Real-time Polymerase Chain Reaction Approach Research Articles

Comparative Analysis of Gene Expression at Sea Level and High Altitude: A Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Approach

Comparative Analysis of Gene Expression at Sea Level and High Altitude: A Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Approach

Hsa_circ_0000190 Promotes NSCLC Cell Resistance to Cisplatin via the Modulation of the miR-1253/IL-6 Axis.

This study explored the mechanistic basis for nonsmall cell lung cancer (NSCLC) cisplatin (DDP) treatment resistance in an effort to define effective approaches to abrogating the emergence of such chemoresistance. Analyses of NSCLC expression of hsa_circ_0000190, miR-1253, and interleukin 6 (IL-6) were conducted via a quantitative real-time polymerase chain reaction (qPCR) approach, while the ability of these tumor cells to resist DDP treatment was evaluated with a CCK-8 assay. Interactions between different RNA molecules were assessed using both RNA immunoprecipitation and dual-luciferase reporter assays. NSCLC cell lines and tissues resistant to DDP were found to express higher levels of hsa_circ_0000190, and knocking down this circRNA in NSCLC cells was associated with greater sensitivity to DDP exposure. Further research identified miR-1253 as a hsa_circ_0000190 target, with the ability of hsa_circ_0000190 knockdown to restore DDP sensitivity being largely attributable to the ability of this circRNA to suppress miR-1253 activity. IL-6 was identified as a major miR-1253 target in this context, with miR-1253 regulating chemoresistance in NSCLC cells in part by preventing IL-6 upregulation. Together, these data suggest that hsa_circ_0000190 can promote DDP chemoresistance in NSCLC cells through its ability to modulate miR-1253/IL-6 axis activity, highlighting a novel pathway that can be targeted in an effort to guide the more effective diagnosis and management of DDP-resistant tumors.

An efficient screening system of disease-resistant genes from wild apple, Malus sieversii in response to Valsa mali pathogenic fungus

For molecular breeding of future apples, wild apple (Malus sieversii), the primary progenitor of domesticated apples, provides abundant genetic diversity and disease-resistance traits. Valsa canker (caused by the fungal pathogen Valsa mali) poses a major threat to wild apple population as well as to cultivated apple production in China. In the present study, we developed an efficient system for screening disease-resistant genes of M. sieversii in response to V. mali. An optimal agrobacterium-mediated transient transformation of M. sieversii was first used to manipulate in situ the expression of candidate genes. After that, the pathogen V. mali was inoculated on transformed leaves and stems, and 3 additional methods for slower disease courses were developed for V. mali inoculation. To identify the resistant genes, a series of experiments were performed including morphological (incidence, lesion area/length, fungal biomass), physiological (H2O2 content, malondialdehyde content), and molecular (Real-time quantitative Polymerase Chain Reaction) approaches. Using the optimized system, we identified two transcription factors with high resistance to V. mali, MsbHLH41 and MsEIL3. Furthermore, 35 and 45 downstream genes of MsbHLH41 and MsEIL3 were identified by screening the V. mali response gene database in M. sieversii, respectively. Overall, these results indicate that the disease-resistant gene screening system has a wide range of applications for identifying resistant genes and exploring their immune regulatory networks.

Skeletal muscle-specific DJ-1 ablation-induced atrogenes expression and mitochondrial dysfunction contributing to muscular atrophy.

DJ-1 is a causative gene for Parkinson's disease. DJ-1-deficient mice develop gait-associated progressive behavioural abnormalities and hypoactive forearm grip strength. However, underlying activity mechanisms are not fully explored. Western blotting and quantitative real-time polymerase chain reaction approaches were adopted to analyse DJ-1 expression in skeletal muscle from aged humans or mice and compared with young subjects. Skeletal muscle-specific-DJ-1 knockout (MDKO) mice were generated, followed by an assessment of the physical activity phenotypes (grip strength, maximal load capacity, and hanging, rotarod, and exercise capacity tests) of the MDKO and control mice on the chow diet. Muscular atrophy phenotypes (cross-sectional area and fibre types) were determined by imaging and quantitative real-time polymerase chain reaction. Mitochondrial function and skeletal muscle morphology were evaluated by oxygen consumption rate and electron microscopy, respectively. Tail suspension was applied to address disuse atrophy. RNA-seq analysis was performed to indicate molecular changes in muscles with DJ-1 ablation. Dual-luciferase reporter assays were employed to identify the promoter region of Trim63 and Fbxo32 genes, which were indirectly regulated by DJ-1 via the FoxO1 pathway. Cytoplasmic and nuclear fractions of DJ-1-deleted muscle cells were analysed by western blotting. Compound 23 was administered into the gastrocnemius muscle to mimic the of DJ-1 deletion effects. DJ-1 expression decreased in atrophied muscles of aged human (young men, n=2; old with aged men, n=2; young women, n=2; old with aged women, n=2) and immobilization mice (n=6, P<0.01). MDKO mice exhibited no body weight difference compared with control mice on the chow diet (Flox, n=8; MDKO, n=9). DJ-1-deficient muscles were slightly dystrophic (Flox, n=7; MDKO, n=8; P<0.05), with impaired physical activities and oxidative capacity (n=8, P<0.01). In disuse-atrophic conditions, MDKO mice showed smaller cross-sectional area (n=5, P<0.01) and more central nuclei than control mice (Flox, n=7; MDKO, n=6; P<0.05), without alteration in muscle fibre types (Flox, n=6; MDKO, n=7). Biochemical analysis indicated that reduced mitochondrial function and upregulated of atrogenes induced these changes. Furthermore, RNA-seq analysis revealed enhanced activity of the FoxO1 signalling pathway in DJ-1-ablated muscles, which was responsible for the induction of atrogenes. Finally, compound 23 (an inhibitor of DJ-1) could mimic the effects of DJ-1 ablation in vivo. Our results illuminate the crucial of skeletal muscle DJ-1 in the regulation of catabolic signals from mechanical stimulation, providing a therapeutic target for muscle wasting diseases.

Phenotypic and genotypic characterization of erythromycin-resistant Staphylococcus aureus isolated from bovine subclinical mastitis in Egypt.

Subclinical mastitis (SCM) caused by erythromycin-resistant Staphylococcus aureus is a significant disease in lactating animals. Therefore, it is crucial to understand the genetic factors contributing to erythromycin resistance in S. aureus. This study aimed to estimate the prevalence of S. aureus in milk from subclinical mastitic cattle and buffaloes and tank milk samples as identified by probe-based real-time polymerase chain reaction (PCR) and the genotypic assessment of macrolide and erythromycin resistance profiles, as well as to analyze the phylogenetic relatedness of our local isolates of S. aureus. In total, 285 milk samples were analyzed using the California mastitis test to detect SCM. Milk samples were cultured on different specific Staphylococcus media. The presence of S. aureus was confirmed by Gram staining, the catalase and coagulase tests, the detection of hemolytic activity, DNase agar testing, and biofilm activity in Congo red medium. The genotypic identification of S. aureus (nuc) was performed. The determinants of erythromycin (ermA, ermB, ermC, and ermT) and macrolide resistance (msrA) were screened in all isolates. DNA sequencing of our local isolates of S. aureus was used to analyze their phylogenetic relatedness. Moreover, histopathological examination of tissue specimens of mammary gland was performed. The S. aureus positivity rates were 36.4%, 48.8%, and 63.6% in cattle, buffalo, and bulk tank milk, respectively. Probe-based real-time PCR molecularly confirmed all 62 S. aureus isolates. Thirty-one isolates were subjected to PCR to create profiles of their genotypic erythromycin resistance. ermA, ermB, ermC, and ermT were present in 5 (8%), 26 (41.9%), 18 (29%), and 15 (24.1%) S. aureus isolates, respectively. Moreover, msrA was found in three (4.8%) strains. Eight PCR products were produced using standard PCR for DNA sequencing. Multiple sequence alignment, phylogenetic tree construction, and analysis of nuc in S. aureus revealed a high degree of homology (100%) with S. aureus strains isolated from milk in cases of bovine mastitis in India and Kenya. Histological analysis of udder tissues revealed extensive aggregation of mononuclear inflammatory cells in the interstitial connective tissue, primarily lymphocytes, and macrophages. This study showed a high prevalence of erythromycin resistance in S. aureus isolates. This information is vital for controlling mastitis and the spread of resistance genes between bacterial strains and hosts. Moreover, the probe-based real-time PCR approach is helpful for the rapid screening of S. aureus isolates and the consequent efficient treatment and control of S. aureus mastitis.

The Chinese herbal KangXianYiAi formula inhibits hepatocellular carcinoma by reducing glutathione and inducing ferroptosis

IntroductionHepatocellular carcinoma (HCC) is one of the most common malignant tumors worldwide and has a moderate treatment outcome. The traditional Chinese medicine KangXianYiAi formula (KXYA) has shown a clinically therapeutic effect on liver lesions. Here, we aimed to explore the effect and mechanism of KXYA in treating HCC. MethodsThe rat HCC model induced with diethylnitrosamine was treated with KXYA. After treatment, the rat's serum and the liver tissues were used to detect biochemical indices and perform pathological examinations, second-generation sequencing (RNA-seq), and phosphoproteomics analysis (PPOA). The hepatoma cells were treated with KXYA in vitro and a nude-mice model. The expression levels of genes were detected by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR), Western blotting (WB), and immunohistochemistry approaches. The expression and prognosis data of genes in HCC were obtained from the public database. ResultsCompared to the model, KXYA treatment significantly downregulated the rats’ serum hepatitis index, inflammatory damage, collagen deposition, and malignant proliferation of hepatocytes. And through the RNA-seq and PPOA analysis of rat liver tissues, we obtained the key pathways of KXYA including glutathione metabolism and ferroptosis. The qRT-PCR and WB results suggested that the key targets of UGDH, AKR1B10, and SLC7A11 were abnormally upregulated in HCC and downregulated to normal levels after KXYA treatment. In the public database, UGDH, AKR1B10, and SLC7A11 expression was significantly upregulated in HCC and indicated a poor prognosis. DiscussionOur study showed that KXYA treatment could effectively inhibit the progression of HCC by reducing glutathione and inducing ferroptosis of liver cancer cells.

Jian-Pi-Yi-Shen formula restores iron metabolism from dysregulation in anemic rats with adenine-induced nephropathy

Ethnopharmacological relevanceJian-Pi-Yi-Shen (JPYS) is a herbal decoction being used to relieve the symptoms of chronic kidney disease (CKD) and its complications, including anemia, for over twenty years. Nonetheless, it is unclear how JPYS influences renal anemia and iron metabolism. Aim of the studyAn analysis of network pharmacology, chemical profiling, and in vivo experiments was conducted to identify the impact of JPYS on JAK2-STAT3 pathway and iron utilization in renal anemia and CKD. Materials and methodsThe chemical properties of JPYS and its exposed ingredients were detected in vivo. And based on the aforesaid chemical compounds, the potential targets and signaling pathways of JPYS for renal anemia treatment were predicted by network pharmacology. Afterward, an adenine-feeding animal model of CKD-related anemia was developed to verify the mechanism by which JPYS modulates iron recycling to treat renal anemia. Renal injury was estimated by serum creatinine (Scr), blood urea nitrogen (BUN), histopathological examinations and fibrosis degree. Western blot, enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR) and immunohistochemistry approaches were utilized to assess the levels of JAK2, STAT3 and iron metabolism-related factors. ResultsThere were 164 active ingredients identified in JPYS, including prototypes and metabolites in vivo were identified in JPYS, and 21 core targets were found through network pharmacology based on topological characteristics. Combined with the core targets and pathway enrichment analysis, the majority of the candidate targets were associated with the JAK2-STAT3 signaling pathways. Experimental results indicated that JPYS treatment significantly decreased the expression of BUN and Scr, restored renal pathological damage, down-regulated fibrosis degree, and improved hematological parameters such as red blood cell, hemoglobin and hematocrit in CKD rats. Furthermore, JPYS significantly restored iron metabolism from dysregulation by increasing the levels of iron and ferritin in the serum, inhibiting the production of hepcidin in liver and serum, and regulating transferrin receptor 1 in bone marrow. Meanwhile, the expression of JAK2 and STAT3 was suppressed by JPYS treatment. ConclusionsBased on these results, JPYS reduces hepcidin levels by inhibiting the activation of JAK2-STAT3 signaling, thereby protecting against iron deficiency anemia.

Salinity significantly affects methane oxidation and methanotrophic community in Inner Mongolia lake sediments.

Methanotrophs oxidize methane (CH4) and greatly help in mitigating greenhouse effect. Increased temperatures due to global climate change can facilitate lake salinization, particularly in the regions with cold semiarid climate. However, the effects of salinity on the CH4 oxidation activity and diversity and composition of methanotrophic community in the sediment of natural lakes at a regional scale are still unclear. Therefore, we collected lake sediment samples from 13 sites in Mongolian Plateau; CH4 oxidation activities of methanotrophs were investigated, and the diversity and abundance of methanotrophs were analyzed using real-time quantitative polymerase chain reaction and high throughput sequencing approach. The results revealed that the diversity of methanotrophic community decreased with increasing salinity, and community structure of methanotrophs was clearly different between the hypersaline sediment samples (HRS; salinity > 0.69%) and hyposaline sediment samples (HOS; salinity < 0.69%). Types II and I methanotrophs were predominant in HRS and HOS, respectively. Salinity was significantly positively correlated with the relative abundance of Methylosinus and negatively correlated with that of Methylococcus. In addition, CH4 oxidation rate and pmoA gene abundance decreased with increasing salinity, and salinity directly and indirectly affected CH4 oxidation rate via regulating the community diversity. Moreover, high salinity decreased cooperative association among methanotrophs and number of key methanotrophic species (Methylosinus and Methylococcus, e.g). These results suggested that salinity is a major driver of CH4 oxidation in lake sediments and acts by regulating the diversity of methanotrophic community and accociation among the methanotrophic species.

The Novel SLRP Family Member Lumican Suppresses Pancreatic Cancer Cell Growth.

The past studies clearly indicated that lumican was important in the context of pancreatic cancer (PC) onset and progression, but failed to clarify the underlying mechanistic basis for such activity. As such, we evaluated the functional importance of lumican in the context of pancreatic ductal adenocarcinoma (PDAC) to understand its mechanistic role in PC. Lumican levels were evaluated in PDAC patient tissues via quantitative real-time polymerase chain reaction, Western blotting, and immunohistochemistry approaches. The role of lumican was additionally assessed via transfecting PDAC cell lines (BxPC-3, PANC-1) with lumican knockdown or overexpression constructs and treating PDAC cell lines with exogenous recombinant human lumican. Lumican expression levels were significantly higher in pancreatic tumor tissues relative to healthy paracancerous tissues. Lumican knockdown in BxPC-3 and PANC-1 enhanced their proliferation and migration, but reduced cellular apoptosis. Alternatively, lumican overexpression and exogenous lumican exposure failed to alter the proliferative activity of these cells. Further, lumican knockdown in BxPC-3 and PANC-1 cells results in marked P53 and P21 dysregulation. Lumican may suppress PDAC tumor growth by regulating P53 and P21, and the function of lumican sugar chains in the context of PC is worth studying in future studies.

Detection of Streptococcus suis using the optimized real-time polymerase chain reaction protocol

The article presents the results of studies on the detection of Streptococcus suis by real-time polymerase chain reaction. Isolation and species identification of the studied isolates of streptococci was carried out according to morphological, cultural, biochemical and biological properties by conventional methods. The study of cultural characteristics of growth was carried out using conventional bacteriological methods on the brain heart infusion broth (BHI) and BHI agar with the addition of 5% sheep blood (blood BHI agar). To confirm biochemical properties as a confirmatory method, API 20 STREP test kit (bioMerieux, France) was used. In addition, to differentiate S. suis from the non-pathogenic species of streptococci, the hemolysis test was used. As a result of the studies, it was found that the use of the real-time PCR (polymerase chain reaction) method makes it possible to detect S. suis in an amount of 1 x 104 genome copies in the sample. All described validation parameters for the qualitative detection of S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results. In Ukraine only a diagnostic test kit for convential PCR has been developed for the detection of swine streptococcosis. This approach is more time consuming and complex in comparison with the real-time PCR approach. We recommend that diagnostic laboratories implement this method in their practice. This will increase the number of effective diagnostic tools available to veterinarians on pig farms when they order laboratory tests. The high analytical sensitivity limit of a test is an essential parameter when screening is the focus, and obtaining false negative results causes a risk of the development of infection process among pig populations within infected herds. Our study showed that microbiological diagnostic methods to determine morphological and cultural properties can identify S. suis at the genus level. Determination of biochemical properties using the API 20 STREP test kit can be used to identify S. suis 1 and 2 serotypes. The conventional method and real-time PCR have 100% specificity and can be used to identify S. suis of different serotypes. Real-time PCR is a 2 to 4 times more sensitive limit than conventional PCR depending on the serotype being studied, and can be used to more accurately identify streptococcal DNA. It was found that the use of the real-time PCR method makes it possible to detect S. suis in an amount of 1 x 104 copies of the genome in the sample. Additionally, it was found that all the studied validation parameters of the qualitative method for determining S. suis DNA by real-time PCR meet international requirements, which guarantees accurate and reliable results.

Can periodontal pockets and caries lesions act as reservoirs for coronavirus?

The periodontal pocket and likely caries lesions may act as a reservoir and source of dissemination and development of systemic infections. While periodontal pockets have been found to harbor several viral species, there is no information on its ability to serve as a reservoir for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We have used a real-time polymerase chain reaction (RT-PCR) approach to evaluate SARS-CoV-2 in periodontal pockets and cavitated caries lesions in a cross-sectional study of 72 participants who were divided into six groups: symptomatic positive COVID-19 cases with periodontal pockets, symptomatic positive with cavitated caries lesions, asymptomatic positive with periodontal pockets, asymptomatic positive with cavitated caries lesions, positive control, and negative control. A total of 180 samples were interrogated by RT-PCR to amplify the SARS-CoV-2 E and S genes. SARS-CoV-2 was present in 41.7% of symptomatic positive COVID-19 cases with periodontal pockets and 16.7% of symptomatic positive with cavitated caries lesions. The mean Ct value of E and S genes in periodontal pockets patients were 36.06±0.46 and 30.06±6.73, respectively, and the mean Ct value for both genes in caries lesions patients were 35.73±4.14, and 34.78±1.93, respectively. The sensitivity, specificity, and accuracy to detect SARS-CoV-2 among periodontal pockets were 20.8% (95% CI 7.13-42.15), 100% (95% CI 73.54-100.0), and 47.2% (95% CI 30.22-64.51), respectively. Among cavitated caries lesions patients, they were 8.3% (95% CI 1.03-27.0), 100% (95% CI 73.54-100.0), and 38.9% (95% CI 23.14-56.54), respectively. SARS-CoV-2 can be detected in periodontal pockets and caries lesions, and these sites may act as reservoirs for the virus. However, the sensitivity of SARS-CoV-2 detection is low compared with other methods. To our knowledge, this report is the first to investigate the relationship between SARS-CoV-2 and periodontal pockets and caries.

Investigation of pork meat in chicken- and beef-based commercial products by ELISA and real-time PCR sold at retail in Kosovo

Food adulteration and fraudulent practices are widely observed in the food industry worldwide and are of great concern for Balkan countries. This study aims at investigating the level of undeclared pork meat in commercial beef and chicken meat products sold in Kosovo by implying one commercial enzyme-linked immunosorbent assay (ELISA) and two confirmatory real-time polymerase chain reaction (PCR) approaches [ready-to-use real-time PCR and real-time PCR with primers specific for pork mitochondrial deoxyribonucleic acid (DNA)]. In supermarkets in the capital city, Prishtina, 62 meat products were randomly sampled, and the three methods were applied. Additionally, these three approaches were evaluated for their practicability, reproducibility, and cost. The results showed that pork was present in 32% of beef- and 8% chicken-based products. ELISA and real-time PCR with pork specific primers showed 100% of reproducibility for beef- and chicken-based products. In contrast, the ready-to-use real-time PCR kit showed 100% reproducibility in chicken-, but only 75% in beef-based samples. ELISA was more rapid than both real-time PCR approaches, but it was more challenging when large numbers of samples were processed. The real-time PCR approach with pork specific primers was the cheapest, while the ready-to-use real-time PCR was the most practical method. Commercial ELISA, in combination with real-time PCR with pork specific primers, provides a reliable and affordable testing methodology that can be implemented for rapid detection and monitoring of pork adulteration in diverse commercial foods.

Telomere length in individuals with early pregnancy losses

Aim. Over the past decade, telomere biology has become an important topic in the field of human reproduction.We focused on the relationship between relative telomere length (RTL) and tendency to early pregnancy loss (EPL) in humans. Methods. RTL was measured in DNA isolated from the blood samples using a real-time polymerase chain reaction approach. RTL was examined in control group (C) (N=209) – women (CW) (N=107) and men (CM) (N=102) who had healthy pregnancies with no history of infertility or miscarriage, and in group with EPL (N=445) – women (EPLW) (N=223) and men (EPLM) (N=212) who had single or more EPL. RTL data were analyzed by gender and reproductive history. Results. Women (CW+EPLW) have significantly higher RTL that men (CM+EPLM) (1.74±0.06 in women and 1.40±0.05 in men, P=0.000053). Average RTL were significantly lower in CM compared to CW (CW: 2.27±0.12 versus CM: 1.15±0.08, P=0.0000001), and were similar in EPLW and EPLM (1.50±0.06 in EPLW and 1.53±0.06 in EPLM, P=0.47). The EPLW group had significantly lower RTL than control (EPLW: 1.50±0.06 versus CW: 2.27±0.12, P=0.0000001). Average RTL were significantly lower in CM compared to EPLM (1.15±0.08 in CM and 1.53±0.06 in EPLM, P=0.00006). Conclusions. Women with no history of EPL have longer telomere than men. Woman with EPL have shorter telomere that women without miscarriage. In EPL group women and men have similar telomere length.Keywords: telomeres, RT-PCR, gender, early reproductive loss.

A Preliminary Study of Antibiotic Resistance Genes in Domestic Honey Produced in China.

Antibiotic resistance genes (ARGs) are emerging contaminants that pose a health risk to humans worldwide. Little information on ARGs in bee honey is available. This study profiles ARGs in bee honey samples produced in China, the biggest producer in the world. Of 317 known ARGs encoding resistance to 8 classes of antibiotics, 212 were found in collected honey samples by a real-time quantitative polymerase chain reaction approach. Occurrence frequencies of genes providing resistance to FCA (fluoroquinolone, quinolone, florfenicol, chloramphenicol, and amphenicol) and aminoglycosides were 21.0% and 18.5%, respectively. Frequencies of genes encoding efflux pumps were 42.5% and those of destructase genes 36.6%, indicating that these two mechanisms were predominant for resistance. Nine plasmid-mediated quinolone resistance genes were detected. Of the nine transposase genes known to be involved in antibiotic resistance, eight were found in the samples examined, with tnpA-4, tnpA-5, and tnpA-6 being more abundant. The abundance of the transposase genes was associated with genes conferring resistance to tetracyclines (r = 0.648, p < 0.01), macrolide-lincosamide-streptogramin B (r = 0.642, p < 0.01), FCA (r = 0.517, p < 0.01), and aminoglycosides (r = 0.401, 0.01 < p < 0.05). This is the first study on the abundance and diversity of ARGs in Chinese bee honey products. These findings suggest that bee honey may be a significant source of ARGs that might pose threat to public health. Further research is required to collect more samples in diverse geographic regions in China to make a more comprehensive judgment of ARG in bee honey.

Culturomic and quantitative real-time-polymerase chain reaction analyses for early contamination of abutments with different surfaces: A randomized clinical trial.

Rough and/or plasma-activated abutments seem to be able to increase soft tissue adhesion and stability; however, limited evidence is available about bacterial contamination differences. The aim was to investigate the oral microbiota on four dental abutments with different surfaces by quantitative real-time polymerase chain reaction (qRT-PCR) and culturomic approach. Forty patients needing a single implant rehabilitation were involved in the study. Forty healing abutments, especially designed for the study, were divided into four groups according to the surface topography (1. machined [MAC], 2. machined plasma of argon treated [plasmaMAC], 3. ultrathin threaded microsurface [UTM], 4. UTM plasma of argon treated [Plasma UTM]). Random assignment was performed according to predefined randomization tables. All patients underwent surgical intervention for implant and contextual healing abutment positioning. After 2months of healing, a sterile cotton swab was used for microbiological sampling for culturomics, while sterile paper points inserted into the sulcus were used for qRT-PCR. At the end of the study, 36 patients completed all procedures and a total of 36 abutments (9 per group) were analyzed. qRT-PCR retrieved data for 23 bacterial species whereas culturomics revealed the presence of 74 different bacteria, most of them not routinely included into oral cavity microbiological kits of analysis or never found before in the oral microenvironment. No statistically significant differences emerged analyzing the four different surfaces (p= 0.053). On the contrary, higher total and specific bacterial counts were detected in the plasma-treated surfaces compared to the untreated ones (p= 0.021). Abutments with different topographies and surface treatments resulted contaminated by similar oral bacterial flora. Abutments with moderately rough surface were not associated with a greater bacterial adhesion compared to machined ones. Conversely, more bacteria were found around plasma-treated abutments. Furthermore, data reported suggested to include new species not previously sought in the routine analyses of the oral bacterial microflora.

SARS-CoV-2 Direct real-time Polymerase Chain Reaction Testing in Laboratories With Shortage Challenges

Aim: In our study, we propose the use of direct real-time polymerase chain reaction (RT-PCR), this test does not require extraction or a preheating step, which saves a lot of cost, labor, processing time and provides a solution for supply shortage. Materials & methods: We assayed 185 nasopharyngeal samples stored in viral transport media. The indirect method was done with RNA extraction step, and the direct RT-PCR was done without an extraction step, both assays were evaluated on a commercially validated severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) kit targeting E gene. Results: Our assay showed a sensitivity of 79%, a specificity of 100% and the agreement between methods was 72%. Conclusion: Overall, this simple direct RT-PCR approach can be utilized as a qualitative diagnostic tool for emergency SARS-CoV-2 surveillance in countries with limited resources and may help laboratories to continue testing and at greater frequency despite supply shortages, although with delay in cycle threshold value in comparison with indirect RT-PCR.

Expression, Affinity, and Functional Characterization of the Specific Binding of Two Putative Pheromone-Binding Proteins in the Omnivorous German Cockroach Blattella germanica.

The German cockroach Blattella germanica (L.) is an important pest in medical, veterinary, and public health. Studies on the olfaction mechanism of hemimetabolous insects have rarely been reported, especially in cockroaches. Pheromone-binding proteins (PBPs) play a vital role in insect sex pheromone recognition, which solubilize and carry the hydrophobic pheromonal compounds through the antennal lymph to receptors. In this study, two potential PBPs (BgerOBP26 and BgerOBP40) were identified on the basis of their biased expression in male antennae using tissue transcriptome data and verified by the quantitative real-time polymerase chain reaction approach. We then expressed and purified the two identified odorant-binding proteins (OBPs) using the Escherichia coli expression system and affinity purification. In vitro binding studies showed that the two OBPs display stronger binding affinities to the female volatile sex pheromone blattellaquinone than to its analogues and contact sex pheromone components. Finally, three-dimensional modeling of the two OBPs and dock conformation with sex pheromone molecules showed BgerOBP26 has a larger odorant cavity and more conservative active amino acid residues than BgerOBP40. These results illuminated the binding characteristics of potential PBPs of B. germanica, which could lay the groundwork for improved understanding of many aspects of the chemical ecology of B. germanica. Moreover, this information complements the understanding of the olfactory molecular mechanism in cockroaches and provides potential gene targets for B. germanica control.

Authentication of Ginkgo biloba Herbal Products by a Novel Quantitative Real-Time PCR Approach.

Ginkgo biloba is a widely used medicinal plant. Due to its potential therapeutic effects, it is an ingredient in several herbal products, such as plant infusions and plant food supplements (PFS). Currently, ginkgo is one of the most popular botanicals used in PFS. Due to their popularity and high cost, ginkgo-containing products are prone to be fraudulently substituted by other plant species. Therefore, this work aimed at developing a method for G. biloba detection and quantification. A new internal transcribe spacer (ITS) marker was identified, allowing the development of a ginkgo-specific real-time polymerase chain reaction (PCR) assay targeting the ITS region, with high specificity and sensitivity, down to 0.02 pg of DNA. Additionally, a normalized real-time PCR approach using the delta cycle quantification (ΔCq) method was proposed for the effective quantification of ginkgo in plant mixtures. The method exhibited high performance parameters, namely PCR efficiency, coefficient of correlation and covered dynamic range (50–0.01%), achieving limits of detection and quantification of 0.01% (w/w) of ginkgo in tea plant (Camellia sinensis). The quantitative approach was successfully validated with blind mixtures and further applied to commercial ginkgo-containing herbal infusions. The estimated ginkgo contents of plant mixture samples suggest adulterations due to reduction or almost elimination of ginkgo. In this work, useful and robust tools were proposed to detect/quantify ginkgo in herbal products, which suggests the need for a more effective and stricter control of such products.

Effect of Silver Diamine Fluoride on Reducing Candida albicans Adhesion on Dentine.

Candida albicans is the most frequent pathogenic fungus in oral cavities. It adheres to dental tissues as part of dental plaques and contributes to caries formation. To evaluate the effect of silver diamine fluoride (SDF) on reducing C. albicans adhesion on dentine surfaces. Flat dentine surfaces were prepared from bovine dental disks, and samples were divided into three groups. The first and second groups were pretreated for 3min with 299mM or 2.99M SDF, respectively, and the third group (control) did not undergo any SDF pretreatment. All samples were washed, inoculated with C. albicans suspension onto their dentine surface, incubated at 30°C for 6h, and washed again to remove any nonadherent cells. The abundance of adherent cells was investigated using colorimetric and real-time polymerase chain reaction approaches. Subsequently, the morphological changes in C. albicans by pretreatment with SDF were observed under a scanning electron microscope (SEM). SDF inhibited candidal growth at concentrations as low as 2.99µM. Dentine disks pretreated with 299mM or 2.99M SDF displayed significantly fewer adhered cells as compared with the control group. Upon pretreatment with SDF, SEM images showed severe morphological changes in the cellular walls, in a dose-dependent manner, suggesting a fungicidal effect of SDF against the yeast. SDF should be considered for clinical applications aimed at inhibiting dental plaque caused by C. albicans, particularly in children and elderly individuals.

High-salt diet decreases mechanical thresholds in mice that is mediated by a CCR2-dependent mechanism

BackgroundThough it is well-known that a high-salt diet (HSD) is associated with many chronic diseases, the effects of long-term high-salt intake on physiological functions and homeostasis remain elusive. In this study, we investigated whether and how an HSD affects mouse nociceptive thresholds, and myeloid cell trafficking and activation.MethodsHealthy C57BL/6 male and female mice were fed an HSD (containing 4% NaCl in chow and 1% NaCl in water) from the time of weaning for 3 to 4 months. Circulating monocytes, nerve macrophages, spinal microglia, and associated inflammatory responses were scrutinized using flow cytometry, immunohistochemistry, and quantitative real-time polymerase chain reaction (qPCR) approaches. Mouse pain sensitivity to mechanical stimuli was monitored with von Frey tests along the experimental duration.ResultsMice on an HSD have reduced mechanical thresholds. They feel more pain than those on a normal diet (ND), e.g., regular laboratory chow (0.3% NaCl in chow). An HSD induced not only a remarkable expansion of circulating monocytes, CCR2+Ly6Chi inflammatory monocytes in particular, but also an accumulation of CD11b+F4/80+ macrophages in the peripheral nerves and an activation of Iba-1+ spinal microglia. Replacing an HSD with a ND was unable to reverse the HSD-induced mechanical hypersensitivity or rescue the altered immune responses. However, treating HSD-fed mice with a chemokine receptor CCR2 antagonist effectively normalized the pain thresholds and immune cell profile in the periphery and spinal cord. An HSD failed to alter pain thresholds and myeloid cell activation in CCR2-deficient mice. Spinal microglial activation is required for HSD-induced mechanical hypersensitivity in male, but not in female mice.ConclusionOverall, this study provides evidence that an HSD has a long-term impact on physiological function. CCR2-mediated cellular response, including myeloid cell trafficking and associated inflammation, plays pivotal roles in salt-dietary modulation of pain sensitivity.