein intake was marginally associated with psychological distress (OR = 2.59; 95% CI: 0.91, 7.34; P = 0.07). KYN to serotonin ratio was inversely associated with animal protein consumption (ß = 1.10; 95% CI: -0.13, 2.33; P = 0.07). Firmicutes/Bacteriodetes ratio (β = -1.27 × 103, SE = 5.99 × 102, P = 0.03) was lower in the top tertile of plant protein. A partially negative correlation was found between dietary animal protein and Prevotella abundance (β = -9.20 × 1018, SE = 5.04 × 1018, P = 0.06). Overall, significant inverse associations were found between a diet high in plant protein with mental disorders, KYN levels, and Firmicutes to Bacteroidetes ratio while adhering to higher animal protein could predispose women to psychological stress.

Prevalence of the Potato virus Y strain composition impacting Michigan seed potato production

Potato virus Y (PVY) is a complex of multiple strains that cause a variety of foliar and tuber symptoms. Several current strains have potential to result in variety-dependent yield and quality losses. Understanding the local PVY strain population and variety responses are critical for detection and management. From 2019-2023, PVY strain composition was monitored across seven Michigan seed potato-producing counties growing prevalent chip, russet, and tablestock market classes. Using multiplex reverse-transcriptase polymerase chain reaction methods, PVY strains were identified from potato tuber or leaf samples collected through the certification program (N = 78 positive lots). PVYN-Wi remained most prevalent in Michigan, however, tuber necrotic strains PVYNTN and PVYE also continued to be detected. These, as well as some PVYN-Wi isolates, are known to cause necrotic symptoms in tubers, causing yield and quality reductions for susceptible potato cultivars. As in other U.S. potato-growing regions, PVYN-Wi and PVYNTN were most prevalent in Michigan seed potato lots. Of note, mild to severe tuber necrosis was observed in diagnostic samples positive with local PVYN-Wi strains. Parental strain PVYO appeared to re-emerge in 2023 and should be considered in certification and management. Mild or asymptomatic reactions may still lead to yield or quality losses and may pose a risk to more susceptible cultivars. Due to the diversity of strains and varieties prevalent in the Great Lakes region, continued uniform certification is recommended. Information gathered from this and future survey efforts will be valuable for growers, inspectors, and future breeding and selection efforts.

Epidemiology of pediatric meningitis and encephalitis in Japan: a cross-sectional study.

Information on the epidemiology and cause-specific clinical features of pediatric meningitis and encephalitis is limited, owing to conventional laboratory methods' limitations in identifying causative pathogens. The FilmArray Meningitis/Encephalitis (FA-M/E) panel, a molecular diagnostic tool, can detect 14 pathogens within 1 hour. We investigated meningitis and encephalitis epidemiology among children in Japan and FA-M/E panels' utility in their clinical management. This cross-sectional study was conducted among children aged 0-18 years admitted to seven regional hospitals in western Japan between October 2022 and September 2023. Cerebrospinal fluid (CSF) samples were collected from 221 children and tested using the FA-M/E panel. Clinical and microbiological data were reviewed retrospectively. Fifty-eight patients tested positive using the FA-M/E panel. Viral and bacterial pathogens were detected in 49 and nine cases, respectively. Human parechovirus and enterovirus occurred mainly in epidemic clusters during the summer and were primarily detected in young infants. Patients who tested positive for human parechovirus had a significantly higher frequency of sepsis-like manifestations and a lower frequency of CSF pleocytosis than did those who tested positive for enterovirus. CSF pleocytosis was absent in 30 patients who tested positive using the FA-M/E panel. Among the patients who tested positive for bacteria, three of the nine were not diagnosed using conventional culture methods, owing to prior antimicrobial therapy. The FA-M/E panel can identify bacterial and viral pathogens causing pediatric meningitis and characterize the epidemiology in local communities.IMPORTANCECulture and polymerase chain reactions have traditionally been used to identify microorganisms causing pediatric meningitis and encephalitis. However, the methods currently used to identify the causative microorganisms are limited, particularly in general hospitals. The FilmArray Meningitis/Encephalitis (FA-M/E) panel, a fully automated genetic testing system, can detect 14 pathogens using the multiplex polymerase chain reaction method. This study described the epidemiology of pediatric meningitis and encephalitis in Japan. The microorganisms causing acute meningitis and encephalitis in children in Japan were identified using the FilmArray Meningitis/Encephalitis panel. Testing cerebrospinal fluid using the FA-M/E panel is useful for the identification of the pathogen in children with community-acquired acute meningitis and encephalitis. This increases knowledge on the epidemiology and clinical manifestations of acute meningitis and encephalitis caused by specific pathogens and can be used to facilitate optimal patient management.

Molecular genetic methods for quality control of inactivated vaccines using a Chikungunya virus model: vaccine strain identification and completeness of virus inactivation

INTRODUCTION. The completeness of virus inactivation and the identity of the vaccine strain are essential parameters for the safety and quality of inactivated virus vaccines, which should be controlled during vaccine development and production. Currently, the most promising quality control methods for inactivated virus vaccines are molecular genetic methods, which provide rapid results with high sensitivity and specificity.AIM. The aim of this study was the development of a real-time quantitative polymerase chain reaction (qPCR) method and an integrated cell culture real-time quantitative polymerase chain reaction (ICC-qPCR) method to assess the completeness of virus inactivation, as well as a reverse-transcription polymerase chain reaction assay coupled with restriction fragment length polymorphism analysis (RT-PCR-RFLP) to confirm the identity of the vaccine virus strain.MATERIALS AND METHODS. This study used RNA of CHIKV genotypes (three strains of each of the four CHIKV genotypes, including Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL), which were identified by sequencing prior to analysis). Additionally, the study used the Nika21 CHIKV strain (ECSA genotype), the Nika21 CHIKV strain inactivated with β-propiolactone, and the Nika21 CHIKV strain antigen adsorbed on aluminium hydroxide. The methods used included real-time qPCR, RT-PCR-RFLP, and virus neutralisation.RESULTS. The study identified a 218 bp fragment of the nsP1 gene (positions 789 to 1006) with restriction endonuclease recognition sites. These sites were present or absent in combinations specific to each of the four CHIKV genotypes. The authors selected primers for amplification of the specified gene region and tested the conditions for real-time qPCR and RT-PCR-RFLP. The study demonstrated the possibility of using the ICC-qPCR method to confirm the completeness of virus inactivation and the RT-PCR-RFLP method to identify the vaccine strain.CONCLUSIONS. The study showed the advantages of using the ICC-qPCR method to confirm the completeness of antigen inactivation and the RT-PCR-RFLP method to identify the vaccine strain. These methods are more sensitive and faster than traditional culture methods. ICC-qPCR and RT-PCR-RFLP can be used at any stage of the production process for inactivated vaccines.

Identification and genotyping of Chikungunya virus using reverse transcription polymerase chain reaction and restriction fragment length polymorphism methods

INTRODUCTION. Chikungunya virus (CHIKV) genotyping involves sequencing fractions of genes encoding E1, E2, and nsP1 proteins or the entire genome of the virus. Available reagent kits or polymerase chain reaction protocols cannot be used for CHIKV genotyping, and nucleic acid sequencing requires expensive equipment and materials, which are not always available. Therefore, it seems promising to use a simpler and more cost-effective restriction fragment length polymorphism (RFLP) method, which has not previously been used for CHIKV genotyping.AIM. This study aimed to investigate the possibility of using reverse transcription polymerase chain reaction (RT-PCR) and RFLP for CHIKV identification and genotyping.MATERIALS AND METHODS. The experimental study used RNA from CHIKV strains of four genotypes, including the Asian, West African (WAf), and East/Central/South African (ECSA) genotypes, and the Indian Ocean Lineage of the ECSA genotype (ECSA-IOL). The study used RT-PCR followed by DNA restriction and restriction fragment length analysis.RESULTS. The nsP2 gene fragment of 648 bp in length (positions 3806 to 4453) contains recognition sites for the restriction endonucleases PspEI, PvuII, and DraI. The presence or absence of these sites generates a different combination specific to each of the four CHIKV genotypes. The authors designed primers for amplification of the selected gene region and performed RTPCR and RFLP.CONCLUSIONS. The RFLP method can be used for rapid CHIKV identification and genotyping. The method provides results within a few hours and does not require high-tech equipment.

Characterization of Hibiscus Chlorotic Ringspot Virus-Derived vsiRNAs from Infected Hibiscus rosa-sinensis in China.

Lots of progress have been made about pathogen system of Hibiscus rosa-sinensis and hibiscus chlorotic ringspot virus (HCRSV), however, interactions between H. rosa-sinensis and HCRSV remain largely unknown. Hereon, firstly, HCRSV infection in H. rosa-sinensis from Zhangzhou city of China was confirmed by traditional electron microscopy, modern reverse transcription polymerase chain reaction and RNA-seq methods. Secondly, sequence feature analysis showed the full-length sequence of HCRSV-ZZ was 3,909 nucleotides (nt) in length and had a similar genomic structure with other carmovirus. It contains a 5' untranslated region (UTR), followed by seven open reading frames encoding for P28, P23, P81, P8, P9, P38, and P25, and the last a 3-terminal UTR. Thirdly, HCRSV- ZZ-derived vsiRNAs were identified and characterized for the first time from disease H. rosa-sinensis through sRNA-seq to reveal interactions between pathogen ant plant host. It was shown that the majority of HCRSV-ZZ-derived vsiRNAs were 21 nt, 22 nt, and 20 nt, with 21 nt being most abundant. The 5&apos;-terminal nucleotide of HCRSV-ZZ vsiRNAs preferred U and C. HCRSV-ZZ vsiRNAs derived predominantly (72%) from the viral genome positive-strand RNA. The distribution of HCRSV-ZZ vsiRNAs along the viral genome is generally even, with some hot spots and cold spots forming in local regions. These hot spots and cold spots could be corresponded to the regions of stem loop secondary structures forming in HCRSV-ZZ genome by nucleotide paring. Taken together, our findings certify HCRSV infection in H. rosa-sinensis and provide an insight into interaction between HCRSV and H. rosa-sinensis and contribute to the prevention and treatment of this virus.

First evidence of WW superfemale for hybrid sturgeon, the bester (Huso huso × Acipenser ruthenus)

Sex control offers a promising strategy for cost-effective aquaculture in certain fish species. Sturgeons, which utilize the ZZ/ZW sex determination system, are particularly valuable for producing high-priced black caviar derived from female roes, making all-female production highly desirable. However, this goal has yet to be fully realized. In this study, we identified a short genomic region specific to the Z and W genomic scaffolds of Acipenser ruthenus and developed a polymerase chain reaction (PCR) method to distinguish between the Z and W alleles. Using PCR, 1280 A. ruthenus, 66 A. baerii baerii, and 31 F2 hybrids of besters (Huso huso × A. ruthenus) were genotyped, revealing either a single Z amplicon or dual Z plus W amplicons in an approximately 1:1 ratio. This indicates successful discrimination of the ZZ/ZW genotypes. Additionally, gonadal sex was identified in 145 of the genotyped individuals, all of which corresponded to their genetic sex. Gynogenetic individuals of the F3 bester hybrid were also genotyped, with 25 of 112 (22.3 %) exhibiting only W amplicons, identifying them as WW superfemales. These findings contribute to the advancement of all-female sturgeon production for aquaculture, facilitating the efficient farming of these highly prized species.

Identification of a novel RHCE*Ce (829G > A) allele associated with absence of C and e antigens expression.

The Rh blood group antigens are encoded by the RHD and RHCE genes, which possess a remarkable degree of polymorphism owing to their high homologous structures. These variants of the RH genes can lead to absence or weak expression of antigens. Analysis of RHCE genotyping by Polymerase Chain Reaction (PCR-SSP) method specific to detect c.48G, c.48C, 109 bp insertion of IVS2, c.201A and c.307C and RhCE phenotyping, were conducted in 316 Chinese patients in previous study. One patient with discrepancy typing result was collected for further RhCE serologic typing using microcolumn gel method and tube method in saline using monoclonal antibodies. PacBio sequencing was performed for RHCE, RHD and RHAG complete sequence analysis. 3D molecular models of the protein with the wild-type and mutant residue were generated using the DynaMut web server. The effect of the mutation on the protein function was predicted by PolyPhen-2 software. One male patient of Chinese Han was detected with RHCE*C allele showed by PCR-SSP method but ccEE phenotype. Further PacBio sequencing identified one normal RHCE*cE allele and one RHCE*Ce allele carried a novel c.829G > A (p.Gly277Arg) variant, which the encoded amino acid located in the ninth transmembrane segment of RhCE protein. Crystallisation analysis of 3D molecular models revealed that the substitution at Arg277 leads to the formation of additional hydrogen bonds, including weak hydrogen bonds between multiple atoms. It also results in hydrophobic ion interactions between Arg277 and Ala244. This mutation is predicted to have a damaging effect on protein function. One novel RHCE*Ce allele with c.829G > A (p.Gly277Arg) variant was identified to resulting in the absence or weak expression of C and e antigens.

Spore PCR and qPCR Methods for Rapid Detection of Five Colletotrichum Species Responsible for Pepper Anthracnose in Korea

Pepper anthracnose, caused by &lt;i&gt;Colletotrichum&lt;/i&gt; spp., leads to a decrease in the quantity of pepper fruit production. Molecular diagnosis is crucial for rapid identification of pathogens and determination of fungicide resistance. However, the traditional process of isolating the pathogen, extracting genomic DNA, and analyzing the gene sequence is time-consuming, which delays rapid diagnosis. In this study, we introduced a method using conidia of &lt;i&gt;Colletotrichum&lt;/i&gt; spp. instead of genomic DNA, eliminating the need for DNA extraction or special processing for diagnosis. To elucidate this method, sensitivity was assessed through polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) tests using internal transcribed spacer-based primer pairs. Both PCR and qPCR tests showed that detection is feasible with just one conidia, with over 1,000 conidia yielding results comparable to approximately 1 pg of genomic DNA. For amplifying the cytochrome b gene for quinone-outside inhibitor fungicide susceptibility testing, detection from a single conidium is achievable, but a stable PCR product is obtained by increasing the number of cycles to 35. Additionally, the addition of 10% grinding fresh chili pepper paste to V8-Juicea gar medium, which is known for inducing conidia rapidly from the isolates, resulted in 3.2 to 6.0 times more conidia compared to the commonly used potato dextrose agar medium, enhancing the potential for swift testing. Taken together, this study presents a direct utilization of pepper anthracnose conidia through PCR or qPCR, offering a valuable technique for amplifying target genes, such as the minimum conidial amount and barcode genes, for molecular identification of anthracnose disease in pepper through PCR and qPCR analysis.

PENGGUNAAN PRIMER GEN CYTOCHROME OXIDASE 1 DALAM REAKSI POLYMERASE CHAI REACTION (PCR) UNTUK IDENTIFIKASI KANDUNGAN BABI PADA MAKANAN

Muslims make up the majority of the population in Indonesia so halal food safety is of paramount importance. From a religious and health perspective, adulteration and contamination of Sus scrofa meat is considered a violation of halal norms. Sus scrofa is often used to adulterate beef to take advantage of the physical resemblance and price difference. The need to identify Sus scrofa meat contamination in food products was highlighted in this study. After a successful isolation process, the Polymerase Chain Reaction (PCR) method was used to validate the presence of porcine DNA. PCR utilizes primers specifically made to replicate specific DNA sequences associated with pigs. PCR testing provides precise results for pig DNA, ensuring the accuracy of detection of pork contamination in foodstuffs. This research contributes to efforts to maintain halal food safety in Indonesia by emphasizing the important role of PCR in the detection process of Sus scrofa meat.

Aspergillus aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis as Four New Pathogens of Eye Infection in DNA Barcoding Markers

Background: The use of a morphological method to determine Aspergillus species in routine laboratory investigation has always been challenged with low sensitivity and low specificity. Time-consuming specimen preparation and insufficient expertise are the crucial reasons. DNA barcoding markers have been introduced for identification as another gold standard method. Objective: To investigate DNA barcoding markers in identifying Aspergillus strains isolated from patients with eye infection. Methods: This study extracted 12 DNA samples of 12 Aspergillus strains. DNA barcoding genes, i.e., ITS, betatubulin (BT2), calmodulin (CAL), and actin (ACT) genes, were amplified and sequenced by polymerase chain reaction and Sanger's sequencing methods. All gene sequences were compared to those of the type or reference species collected from public databases. The species were identified with phylogenetic analyses in free applications from public websites. Results: ITS, BT2 and CAL, and ACT identified 5, 6, and 3 species and 3, 2, and 1 species complex of Aspergillus, respectively. DNA barcoding with multi-locus sequence typing revealed four new pathogens of human eye infection, including A. aureoterreus, A. pseudonomiae, A. siamensis, and A. flocculosus-ochraceopetaliformis. Conclusion: The DNA barcoding markers identified Aspergillus species with better specificity than the conventional method. The markers and techniques need to be used as tools for routine Aspergillus identification.

Peripheral Brain-Derived Neurotrophic Factor (BDNF) and Its Regulatory miRNAs as Biological Correlates of Impulsivity in Young Adults.

Background: Impulsivity assessment may serve as a valuable clinical tool in the stratification of suicide risk. Acting without forethought is a crucial feature in the psychopathology of many psychiatric disturbances and corresponds with suicidal ideations, behaviors, and attempts. Methods: We present data on biological and psychological correlates of impulsivity among young adults (n = 47). Psychological analysis included both the self-description questionnaire-Barratt Impulsiveness Scale (BIS-11)-and neuropsychological behavioral tests, including the Iowa Gambling Task (IGT), the Simple Response Time task (SRT), and the Continuous Performance Test (CPT). mRNA and micro-RNA were isolated from peripheral blood mononuclear cells (PBMC). Expression levels of Brain-Derived Neurotrophic Factor (BDNF) mRNA and its regulatory micro RNAs, mir-1-3p, mir-15a-5p, mir-26a-5p, mir-26b-5p, and mir-195-5p, were analyzed using the quantitative reverse transcription polymerase chain reaction (RT-qPCR) method. proBDNF and BDNF plasma protein levels were quantified using enzyme-linked immunosorbent assay (ELISA). Results: Significant correlations between BDNF mRNA and mir-15a-5p as well as proBDNF levels and mir-1-3p were detected. proBDNF protein levels correlated with motor and perseverance, while mir-26b correlated with cognitive complexity subdimensions of the BIS-11 scale. Correlations between BDNF, miRNAs, and the results of neuropsychological tests were also detected. Conclusions: The BDNF pathway shows a clinical potential in searching for biomarkers of impulse-control impairment. BDNF-regulatory micro-RNAs are detectable and related to clinical parameters in the studied population, which needs further research.

Enhancing Conventional PCR for Detection of Erwinia amylovora

Polymerase chain reaction (PCR) methods, including conventional PCR (cPCR) and quantitative real-time PCR (qRT-PCR), with both plasmid- and chromosome-targeting primers, are currently the most reliable methods for detecting &lt;i&gt;Erwinia amylovora&lt;/i&gt; due to their high sensitivity and specificity. Despite qRT-PCR’s quantitative advantage, cPCR remains an attractive method to detect this bacterium in initial screenings of suspected host plants, as it is cost-effective and does not require skilled personnel in well-equipped laboratories. This study aimed to significantly improve cPCR robustness via application of bovine serum albumin (BSA) as a PCR facilitator, with a modified EaF/R primer pair, as previously reported. Experiments have shown that simple supplementation with BSA (10 mg/ml) enhances cPCR reactions using templates such as genomic DNA, bacterial cells, and infected symptomless host organs, including immature apple fruits and seedlings, with EaF/R primers. The cPCR method described in this study is simple, specific, and reliable, and can be applied in routine assays to diagnose fire blight.

Development of a Multiplex Polymerase Chain Reaction Assay for Detecting Five Previously Unreported Papaya Viruses for Quarantine Purposes in Korea

There are concerns about the introduction and spread of plant pests and pathogens with globalization and climate change. As commercial control agents have not been developed for plant viruses, it is important to prevent virus spread. In this study, we developed a multiplex polymerase chain reaction (PCR) detection method to rapidly diagnose and control three DNA (papaya golden mosaic virus, Lindernia anagallis yellow vein virus, and melon chlorotic leaf curl virus) and two RNA (papaya leaf distortion mosaic virus and lettuce chlorosis virus) viruses that infect papaya. Specific primer sets were designed for the virus coat protein. Performing PCR, clear bands were observed with no non-specific reaction. Our multiplex PCR method can simultaneously detect small amounts of DNA/RNA to diagnose five viruses infecting papaya and prevent the spread of the virus.

Expression of c-jun/c-fos mRNA Indicates Persistent Myocardial Stretch During Asphyxia-Induced Cardiac Arrest.

Right ventricular dysfunction is a key clinical issue for the viability of donation-after-circulatory-death (DCD) heart transplantation. DCD hearts with volume overload have the potential to exhibit aggravated right ventricular dysfunction following heart transplantation. The c-jun/c-fos mRNAs are genes that immediately respond to myocardial cell stretch. We assessed myocardial cell stretch during asphyxia-induced cardiac arrest by measuring c-jun/c-fos mRNA expression levels. The trachea was dissected and ligated to initiate asphyxiation in anesthetized Wistar rats under paralyzed ventilation. The hearts were harvested at 4 time points: 0, 15, 30, and 45 minutes after the termination of ventilation. Free walls of the right and left ventricles and the interventricular septum were sectioned. Total RNA was extracted from these tissues, and cDNA was synthesized using reverse transcription. The c-jun/c-fos mRNA expression levels were quantified using the droplet digital polymerase chain reaction method. In the left ventricle, c-jun/c-fos expression levels rapidly increased at 15 minutes, but the expression levels returned to the baseline level at 30 minutes after tracheal ligation. In contrast, in the right ventricle, c-jun/c-fos expression levels gradually increased and peaked 30 minutes after tracheal ligation. Myocardial cell stretching in the right ventricle is prolonged after asphyxia-induced cardiac arrest compared to that in the left ventricle, which may lead to right ventricular dysfunction after DCD heart transplantation.

Propidium monoazide (PMA) qPCR assay compared to the plate count method for quantifying the growth of Salmonella enterica serotypes in vacuum-packaged turkey breast combined with a mathematical modeling approach

This study compares the plate count (PC) and the Propidium Monoazide-quantitative Polymerase Chain Reaction (PMA-qPCR) methods to assess the growth of a cocktail of three serotypes of Salmonella enterica (Heidelberg, Typhimurium, and Enteritidis) in cooked, sliced, and vacuum-packaged turkey breast (STB) under isothermal storage temperatures (8 °C–20 °C), using predictive models. Standard curves were developed for PMA-qPCR, demonstrating high efficiency (101%) and sensitivity, with quantification limits ranging from 1 to 2 log10 CFU/g for all temperatures studied. Comparative analysis revealed a significant correlation (R2 = 0.99; 95% CI) between the PC and PMA-qPCR methods; however, the agreement analysis indicated a mean difference (Bias) of −0.11 log10 CFU/g (p < 0.05), suggesting underestimation by the PC method. This indicates the presence of stressed or viable but nonculturable (VBNC) cells, detectable by PMA-qPCR but not by PC. The Baranyi and Roberts model showed a good ability to describe the behavior of S. enterica cocktail in STB for PC and PMA-qPCR data under all isothermal conditions. The exponential secondary model more accurately represented the temperature dependence of the maximum specific growth rate compared to the Ratkowsky square root model, with R2 values ≥ 0.984 and RMSE values ≤ 0.011 for both methods. These results suggest that combining PMA-qPCR with predictive modeling allows for a more accurate prediction of S. enterica growth, compared to PC method. In the event of cold chain disruptions of meat products, the use of PMA-qPCR method allow the quantification of VBNC cells, that can still pose a health risk to consumers, especially in ready-to-eat products.

Comparative assessment of acute neurotoxicity of real-world ultra-fine black carbon emitted from residential solid fuel combustion

Incomplete combustion of residential solid fuel is one of the main anthropogenic sources for black carbon (BC). Fresh BC, mainly enriched in ultra-fine fraction of particles, can directly cross blood-brain barrier and are reported to be associated with neurodegenerative diseases. Because of the difficulties in collection and purification of BC from ambient particles, there are still significant knowledge gaps in understanding neurotoxicity caused by real-world BC. The purpose of this study is to compare the neurotoxic effects caused by BCs emitted from combustion of six residential solid fuels, and try to reveal associated biological mechanisms in SH-SY5Y cells. Two straw BC (Wheat-BC and Corn-BC) showed highest neurotoxic effects followed by wood BC (Pine-BC and Aspen-BC) and coal BC (Xvzhou and Longkou Coal), as indicated by viability, lactic dehydrogenase, malondialdehyde, adenosine triphosphate and acetylcholine levels. Coal BC caused nearly no toxicity in human neuroblastoma (SH-SY5Y) cells within highest dose of 200 μg/mL. RNA sequence and bioinformatics analysis were applied to effectively identify differential genes and signaling pathways. Based on Gene Ontology (GO) annotation, Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment, Protein Protein Interaction network (PPI network) construction, we found biomass BC affected mitochondrial function, interfered with cellular metabolic processes, disturbed redox homeostasis, and finally resulted in cellular damages. Coal-BC mainly caused cytokine/chemokine related inflammatory responses. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting methods were further applied to find out related signaling pathways. Biomass BC activated IL6R/JAK3/STAT3 and JAK3/STAT6 pathways leading to oxidative stress and inflammatory responses. Coal BC activated JAK3/STAT3 pathway leading to chemokine related responses. This study revealed the heterogeneity in neurotoxicity of BCs from different combustion sources and provided important data for health risk assessment. BC-related neurotoxicity should be considered when making air pollution emission control strategies, with residential biomass receiving more policy attention.