Polymerase Chain Reaction Method Research Articles

Development and Validation of In-House Conventional and Multiplex PCR Methods for the Detection and Identification of Lophomonas spp.: An Innovative Approach.

Pulmonary lophomoniasis is an emerging disease caused by the protozoan pathogen Lophomonas spp. Recently, a conventional polymerase chain reaction (PCR) method has been developed. However, its sensitivity and specificity remain to be fully established. Therefore, this study aimed to develop in-house conventional and multiplex PCR for the detection and identification of Lophomonas infections. Additionally, we attempted to compare the diagnostic performance of these novel PCR tests with the current microscopic examination method using BAL samples. We studied 120 bronchoalveolar lavage (BAL) specimens of the patients clinically suspected of having lophomoniasis. The specimens were examined using three methods: microscopic examination (Giemsa staining), in-house conventional PCR, and multiplex-PCR. Moreover, multiplex-PCR was used for the simultaneous identification of two species of Lophomonas. Out of the 120 BAL specimens tested, 30 (25%) tested positive through microscopic wet mount examination. Among the three techniques, multiplex-PCR was the most sensitive (100%, 95% CI, 88.3-100), while Giemsa staining had the lowest sensitivity (86.2%, 95% CI, 69.4-94.5). The data reveal a strong agreement between multiplex-PCR and conventional PCR (κ = 0.96), while the lowest agreement was found between multiplex-PCR and microscopy methods (κ = 0.16). The study also confirmed the presence of L. blattarum species in all samples using multiplex-PCR. This study demonstrates that the in-house multiplex-PCR is a robust and accurate diagnostic test for the detection and identification of Lophomonas species. Therefore, our findings suggest that this method may be a powerful tool to overcome some diagnostic pitfalls for lophomoniasis.

The Impact of Orthodontic Adhesives on the Subgingival Microbiota During Early Fixed Appliance Therapy: A Pilot Study

Background and Objectives: The most common method for treating malocclusions today is fixed orthodontic therapy, during which brackets and tubes are bonded to the surface of the teeth, which makes oral hygiene difficult to maintain. Increased plaque retention, gingival bleeding, and gingivitis can be diagnosed in the early phases of treatment. The periodontal response to plaque accumulation can be explained by quantitative and qualitative changes in the subgingival microbiota. The aim of our research was to investigate the changes in the subgingival microbiota that occurred within 6–8 weeks after bonding when two different orthodontic adhesives were used. Materials and Methods: Thirty patients were followed; molar tubes were bonded with a composite (C) in fifteen cases, and in the other fifteen cases, they were bonded with glass ionomer cementum (GIC). A microbiological sample was taken from the gingival sulcus of the maxillary first molars at the time of appliance placement (T1) and 6–8 weeks (T2) after bonding. Bacterial DNA detection was performed using the micro-IDent®plus11 (Hain Lifescience GmbH, Germany) PCR (polymerase chain reaction) method. The statistical analysis included McNemar’s test to analyze the paired binary data and Fisher’s Exact Test to compare the prevalence of each of the 11 bacteria at T1 and, ultimately, T2 between the two studied groups. The Bonferroni correction was also applied. Results: When analyzing GIC vs. C at T1 and T2, none of the studied pathogens showed significant differences. Conclusions: Given the lack of statistical significance, these trends do not confirm a definitive impact of the procedure on bacterial presence. The increased presence of periodontal pathogens might suggest that bonding does not reduce the bacterial loading of subgingival microbiota. Less protective effects of the GIC intervention against Tannerella forsythia and Eubacterium nodatum bacteria were detected.

PCR targeting 16S rRNA, cfb, and hylB for group B Streptococcus detection in women with 35 to 37 weeks of pregnancy.

Polymerase chain reaction (PCR) is a rapid molecular technique for pathogen detection, but its application in detecting group B Streptococcus (GBS) requires further exploration. This study assessed the diagnostic performance of the PCR method for GBS detection in pregnant women with 35 to 37 weeks of pregnancy. This study enrolled 116 women at 35-37 weeks of pregnancy and simultaneously collected two vaginal swab samples for GBS detection via culture and PCR methods. Through the culture method, 21 (18.1%) samples were positive for GBS. Moreover, 24 (20.7%), 23 (19.8%), and 23 (19.8%) samples were positive for GBS according to PCR methods targeting 16S rRNA, cfb, and hylB, respectively. The results of PCR-based GBS detection of 16S rRNA (Kappa value = 0.917, p < 0.01), cfb (Kappa value = 0.944, p < 0.01), and hylB (Kappa value = 0.944, p < 0.01) were consistent with those of the culture method. The sensitivity and specificity were 1.000 and 0.968 for the PCR method targeting 16S rRNA, 1.000 and 0.979 for cfb, and 1.000 and 0.979 for hylB, respectively. PCR targeting 16S rRNA, cfb, and hylB may serve as a reliable diagnostic method for GBS detection in women with 35 to 37 weeks of pregnancy.

CRISPR‐Based Environmental Biosurveillance Assisted via Artificial Intelligence Design of Guide‐RNAs

ABSTRACTEnvironmental biosecurity challenges are intensifying as climate change and human activities accelerate the spread of invasive species, disrupting ecosystem composition, function, and essential services. Environmental DNA (eDNA) has transformed traditional biosurveillance by detecting trace DNA fragments left by organisms in their surroundings, primarily by applying quantitative polymerase chain reaction (qPCR) methods. However, qPCR presents challenges, including limited portability, reliance on precise thermal cycling, and susceptibility to inhibitors. To address these challenges and enable field‐deployable monitoring, isothermal amplification techniques such as recombinase polymerase amplification (RPA) paired with clustered regularly interspaced short palindromic repeats and associated proteins (CRISPR‐Cas) have been proposed as promising alternatives. CRISPR‐Cas technology also presents challenges, including searching and optimizing a guide RNA (gRNA) that is highly sensitive and has no off‐target interactions for use as an effective environmental biosurveillance tool. We present here the development of SENTINEL (Smart Environmental Nucleic‐acid Tracking using Inference from Neural‐networks for Early‐warning Localization) that harnesses the programmability, specificity and sensitivity of a one‐pot RPA‐CRISPR‐Cas13a reaction by integrating an accessible and pre‐trained neural network to accelerate assay design for rapid deployment. We challenged SENTINEL with waterborne eDNA from two marine sites invaded by species not native to New Zealand as proof‐of‐concept fluorescence‐based tests: Sabella spallanzanii (Mediterranean fanworm) and Undaria pinnatifida (Wakame). Off‐target effects were explored by challenging the SENTINEL assays on gDNA from a suite of co‐occurring species. SENTINEL presented a robust, streamlined method incorporating the trained neural network, achieving a sensitivity down to 10 attomolar using recombinant DNA and down to ~0.34 copies/μL for eDNA samples with 1 h, costing 3.5 USD per sample. There was a 100% agreement between SENTINEL results and qPCR‐based analysis of the eDNA samples. SENTINEL displayed no off‐target activity when challenged against 23 gDNA samples from co‐occurring species. Thus, our study showcases SENTINEL's potential as a robust platform for eDNA screening applications.

Assistance in Mastering PCR Methods for Olympiad Students at MAN Insan Cendekia in Kendari City

Madrasah Aliyah Negeri Insan Cendekia (MAN IC) in Kendari City is one of the OSN (National Science Olympiad) participants in the subject of biology, which is required to understand the idea and practice of PCR (Polymerase Chain Reaction). However, there are insufficient skilled PCR teachers at the school, and no PCR facilities are currently accessible. The goal of this mentoring is to provide an in-depth understanding of the concept of PCR as well as full training on how to use PCR effectively. The method consists of two components: community education and training. The introduction of the PCR concept in this community service includes genetic material, DNA concepts, genes, genomes, DNA isolation, and DNA replication, while the training material includes solution taking techniques, PCR solution preparation, PCR usage procedures, PCR result visualization with electrophoresis, and data interpretation. The PCR has three main steps consisting of denaturation, annealing and extension which are carried out for 30-35 cycles with different temperatures at each step. The evaluation results showed that this mentoring helped the student understand PCR and was beneficial for the OSN competition due to its relevance to the competition syllabus. Moreover, the student also received a bronze medal in the biology competition.

Development and optimization of multiplex PCR for rapid detection of type I-F1 and type I-F2 Cas cluster genes in Acinetobacter baumannii.

Development and optimization of multiplex PCR for rapid detection of type I-F1 and type I-F2 Cas cluster genes in Acinetobacter baumannii.

SNP Variety of UMOD Gene in Patients with Hypertension Disease in Hilla Province

A major risk factor for cardiovascular illnesses, hypertension—also known as high blood pressure—is shaped by both environmental and hereditary elements. Recent research have shown several genetic variations linked to hypertension, among which the rs13333226 variant found in the Uromodulin gene (UMOD). This paper explores the biological relevance of Uromodulin, consequences of the rs13333226 variation, and possible function in hypertension. Fifty blood samples in all came from diabetic patients visiting the Diabetes Center at Merjan Teaching Hospital in Babylon, Iraq. Five samples in all were also gathered to act as a control group. PCR magnificues DNA taken from blood samples. The SOD2 gene was genotyped using a polymerase chain reaction (PCR) method then applying the DNA single-stranded conformation polymorphism (SSCP) method. The single-stranded conformation polymorphism (SSCP) technique thus verified the genuineness of these DNA polymorphisms. The findings show several haplotypes seen in SOD2. The findings showed that in the sick group the DNA polymorphism distribution was 84% and 16%; in the control group it was 70% and 30%. Understanding hypertension depends much on the rs13333226 variation on the Uromodulin gene. Clarifying the link between this variation and blood pressure control would help scientists open the path for better risk evaluation and focused treatments in the control of hypertension. Complete knowledge of the consequences of this variation and its possible influence in cardiovascular health depends on ongoing research.

Applications of Bacterial Biotechnology in the Bioremediation of Water Contaminated with Heavy Metals

Background: Heavy metals contaminated soil and water samples were collected from the Tigris River Power Plant, Baghdad. The bacterial isolation was performed by using the culturing technique on proper nutrient media prepared with low a concentration of heavy metals such as lead and mercury. The isolated strains were tested for their growth capability in heavy metal-polluted media by the polymerase chain reaction method for the identification of genes that provide resistance or degradation of heavy metals. Isolation and determination of heavy metal-resistant bacterial strains from genera like Pseudomonas, Bacillus and Streptomyces, which showed high tolerance to lead and cadmium. Methods: The aim to analyze environmental factors like pH, temperature, carbon sources and nitrate that would affect bacterial growth. Efficiency of bacteria in the removal of heavy metals from contaminated water was also determined by atomic absorption spectroscopy by comparing the concentration of metals before and after treatment. Result: The study showed that significant soil and water contamination was present around the power plant area, emanating from industrial activity: lead concentration ranged from 100-500 ppm and cadmium from 5-30 ppm in soil. Water concentrations were above the safety limits set by WHO for lead and cadmium, ranging between 0.1-1 ppm and 0.01-0.1 ppm, respectively, showing slight decreases after discharge into the Tigris River but remaining above natural levels. Some of the bacterial strains, like Pseudomonas, Bacillus and Streptomyces, which are resistant to heavy metals (Pb: 50-100 ppm and Cd: 5-20 ppm), were isolated. Genetic detection identified genes that code for resistance, including czcA and merA; however, it lacked the gene pbrT. Thus, the capability for lead removal is very limited.

Inhibition of LPL suppresses the osteoclast differentiation of bone-marrow-derived macrophages by promoting the ACSL4 ubiquitination.

Inhibition of LPL suppresses the osteoclast differentiation of bone-marrow-derived macrophages by promoting the ACSL4 ubiquitination.

Investigation of the Frequency of Cryptosporidium spp. in Immunosuppressed Patients with Diarrhea.

We aimed to investigate the frequency of Cryptosporidium spp. in immunosuppressed patients who were admitted with diarrhea by microscopic, serological, and molecular methods and to evaluate the results in comparison with the direct fluorescent antibody (DFA) method. We analyzed 90 stool samples from immunosuppressed patients with diarrhea. All stool samples were examined using modified acid-fast staining, polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) tests for the detection of Cryptosporidium spp. samples examined with the DFA method were selected randomly, including samples found positive in other diagnostic tests. Stool samples that were positive in any of these diagnostic tests were evaluated by immunochromatographic card test. Our study included 90 samples, of which 44 (48.8%) and 46 (51.2%) were from male and female patients, respectively. The mean age of the patients was 37.8±27.5. Thirty-one (34.4%) of the samples were from pediatric patients and their mean age was 5.3±4.34. The methods used did not show a statistically significant difference between the positivity status and patient age and gender (p>0.05). Cryptosporidium spp. positivity was detected in five samples (5.6%), two samples (2.2%), and one sample (1.1%) by using the modified acid-fast staining, PCR, and ELISA methods, respectively. Six of 51 samples to which the DFA method was applied were detected positive. In 4 of the 7 stool samples that were found positive by any of these methods, Cryptosporidium spp. was detected positive by the immunochromatographic card test. When the DFA method is accepted as the gold standard method in the diagnosis of Cryptosporidium spp., the modified acid-fast method can be used in routine diagnosis with high sensitivity and specificity. Besides, it immunochromatographic diagnostic tests thought to be useful in laboratories with intensive workflow due to their practical use and rapid results.

Identification of the Parasitoid Wasp Dinocampus coccinellae (Schrank) (Hymenoptera: Braconidae) in Pistachio Orchards Using DNA Barcoding

Identification of Dinocampus coccinellae (Schrank) (Hymenoptera: Braconidae), a parasitoid of various species within the Coccinellidae family, is essential for the development of effective biological control strategies in pistachio orchards. This study aimed to accurately identify D. coccinellae utilizing DNA barcoding techniques. Research was conducted throughout 2023 on coccinellid specimens collected regularly from pistachio orchards in Siirt. Among the collected Coccinellidae species, D. coccinellae DNA was successfully identified in the Hippodamia variegata specimen among a total of 11 individuals. The mitochondrial COI gene region of D. coccinellae was analyzed by DNA barcoding analysis using Polymerase Chain Reaction (PCR) and sequencing methods. PCR results and DNA sequencing confirmed the accurate identification of D. coccinellae, demonstrating high similarity with reference sequences in the NCBI database. The sequencing results exhibited high accuracy and reliability, with a similarity rate of 97.13% and an E-value of 0.0. Phylogenetic relationships were assessed using the Maximum Likelihood (ML) method. Phylogenetic analyses clearly illustrated the relationships of D. coccinellae with other Hymenoptera species. The phylogenetic tree indicated that D. coccinellae is most closely related to Meteorus obfuscatus, with a genetic distance of 0.10, providing insights into interspecies relationships.

Development of a rapid diagnostic method for Dickeya dadantii, the bacterial canker pathogen of peach, using loop-mediated isothermal amplification (LAMP).

Given that accurate diagnosis of bacterial canker in peach trees that cause rapid death is difficult based on disease symptoms alone, a rapid genetic diagnosis technique is required. In this study, we aimed to develop a rapid and efficient method to identify Dickeya dadantii, the causative agent, using the loop-mediated isothermal amplification (LAMP) method. Two types of DNA were extracted from the thin cambium slices of peach tree trunks and branches. Purified DNA was obtained using a commercial kit. Crude DNA was boiled and diluted with the Tris-HCl buffer. Then, 119 peach tree cambium samples were analyzed to detect D. dadantii using the previously reported polymerase chain reaction method and newly designed LAMP primers. Notably, LAMP exhibited equivalent or superior positivity rates compared to polymerase chain reaction for both DNA types, particularly crude DNA. Additionally, D. dadantii was detected in the resin formed on the peach tree trunks and branches. Overall, the developed method shows potential for the non-invasive diagnosis of infections and mitigation of infection risk for effective fruit tree management.

Prevalence, Antimicrobial Resistance Patterns, and Emerging Carbapenemase-Producing Enterococcus Species from Different Sources in Lagos, Nigeria.

Background: Enterococcus species present significant health risks due to their widespread presence in humans, animals, and the environment. This study examined the patterns of antimicrobial resistance (AMR) and the presence of carbapenemase-producing Enterococcus species from various sources. Methods: Between November 2023 and February 2024, 500 samples were collected in Lagos State, including 350 clinical human samples, 50 environmental samples, and 100 animal samples. The samples were processed, and Enterococcus isolates were identified and subjected to antimicrobial susceptibility tests (AST) by standard methods. Furthermore, carbapenemase (blaKPC and oxa-48) and virulence genes (gelE) were detected by real-time polymerase chain reaction (RT-PCR) methods using specific primers. Results: The overall prevalence of Enterococcus isolates was 4.6% (23/500), including 18 E. faecalis and 5 E. faecium. The source prevalence was 24% (12/50) from the environmental samples, 5% (5/100) from animal sources, and 1.7% (6/350) from the clinical samples. All Enterococcus isolates were 100% resistant to ciprofloxacin, erythromycin, imipenem, vancomycin, and ampicillin. However, 91% were susceptible to gentamicin. Six (6) distinct resistance profiles were observed, with the pattern AMP-ERY-TGC-CIP-TS-VA-CHL-AUG-MEM-IMI being the most frequent in 12 E. faecalis (4 isolates from humans, 2 from animals, and 6 from the environment). Notably, 39.1% (9/23) of multiple-drug resistant Enterococcus isolates harbored the gelE virulence gene, including seven E. faecalis (five environmental and two human) and two E. faecium from animal sources. The E. faecalis strains HB003 and HB050, from human bacteremia cases carrying gelE, were the first in Nigeria to produce blaKPC and oxa-48 carbapenemase genes. Conclusions: This study revealed the emergence of carbapenemase-producing Enterococcus species in our environment. A one-health approach and further molecular studies are essential to mitigate the spread and understand the transmission dynamics.

A longitudinal study assessing Aspergillus polymerase chain reaction (PCR) performance through participation in external quality assessment schemes from 2019 to 2022.

Aspergillus polymerase chain reaction (PCR) now employs standardised methodology and commercial options, with clinical performance validated through meta-analyses. The long-term availability of external quality assessment schemes drives performance and provides a currently untapped source of technical data. The Fungal PCR initiative and the Quality Control for Molecular Diagnostics (QCMD) combined forces to evaluate data from the Aspergillus PCR QCMD schemes between 2019 and 2022. Analysis showed inferior detection of non-fumigatus Aspergillus species (i.e.,Aspergillus niger) compared with A. fumigatus. Longitudinal evaluation showed the use of 'in-house' PCR methods by new participants when testing plasma generated lower positivity rates compared to commercial assays.

Utility of hub and spoke model for estimating the burden of Group B Streptococcus infection and its antibiotic resistance profiling among pregnant tribal women

Objectives Group B Streptococcus (GBS) is the leading cause of maternal sepsis and neonatal meningitis. The objective is to determine the feasibility of using the hub-spoke model for the collection and transportation of recto-vaginal swab samples was evaluated in addition to the GBS burden among tribal females, convenient diagnostic method and antibiotic susceptibility profiling. Materials and Methods Recto-vaginal samples were collected from the 35–37 weeks pregnant women using pre-sterile HiCulture transport swab and were transferred into Lim Broth. Direct plating was carried out by inoculating swabs onto chromogenic agar. Positive colonies were subjected to antibiotic susceptibility testing using the Kirby–Bauer disc diffusion method. Polymerase chain reaction (PCR) was also performed. Statistical analysis Statistical tests were performed using Microsoft excel. P &lt; 0.05 was considered statistically significant. Results The socio-demographic and clinical profiling of the 808 pregnant females suggested vaginal discharge (89.23%) to be the most common symptom at the time of presentation. GBS was detected in 116 (14.35%) and 52 (6.43%) by PCR and culture-based methods, respectively. The sensitivity and specificity of the PCR assay were estimated to be 100% and 91.53%, respectively. Obtained GBS isolates were found to be 100% sensitive to quinupristin – dalfopristin, linezolid, cefepime, and vancomycin, whereas 10%, 23%, 27%, and 23% of the isolates were found to be resistant for penicillin, clindamycin, erythromycin, and levofloxacin, respectively. Conclusions Hub-spoke model for GBS screening must be emphasized in rural settings to identify pregnant women requiring prophylactic antibiotic therapy. PCR could be used as an efficient tool for identifying GBS.

MiR-204-5p promoted maternally expressed gene 3 (MEG3) through SP1-mediated DNMT1 pathway in trophoblast cells in recurrent miscarriage.

Recurrent miscarriage (RM) affects 1-2% of couples. Maternally expressed gene 3 (MEG3) is aberrantly expressed in RM patients. To investigate a novel regulatory mechanism, we examined the miR-204-5p/Specificity protein 1 (SP1)/DNA methyltransferase 1 (DNMT1)/MEG3 axis in the trophoblast cell line HTR-8/SVneo. Human trophoblast cell line HTR-8/SVneo was used and cells were transfected with siRNA targeting SP1, miR-204-5p mimics, pcDNA3.1-DNMT1, or their negative controls (NCs). The methylation inhibitor, 5-azadC, was used to treat the cells transfected with pcDNA3.1-SP1. The reverse transcription quantitative polymerase chain reaction (RT-qPCR) method was used to examine the relative RNA levels of SP1, DNMT1 and MEG3. Western blot assay was performed to measure the protein levels of SP1 and DNMT1. The dual-luciferase reporter gene assay was used to validate the miR-204-5p bindings to SP1. Functional assays were utilized to assess cell apoptosis, colony formation, migration, and invasion. SP1 knockdown inhibited DNMT1 and increased MEG3 expression. The expression of MEG3 was enhanced by methylation inhibition through 5-azadC, but SP1 upregulation reversed this effect. SP1 knockdown increased apoptosis and decreased migration and invasion, which was reversed by DNMT1 overexpression. SP1 was targeted and inhibited by miR-204-5p. miR-204-5p also inhibited DNMT1, and enhanced the expression of MEG3. miR-204-5p inhibited cell proliferation, migration and invasion, and promoted apoptosis. Overexpression of SP1 partially reversed these effects by activating DNMT1 and inhibiting MEG3. miR-204-5p promoted MEG3 expression in trophoblast cells via SP1-mediated DNMT1 inhibition, leading to reduced cell migration, proliferation and invasion, as well as increased apoptosis. This study reveals a novel regulatory axis in trophoblast cells, providing insights into potential regulatory mechanisms in RM.

Rapid and Sensitive Detection of RNA Viruses through Imaging of Marker Molecules Derived from Designed Circular DNA Probes.

In the absence of treatment, detection of pathogenic RNA viruses and quarantine of infected individuals are critical for controlling the infection spread. Conventional antibody-based methods often lack the required accuracy and sensitivity, due to the need for a substantial amount of viral antigens. Similarly, traditional reverse transcription real-time polymerase chain reaction methods face challenges in providing timely results due to their multiple thermal nucleic acid amplification steps. To overcome these limitations, a new method based on imaging of virus-specific DNA markers is developed. This approach employs specially designed single-stranded circular DNA probes that capture virus-derived RNA fragments generated by RNase digestion of the viral genome. These fragments serve as primers for a subsequent single-step DNA filling reaction, producing double-stranded virus-specific marker molecules. These individual markers are recognized through fluorescence imaging following linearization by enzyme cleavage and subsequent fluorescence staining. This method can detect viruses at a genome equivalent level of 14 within 40 min. In addition, the molecule-level imaging-based method effectively detects human immunodeficiency virus-1 in clinical samples. This diagnostic approach does not require sophisticated thermal controls nor extensive nucleic acid amplifications, allowing for accurate, sensitive, and rapid detection without the need for large equipment, offering substantial potential for point-of-care applications.

Evaluation of the Effects of Thymoquinone on RAGE/NOX4 Expressions and Brain Tissue Morphometry in Experimental Alzheimer's Disease Induced by Amyloid Beta 1-42 Peptide.

The onset of Alzheimer's disease (AD) is attributed to widespread amyloid beta (Aβ) plaque accumulation, tau hyperphosphorylation, oxidative stress, and neuroinflammation. However, the underlying mechanism of AD remains unclear, and no curative treatment currently exists. The aim was to investigate the effect of thymoquinone by suppressing the RAGE/NOX4 pathway in AD. Mice (n = 60) were divided into five groups, and an experimental AD model induced by an Aβ1-42 peptide was established in two groups. We also administered 5 mg/kg thymoquinone (TMQ) to the mice for its properties to slow or treat neurodegeneration in AD. Behavioral tests for memory and emotional states, micro-computed tomography (Micro CT) to assess brain volume, ELISA to measure malondialdehyde (MDA) levels, hematoxylin and eosin staining (H&E) to evaluate neuronal degeneration were used. Immunohistochemical (IHC), Western blot (WB), and real-time polymerase chain reaction (PCR) methods were used to evaluate the inhibitory effect of TMQ on a receptor for advanced glycation end products (RAGE)/nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4 (NOX4) signaling in AD. The results showed that TMQ may have ameliorative effects on memory, spatial learning, learning ability, and anxiety in AD. We showed that TMQ has an antioxidative effect by decreasing MDA levels by the ELSIA method (p < 0.05). There was a marked increase in neuronal degeneration in AD mice compared to other groups (p < 0.05). We concluded that TMQ could ameliorate neuronal degeneration in AD by H&E staining and suppress RAGE/NOX4 signaling by IHC and WB analysis. We concluded that TMQ could be therapeutic in AD by reducing AB expression level by IHC analysis (p < 0.05). Real-time PCR analysis showed that APP (p < 0.05), RAGE, and NOX4 (p < 0.05) gene expressions could be reduced by TMQ. In conclusion, TMQ has a high therapeutic potential in AD and an effective preventive and therapeutic strategy can be developed with more comprehensive studies on TMQ.

Specific detection of genetically modified potatoes containing asparagine synthetase-1 and polyphenol oxidase 5 genes derived from potato

ABSTRACT Several genetically modified (GM) potatoes have been developed by introducing endogenous genes derived from potatoes, such as asparagine synthetase-1 (Asn1) and polyphenol oxidase 5 (Ppo5), to improve quality. Therefore, it is difficult to distinguish between GM and non-GM potatoes. In this study, we developed a sequence-specific polymerase chain reaction (PCR) detection method to identify innate and inserted genes. We designed four Asn1 gene-specific primers and eight construct-specific detection primers to evaluate GM potatoes (E12, X17, and Y9) and non-GM crops. Consequently, PCR products corresponding to the original endogenous potato genes and the inserted genes were clearly distinguished and simultaneously identified. In addition, the PCR method demonstrated sufficient sensitivity to identify GM content at levels as low as 0.5%. Thus, this study offers an effective detection method for monitoring or screening approved and unapproved GM potato events using Asn1 and Ppo5 transgenes in foods and feeds.

Frequency, socio-economic characteristics, and risk factors of oral cavity parasites in diabetes mellitus patients from Lorestan Province, Iran; a case-control study.

Numerous studies identify diabetes mellitus (DM) as one of the most significant risk factors for the development of periodontal diseases (gum diseases). Individuals with diabetes experience gingival destruction more rapidly and severely due to the accumulation of microbial plaque in the mouth. Entamoeba gingivalis and Trichomonas tenax are parasites commonly found in the human oral cavity. This study aims to determine, the frequency, socio-economic characteristics, and risk factors of E. gingivalis and T.tenax in DM patients from Lorestan Province, Iran as a case-control study. The current case-control study involved 500 DM patients who were referred to health centers in Lorestan province, Iran between December 2022 and June 2024. Furthermore, a control group comprising 500 healthy persons without DM (non-DM) who were referred to health centers during the same study period was incorporated into the research. The prevalence of parasites in the oral cavity was determined using microscopic analysis and conventional polymerase chain reaction (PCR) techniques. A questionnaire was administered to collect demographic information, including age, gender, place of residence, education level, occupation, monthly income, tooth brushing practices, and mouthwash usage. Out of a total of 500 DM patients, 136 (27.2%) and 146 (29.2%) patients had the oral cavity parasites (E. gingivalis and T. tenax) by microscopic and PCR analysis, respectively. While, in non-DM, 61 (12.2%) and 65 (13.0%) tested positive for parasites using microscopic and PCR methods, respectively (P<0.001). Among several factors, income (P = 0.001, OR = 5.491, 95% CI: 4.089 to 9.723), place of residence (P = 0.006, OR = 1.982, 95% CI: 1.222), education (P = 0.002, OR = 3.577 (1.618, 5.907)), and use mouthwash demonstrated a significant protective effect on the oral cavity parasites. This research for the first time in Iran highlighted a considerable prevalence of oral cavity parasites in DM patients in Lorestan province, Western Iran. Dental professionals should maintain a heightened awareness of these risk factors to effectively identify and address oral health challenges within this population, thereby reducing the incidence of oral diseases and infections.