Polymerase Chain
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2023 ◽  
Vol 83 ◽  
Author(s):  
A. Belmok ◽  
T. Rodrigues-Oliveira ◽  
F.A.C. Lopes ◽  
R.H. Krüger ◽  
C.M. Kyaw

Abstract Polymerase chain reaction (PCR) assays targeting 16S rRNA genes followed by DNA sequencing are still important tools to characterize microbial communities present in environmental samples. However, despite the crescent number of deposited archaeal DNA sequences in databases, until now we do not have a clear picture of the effectiveness and specificity of the universal primers widely used to describe archaeal communities from different natural habitats. Therefore, in this study, we compared the phylogenetic profile obtained when Cerrado lake sediment DNA samples were submitted to 16S rDNA PCR employing three Archaea-specific primer sets commonly used. Our findings reveal that specificity of primers differed depending on the source of the analyzed DNA. Furthermore, archaeal communities revealed by each primer pair varied greatly, indicating that 16S rRNA gene primer choice affects the community profile obtained, with differences in both taxon detection and operational taxonomic unit (OTU) estimates.


2021 ◽  
Author(s):  
Hanako Sato ◽  
Akira Yano ◽  
Yu Shimoyama ◽  
Toshiro Sato ◽  
Yukiko Sugiyama ◽  
...  

Abstract Background:Disruption of the indigenous microflora is likely to relate with frailty caused by undernutrition. However, relationship between undernutrition and oral flora, especially normal indigenous bacteria, is not obvious. The aim of this study was to elucidate the associations of nutritional and oral health conditions with bacteria and fungi prevalence in oral cavity of older individuals.Methods:Forty-one institutionalized older individuals with an average age ± standard deviation of 84.6 ± 8.3 years were enrolled in this study. Body mass index (BMI) and Oral health assessment tool (OHAT) were used to represent nutritional and oral health status. Amounts of total bacteria, streptococci, and fungi in oral specimens collected from the tongue dorsum were determined by quantitative polymerase chain reaction (PCR) assay results. Results:There found a significant correlation between BMI and streptococcal amount (ρ=0.526, p<0.001). In addition, the undernutrition group (BMI <20) showed a significantly lower average number of oral streptococci (p=0.003). In logistic regression models, streptococcal amount was the significant variable accounting for “not undernutrition” [odds ratio 5.68 and 95% confidential interval (CI) was 1.64-19.7 (p=0.06)]. On the other hand, participants with poor oral health condition (OHAT ≥5) harbored significantly higher level of fungi (p=0.028). Conclusion:Oral streptococci were associated with systemic nutritional condition and oral fungi were associated with oral health condition. Thus, it is necessary to examine oral indigenous bacteria as well as etiological microorganisms in order to understand the relationship of frailty with oral microflora in older individuals.


Cartilage ◽  
2021 ◽  
pp. 194760352110424
Author(s):  
Elizabeth Vinod ◽  
Kawin Padmaja ◽  
Abel Livingston ◽  
Jithu Varghese James ◽  
Soosai Manickam Amirtham ◽  
...  

Purpose Chondrocytes, isolated from articular cartilage, are routinely utilized in cell-based therapeutics for the treatment of cartilage pathologies. However, restoration of the biological tissue faces hindrance due to the formation of primarily fibrocartilaginous repair tissue. Chondroprogenitors have been reported to display superiority in terms of their chondrogenic potential and lesser proclivity for hypertrophy. In line with our recent results, comparing chondroprogenitors and chondrocytes, we undertook isolation of progenitors from the general pool of chondrocytes, based on surface marker expression, namely, CD166, CD34, and CD146, to eliminate off-target differentiation and generate cells of stronger chondrogenic potential. This study aimed to compare chondrocytes, chondroprogenitors, CD34−CD166+CD146+ sorted chondrocytes, and CD34−CD166+CD146− sorted chondrocytes. Methods Chondrocytes obtained from 3 human osteoarthritic knee joints were subjected to sorting, to isolate CD166+ and CD34− subsets, and then were further sorted to obtain CD146+ and CD146− cells. Chondrocytes and fibronectin adhesion-derived chondroprogenitors served as controls. Assessment parameters included reverse transcriptase polymerase chain reaction for markers of chondrogenesis and hypertrophy, trilineage differentiation, and total GAG/DNA content. Results Based on gene expression analysis, CD34−CD166+CD146+ sorted chondrocytes and chondroprogenitors displayed comparability and significantly higher chondrogenesis with a lower tendency for hypertrophy when compared to chondrocytes and CD34−CD166+CD146− sorted chondrocytes. The findings were also reiterated in multilineage potential differentiation with the 146+ subset and chondroprogenitors displaying lower calcification and chondroprogenitors displaying higher total GAG/DNA content compared to chondrocytes and 146− cells. Conclusion This unique progenitor-like population based on CD34−CD166+CD146+ sorting from chondrocytes exhibits efficient potential for cartilage repair and merits further evaluation for its therapeutic application.


2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Takahiro Matsuo ◽  
Akira Saito ◽  
Fujimi Kawai ◽  
Kazuhiro Ishikawa ◽  
Ryo Hasegawa ◽  
...  

Abstract Background Entamoeba histolytica (E. histolytica) is rarely identified as a cause of amebic pericarditis. We report a case of amebic pericarditis complicated by cardiac tamponade, in which the diagnosis was missed initially and was made retrospectively by polymerase chain reaction (PCR) testing of a stored sample of pericardial fluid. Furthermore, we performed a systematic review of the literature on amebic pericarditis. Case presentation A 71-year-old Japanese man who had a history of sexual intercourse with several commercial sex workers 4 months previously, presented to our hospital with left chest pain and cough. He was admitted on suspicion of pericarditis. On hospital day 7, he developed cardiac tamponade requiring urgent pericardiocentesis. The patient’s symptoms temporarily improved, but 1 month later, he returned with fever and abdominal pain, and multiple liver lesions were found in the right lobe. Polymerase chain reaction of the aspiration fluid of the liver lesion and pericardial and pleural fluid stored from the previous hospitalization were all positive for E. histolytica. Together with the positive serum antibody for E. histolytica, a diagnosis of amebic pericarditis was made. Notably, the diagnosis was missed initially and was made retrospectively by performing PCR testing. The patient improved with metronidazole 750 mg thrice daily for 14 days, followed by paromomycin 500 mg thrice daily for 10 days. Conclusions This case suggests that, although only 122 cases of amebic pericarditis have been reported, clinicians should be aware of E. histolytica as a potential causative pathogen. The polymerase chain reaction method was used to detect E. histolytica in the pericardial effusion and was found to be useful for the diagnosis of amebic pericarditis in addition to the positive results for the serum antibody testing for E. histolytica. Because of the high mortality associated with delayed treatment, prompt diagnosis should be made.


Author(s):  
Ying Fang ◽  
Ting Lei ◽  
Yanmei Wu ◽  
Xuehua Jin

The calla lily (Zantedeschia hybrida) is a valued ornamental plant due to its unique shape and color variations. To determine the mechanisms responsible for color development in the calla lily spathe, we conducted a comparative transcriptomic analysis of the spathes of the black [Black Girl (B)], pink [Romantic (P)], and white [Ventura (W)] cultivars. The gene expression patterns in six spathe colors, including the preceding three colors as well as the amaranth [Promise (N)], red [Figo (F)], and yellow [Sun Club (Y)] cultivars were analyzed by real-time quantitative polymerase chain reaction (PCR). Transcriptomic analysis identified 25,165 differentially expressed genes. The transcription abundance and expression level of genes annotated as anthocyanidin reductase (ANR1, ANR2), basic-helix-loop-helix (bHLH1), and glutathione S-transferases (GST1) were significantly upregulated in B, and the expression of anthocyanidin synthase (ANS) was highest in B except for N. However, chalcone isomerase (CHI2) and dihydroflavonol 4-reductase (DFR1, DFR2) were expressed at significantly lower levels in P, W, and Y. Correlation analysis revealed that bHLH1 might act as a positive regulator of ANS expression, promoting anthocyanin synthesis. Moreover, GST1-encoded proteins may be related to the accumulation and transport of both anthocyanin and procyanidin in the calla lily spathe. It is speculated that the formation of the black spathe is related to the accumulation of anthocyanins and procyanidins. However, the low expression of CHI2, DFR1, and DFR2 may result in the inhibition of anthocyanin synthesis, which may lead to lightening of the spathe color. This preliminary study revealed the mechanism responsible for calla lily spathe color, identifying the key genes involved, thus providing effective gene resources and a theoretical basis for flower color molecular breeding.


PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257276
Author(s):  
Collins Okoyo ◽  
Edward Githinji ◽  
Ruth W. Muia ◽  
Janet Masaku ◽  
Judy Mwai ◽  
...  

Background In Kenya, health service delivery and access to health care remains a challenge for vulnerable populations, particularly pregnant women and children below five years. The aim of this study, therefore, was to determine the positivity rate of Plasmodium falciparum parasites in pregnant women and children below five years of age seeking healthcare services at the rural health facilities of Kwale and Siaya counties as well as their access and uptake of malaria control integrated services, like antenatal care (ANC), offered in those facilities. Methods Cluster random sampling method was used to select pregnant women and children below five years receiving maternal and child health services using two cross-sectional surveys conducted in eleven rural health facilities in two malaria endemic counties in western and coastal regions of Kenya. Each consenting participant provided single blood sample for determining malaria parasitaemia using microscopy and polymerase chain reaction (PCR) techniques. Results Using PCR technique, the overall malaria positivity rate was 27.9% (95%CI: 20.9–37.2), and was 34.1% (95%CI: 27.1–42.9) and 22.0% (95%CI: 13.3–36.3) in children below five years and pregnant women respectively. Additionally, using microscopy, the overall positivity rate was 39.0% (95%CI: 29.5–51.6), and was 50.4% (95%CI: 39.4–64.5) and 30.6% (95%CI: 22.4–41.7) in children below five years and pregnant women respectively. Siaya County in western Kenya showed higher malaria positivity rates for both children (36.4% and 54.9%) and pregnant women (27.8% and 38.5%) using both PCR and microscopy diagnosis techniques respectively, compared to Kwale County that showed positivity rates of 27.2% and 37.9% for children and 5.2% and 8.6% for pregnant women similarly using both PCR and microscopy techniques respectively. Pregnant women presenting themselves for their first ANC visit were up to five times at risk of malaria infection, (adjusted odds ratio = 5.40, 95%CI: 0.96–30.50, p = 0.046). Conclusion Despite evidence of ANC attendance and administration of intermittent preventive treatment with sulfadoxine-pyrimethamine (IPTp-SP) dosage during these visits, malaria positivity rate was still high among pregnant women and children below five years in these two rural counties. These findings are important to the Kenyan National Malaria Control Programme and will help contribute to improvement of policies on integration of malaria control approaches in rural health facilities.


2021 ◽  
Author(s):  
Fei Liu ◽  
Ye Han ◽  
Dongbao Li ◽  
Jun Zhou ◽  
Jingjing Xu ◽  
...  

Abstract Gastric cancer (GC) is one of the most common malignant tumors with a leading cause of cancer-related mortality worldwide. Exosomal miRNAs are considered as promising non-invasive biomarkers for the diagnosis of malignant tumors. In this study, we aimed to investigate the expression of exosomal miR-17-92 cluster and develop a potential biomarker for the diagnosis of GC. Exosomal RNAs were extracted and the expression profile of miR-17-92 cluster was detected using quantitative polymerase chain reaction (qRT-PCR). The ROC (receiver-operating characteristic) curve and AUC (area under the ROC curve) analysis were used to explore the diagnostic utility of miRNAs. Statistical was used to analyze the expression of serum exosomal miR-17-92 cluster with the clinical pathological parameters of GC patients. The results showed that the expressions of four members of the exosomal miR-17-92 cluster in the serum samples of GC patients were significantly upregulated compared with those of healthy controls. The AUC for serum exosomal miR-17, miR-18, miR-19a and miR-92 was 0.750 (95%CI=0.626-0.874, sensitivity=84.7%, specificity=70.0%), 0.736 (95%CI=0.590-0.881, sensitivity=88.9%, specificity=65.0%), 0.700 (95%CI=0.562-0.838, sensitivity=62.5%, specificity=80.0%), 0.689 (95%CI=0.567-0.811, sensitivity=45.8%, specificity=90.0%), respectively. The AUC for the newly combined panel consisting of miR-17, miR-18, miR-19a and miR-92 was 0.808 (95%CI=0.680-0.937), with sensitivity of 90.3% and specificity of 70.0%, which showed much higher clinical diagnostic value for GC than any of the four alone or any pair. Besides, the AUC for the newly developed panel consisting of the two traditional tumor biomarkers including CEA (carcinoembryonic antigen) and CA19-9 (carbohydrate antigen 19-9) and the four miR-17-92 cluster members was 0.881 (95%CI, 0.765-0.998) with sensitivity of 91.7% and specificity of 90.0%, which showed the greatest powerful clinical diagnostic value for GC. Moreover, the elevated exosomal miR-17-92 expressions were closely correlated with tumor size, tumor depth, lymph node metastasis, distant metastasis and TNM stage of GC patients. In conclusion, our findings revealed that circulating exosomal miR-17-92 cluster may be used as a novel potential non-invasive biomarker to improve the diagnostic efficiency in GC.


2021 ◽  
Vol 09 (10) ◽  
pp. E1556-E1560
Author(s):  
Stephan Zellmer ◽  
Alanna Ebigbo ◽  
Maria Kahn ◽  
Anna Muzalyova ◽  
Johanna Classen ◽  
...  

Abstract Background and study aims The European Society of Gastrointestinal Endoscopy (ESGE) has defined COVID-19 infection prevention and control strategies within the endoscopy unit. These include pre-endoscopic questionnaire-based risk-stratification as well as pre-procedure viral testing. Real-life data on the effectiveness of these measures are presented here. Patients and methods Data from the outpatient endoscopic unit of the University Hospital Augsburg between July 1, 2020 and December 31, 2020 including the second pandemic wave were reviewed retrospectively. All patients were assessed with a pre-endoscopic risk-stratification questionnaire as well as viral testing using an antigen point-of-care test (Ag-POCT) in conjunction with a standard polymerase chain reaction (PCR) test. Highly elective procedures were postponed. The theoretically expected number of SARS-CoV-2-positive patients was simulated and compared with the actual number. In addition, endoscopy staff was evaluated with a rapid antibody test to determine the number of infections among the personnel. Results In total, 1029 procedures, 591 questionnaires, 591 Ag-POCTs, and 529 standard PCR tests were performed in 591 patients. 247 procedures in 142 patients were postponed. One Ag-POCT was positive but with a negative PCR test, while one PCR test was positive but with a negative Ag-POCT. This was lower than the theoretically expected number of COVID-19-positive patients (n = 15). One of 43 employees (2.3 %) in the outpatient endoscopy unit was seropositive. Conclusions Pre-endoscopic risk management including questionnaire-based risk stratification and viral testing seems to be an effective tool in combination with personal protective equipment for SARS-CoV-2 infection prevention and control within the endoscopy unit even in a high-prevalence setting.


2021 ◽  
Author(s):  
Tahmaseb Jouzdani ◽  
Amir Sadeghi ◽  
Hamed Tahmasbi ◽  
Ramin Shekouhi ◽  
Maryam Sohooli ◽  
...  

Abstract Background Despite years of research, the etiology of achalasia not well understood. Scientists suppose a role for autoimmunity, in this disorder, and probable viral agent, such as herpes virus (HSV). The aim was to find out the frequency of HSV in esophageal muscle samples in patients with achalasia under Heller's myotomy. Methods In this study, 60 patients with achalasia, after fulfilling the consent form, were underwent Heller’s myotomy surgery. Biopsy samples prepared for polymerase chain reaction (PCR) method for HSV DNA detection. After DNA-extraction, replication performed using specific primers. Results The mean age was 40.62 ± 5.08 years. Thirty-nine patients (65%) were female and 21 (35%) were male. Thirty-eight (63.3%) had no history but the else (36.7%) had a positive history of HSV. HSV-1 was positive in three patients (5%). Two females and one male were HSV-positive. Conclusions HSV-1 frequency is not notable among Iranian patients with achalasia. We suggest exploring other viruses, in special that involving the pathogenesis of achalasia, with a larger sample size.


2021 ◽  
pp. 1-28
Author(s):  
Yoshimasa Kumekawa ◽  
Haruka Fujimoto ◽  
Osamu Miura ◽  
Ryo Arakawa ◽  
Jun Yokoyama ◽  
...  

Abstract Harvestmen (Arachnida: Opiliones) are soil animals with extremely low dispersal abilities that experienced allopatric differentiation. To clarify the morphological and phylogenetic differentiation of the endemic harvestman Zepedanulus ishikawai (Suzuki, 1971) (Laniatores: Epedanidae) in the southern part of the Ryukyu Archipelago, we conducted molecular phylogenetic analyses and divergence time estimates based on CO1 and 16S rRNA sequences of mtDNA, the 28S rRNA sequence of nrDNA, and the external morphology. A phylogenetic tree based on mtDNA sequences indicated that individuals of Z. ishikawai were monophyletic and were divided into clade I and clade II. This was supported by the nrDNA phylogenetic tree. Although clades I and II were distributed sympatrically on all three islands examined (Ishigaki, Iriomote, and Yonaguni), heterogeneity could not be detected by polymerase chain reaction–restriction fragment length polymorphism of nrDNA, indicating that clades I and II do not have a history of hybridisation. Also, several morphological characters differed significantly between individuals of clade I and clade II. The longstanding isolation of the southern Ryukyus from the surrounding islands enabled estimation of the original morphological characters of both clades of Z. ishikawai.


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