• Title/Summary/Keyword: real-time reverse transcriptase-PCR

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Detection of Enteropathogens in Human Immunodeficiency Virus and Non-Human Immunodeficiency Virus-Infected Children with Acute Diarrhea in an Indonesian Tertiary Hospital Using Multiplex Real-Time Polymerase Chain Reaction

  • Dewi Wulandari;Rivaldi Febrian;Pramita Gayatri Dwipoerwantoro;Nia Kurniati
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • 제27권2호
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    • pp.95-103
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    • 2024
  • Purpose: Diarrhea is one of the leading causes of mortality in children living in developing countries. The etiology of acute diarrhea in each healthcare center varies depending on place, time, and population. This study aimed to identify pathogen patterns in human immunodeficiency virus (HIV)-infected and non-HIV children suffering from acute diarrhea, using multiplex real time reverse transcriptase polymerase chain reaction (RT-PCR), in an Indonesian tertiary hospital. Methods: This cross-sectional study was conducted at Dr. Cipto Mangunkusumo National Hospital from March 2019 to April 2020. Results: The study showed that multiplex RT-PCR results were positive in 58.9% of the specimens, with more positive results in HIV-infected children than in non-HIV-infected children (70% vs. 54.7%). Altogether 72 enteropathogens were detected from all specimens. Enteropathogens in non-HIV children with acute diarrhea consisted of bacteria (70.6%) and viruses (29.4%) with a predominance of enteroaggregative Escherichia coli (25.4%), followed by Campylobacter spp. (11.8%), enteropathogenic E. coli (9.8%), Norovirus GII (7.8%), and Clostridium difficile (7.8%). Enteropathogens in HIV-infected children consisted of viruses (57.1%), bacteria (28.6%), and parasites (14.3%) comprising Norovirus GII (24%), Cryptosporidium spp. (14.3%), Campylobacter spp. (14.3%), Norovirus GI (14.3%), and Astrovirus (14.3%). Cryptosporidium spp. was the only parasite found in this study and was found only in HIV-infected children. In non-HIV children with acute diarrhea, most pathogens were invasive bacteria, while in HIV-infected children, more viral and parasite infections occurred, primarily caused by opportunistic pathogens. Conclusion: The pattern of enteropathogens can help clinicians determine further examinations and appropriate empirical antimicrobial therapy for the patient.

Implementation of a Deep Learning-Based Computer-Aided Detection System for the Interpretation of Chest Radiographs in Patients Suspected for COVID-19

  • Eui Jin Hwang;Hyungjin Kim;Soon Ho Yoon;Jin Mo Goo;Chang Min Park
    • Korean Journal of Radiology
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    • 제21권10호
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    • pp.1150-1160
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    • 2020
  • Objective: To describe the experience of implementing a deep learning-based computer-aided detection (CAD) system for the interpretation of chest X-ray radiographs (CXR) of suspected coronavirus disease (COVID-19) patients and investigate the diagnostic performance of CXR interpretation with CAD assistance. Materials and Methods: In this single-center retrospective study, initial CXR of patients with suspected or confirmed COVID-19 were investigated. A commercialized deep learning-based CAD system that can identify various abnormalities on CXR was implemented for the interpretation of CXR in daily practice. The diagnostic performance of radiologists with CAD assistance were evaluated based on two different reference standards: 1) real-time reverse transcriptase-polymerase chain reaction (rRT-PCR) results for COVID-19 and 2) pulmonary abnormality suggesting pneumonia on chest CT. The turnaround times (TATs) of radiology reports for CXR and rRT-PCR results were also evaluated. Results: Among 332 patients (male:female, 173:159; mean age, 57 years) with available rRT-PCR results, 16 patients (4.8%) were diagnosed with COVID-19. Using CXR, radiologists with CAD assistance identified rRT-PCR positive COVID-19 patients with sensitivity and specificity of 68.8% and 66.7%, respectively. Among 119 patients (male:female, 75:44; mean age, 69 years) with available chest CTs, radiologists assisted by CAD reported pneumonia on CXR with a sensitivity of 81.5% and a specificity of 72.3%. The TATs of CXR reports were significantly shorter than those of rRT-PCR results (median 51 vs. 507 minutes; p < 0.001). Conclusion: Radiologists with CAD assistance could identify patients with rRT-PCR-positive COVID-19 or pneumonia on CXR with a reasonably acceptable performance. In patients suspected with COVID-19, CXR had much faster TATs than rRT-PCRs.

Notochordal Cells Influence Gene Expression of Inflammatory Mediators of Annulus Fibrosus Cells in Proinflammatory Cytokines Stimulation

  • Moon, Hong-Joo;Joe, Hoon;Kwon, Taek-Hyun;Choi, Hye-Kyoung;Park, Youn-Kwan;Kim, Joo-Han
    • Journal of Korean Neurosurgical Society
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    • 제48권1호
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    • pp.1-7
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    • 2010
  • Objective : Notochordal cells in the intervertebral disc interact with nucleus pulposus (NP) cells and support the maintenance of disc homeostasis by regulation of matrix production. However, the influence of notochordal cells has not been evaluated in the annulus fibrosus (AF), which is the primary pain generator in the disc. We hypothesized that the notochordal cell has the capacity to modulate inflammatory mediators secreted by AF cells secondary to stimulation. Methods : Notochordal and AF cells were isolated from adult New Zealand white rabbits. AF pellets were cultured with notochordal cell clusters or in notochordal cell-conditioned media (NCCM) for 24 or 48 hours with proinflammatory cytokines at varying concentrations. Gene expression in AF pellets were assayed for nitric oxide synthase (iNOS), cyclo-oxygenase (COX)-2, and interleukin (IL)-6 by real time reverse transcriptase polymerase chain reaction (RT-PCR). Results : AF pellet in NCCM significantly decreased the iNOS and COX-2 messenger ribonucleic acid (mRNA) levels compared to AF pellets alone and AF pellets with notochordal cells (p < 0.05). AF pellet resulted in dose-dependent iNOS and COX-2 expression in response to IL-$1{\beta}$, stimulation, demonstrating that 1 ng/ml for 24 hours yielded a maximal response. AF pellet in NCCM significantly decreased the expression of iNOS and COX-2 in response to 1ng/ml IL-$1{\beta}$, stimulation at 24 hours (p < 0.05). There was no difference in IL-6 expression compared to AF pellets alone or AF pellets with notochordal cell clusters. Conclusion : We conclude that soluble factors from notochordal cells mitigate the gene expression of inflammatory mediators in stimulated AF, as expected after annular injury, suggesting that notochordal cells could serve as a novel therapeutic approach in symptomatic disc development.

Overexpression of Ubiquitin and Amino Acid Permease Genes in Association with Antimony Resistance in Leishmania tropica Field Isolates

  • Kazemi-Rad, Elham;Mohebali, Mehdi;Erfan, Mohammad Bagher Khadem;Hajjaran, Homa;Hadighi, Ramtin;Khamesipour, Ali;Rezaie, Sassan;Saffari, Mojtaba;Raoofian, Reza;Heidari, Mansour
    • Parasites, Hosts and Diseases
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    • 제51권4호
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    • pp.413-419
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    • 2013
  • The mainstay therapy against leishmaniasis is still pentavalent antimonial drugs; however, the rate of antimony resistance is increasing in endemic regions such as Iran. Understanding the molecular basis of resistance to antimonials could be helpful to improve treatment strategies. This study aimed to recognize genes involved in antimony resistance of Leishmania tropica field isolates. Sensitive and resistant L. tropica parasites were isolated from anthroponotic cutaneous leishmaniasis patients and drug susceptibility of parasites to meglumine antimoniate (Glucantime$^{(R)}$) was confirmed using in vitro assay. Then, complementary DNA-amplified fragment length polymorphism (cDNA-AFLP) and real-time reverse transcriptase-PCR (RT-PCR) approaches were utilized on mRNAs from resistant and sensitive L. tropica isolates. We identified 2 known genes, ubiquitin implicated in protein degradation and amino acid permease (AAP3) involved in arginine uptake. Also, we identified 1 gene encoding hypothetical protein. Real-time RT-PCR revealed a significant upregulation of ubiquitin (2.54-fold), and AAP3 (2.86-fold) (P<0.05) in resistant isolates compared to sensitive ones. Our results suggest that overexpression of ubiquitin and AAP3 could potentially implicated in natural antimony resistance.

Protective Effects of Prunus persica Flesh Extract (PPFE) on UV-Induced Oxidative Stress and Matrix Metalloproteinases Expression in Human Skin Cells

  • Park, Hyen-Joo;Park, Kwang-Kyun;Hwang, Jae-Kwan;Chung, Won-Yoon;Kim, Gi-Dae;Lee, Min-Ai;Lee, Sang-Kook
    • Natural Product Sciences
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    • 제18권1호
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    • pp.52-59
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    • 2012
  • In our continuous efforts to procure the active materials from natural products in the protective effects of oxidative stress or UV damage to skin cells we found the Prunus persica flesh extract (PPFE) is considerable to meet the demand to protect the skin damage. PPFE attenuated cell damage induced by hypoxanthine-xanthine oxidase in cultured human keratinocytes, indicating that PPFE has the potential of the scavenging effect of reactive oxygen species (ROS) in human skin cell. Moreover, PPFE significantly suppressed UVA-induced ROS production determined by the oxidation of 2,7-dichlorodihydrofluorescein diacetate (DCFH) using FACS analysis. Additional study revealed that UVA irradiation of HaCaT human keratinocytes increased the gelatinolytic activities of matrix metalloproteinase-2, and -9 (MMP-2, -9) and mRNA expression of MMP-9 analyzing by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and these events were significantly suppressed by the treatment with PPFE. These results suggest that PPFE might be applicable as natural ingredients for skin antiaging agents via UV-induced ROS scavenging activity and suppression of MMP expression in the skin cells.

돌나물추출물에 의한 사람 각질형성세포에서의 Hyaluronan Synthesis 촉진과 인체 피부의 보습력 증진 (Sedum sarmentosum Enhances Hyaluronan Synthesis in Transformed Human Keratinocytes and Increases Water Content in Human Skin)

  • 심관섭;김진화;이동환;나영;이근수;표형배
    • 대한화장품학회지
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    • 제33권1호
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    • pp.17-22
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    • 2007
  • 본 연구에서는 돌나물추출물이 각질형성세포에서 hyaluronan synthase (HAS) mRNA 발현과 hyaluronan (HA) 생성에 미치는 영향을 알아보았다. 또한 돌나물추출물이 가지는 인체 피부에서의 보습력 증진 효과를 평가하였다. 돌나물추출물 처리에 따른 각질형성세포에서의 HAS-1, -2, -3 mRNA 발현 변화는 semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR)을 통해 살펴보았고, HA의 생성량 변화는 enzyme-linked immunosorbent assay (ELISA)를 수행하여 확인하였다. 인체 피부에서의 수분함량 및 피부 수분 손실량에 미치는 영향은 Corneometer와 Tewameter를 이용하여 측정하였다. 그 결과, 돌나물추출물은 사람 각질형성세포의 HAS-2, -3 mRNA의 발현을 증가시켰고, HA의 생성량을 농도의존적으로 증가시킴을 확인하였다. 사람 피부에서는 수분 함량을 유지하는 효과가 우수하였으며, 표피 수분 손실량 또한 감소시켜 피부 보습제로서 매우 우수한 효과를 보였다 이상의 결과를 통해 돌나물은 피부에서 HA 생산을 촉진시키며, 피부 보습력을 증진시키는 화장품 소재로 이용될 수 있을 것으로 사료된다.

갈근(葛根)으로부터 분리된 puerarin의 항당뇨 효과 (Anti-Diabetic Effect of Puerarin Isolated from Puerariae Radix)

  • 임현애;임지선;김정상
    • Current Research on Agriculture and Life Sciences
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    • 제24권
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    • pp.29-35
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    • 2006
  • 본 연구에서는 갈근 및 그의 주 이소플라본인 puerarin의 활성을 세포 수준에서 분석하였다. 먼저 갈근에 함유된 이소플라본의 양을 분석한 결과, puerarin이 총 이소플라본의 90 % 차지하였다. 다음으로는 puerarin의 항당뇨 활성을 검정한 결과 먼저 탄수화물 및 지방소화효소 저해활성에 대해서는 거의 미비한 것으로 나타났으나 인슐린 감수성 및 지방세포의 분화의 유도에 대해서는 농도 의존적으로 작용하는 것으로 관찰되었다. 따라서 puerarin은 지방조직내로 포도당의 흡수를 촉진함으로서 항당뇨 효능을 발휘하는 것으로 추정된다.

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Elevated Expression of Maspin mRNA as a Predictor of Survival in Stage II and III Gallbladder Cancer Cases

  • Baghel, Kavita;Kazmi, Hasan Raza;Raj, Saloni;Chandra, Abhijit;Srivastava, Rajeshwar Nath
    • Asian Pacific Journal of Cancer Prevention
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    • 제15권1호
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    • pp.343-347
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    • 2014
  • Background: Maspin expression is a potential prognostic factor for various malignancies but its relation with gallbladder cancer is unknown and needs to be investigated needs to be investigated. We therefore here focused on maspin mRNA expression in normal, gall stone disease and gallbladder cancer subjects, with particular attention to prognostic importance in individuals with malignancies. Materials and Methods: This study was carried out at the Department of Surgical Gastroenterology, King George's Medical University, Lucknow, India. Gallbladder samples from normal (n=25), gall stone disease (n=25) and cancer patients (n=38) were analysed for maspin mRNA expression by semi-quantitative reverse transcriptase PCR and quantitative real time PCR. Statistical analysis was carried out using the Students t test or ANOVA. Survival analysis was conducted according to the Kaplan-Meier method and correlations were assessed using the Pearson correlation method. p<0.05 was considered statistically significant. Results: Significant increase (p=0.028) in expression of maspin mRNA was observed in gallbladder cancer as compared to gall stone disease, whereas no expression was found in normal tissues. Significant correlation (Pearson's coefficient(r)=-0.798, p<0.0001) was observed between relative quantification of maspin mRNA and survival of cancer patients after surgery, with significantly shorter (p=0.002) survival in patients having relative quantification >1.5 as compared to those having relative quantification <1.5. Similarly, significant differences in patient survival for maspin mRNA expression was observed for stage II (p=0.025) and III (p=0.011) cancer. Conclusions: Higher expression of maspin mRNA in gallbladder cancer has prognostic significance for stage II and III cancer, which needs to be investigated further.

Neurogenic differentiation of human dental stem cells in vitro

  • Lee, Joo-Hee;Um, Soyoun;Song, In-Seok;Kim, Hui Young;Seo, Byoung Moo
    • Journal of the Korean Association of Oral and Maxillofacial Surgeons
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    • 제40권4호
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    • pp.173-180
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    • 2014
  • Objectives: The purpose of this study was to investigate the neurogenic differentiation of human dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), and stem cells from apical papilla (SCAP). Materials and Methods: After induction of neurogenic differentiation using commercial differentiation medium, expression levels of neural markers, microtubule-associated protein 2 (MAP2), class III ${\beta}$-tubulin, and glial fibrillary acidic protein (GFAP) were identified using reverse transcriptase polymerase chain reaction (PCR), real-time PCR, and immunocytochemistry. Results: The induced cells showed neuron-like morphologies, similar to axons, dendrites, and perikaryons, which are composed of neurons in DPSCs, PDLSCs, and SCAP. The mRNA levels of neuronal markers tended to increase in differentiated cells. The expression of MAP2 and ${\beta}$-tubulin III also increased at the protein level in differentiation groups, even though GFAP was not detected via immunocytochemistry. Conclusion: Human dental stem cells including DPSCs, PDLSCs, and SCAP may have neurogenic differentiation capability in vitro. The presented data support the use of human dental stem cells as a possible alternative source of stem cells for therapeutic utility in the treatment of neurological diseases.

Molecular Characterization and Expression Analysis of Insulin-like Growth Factor-1 and Insulin-like Growth Factor Binding Protein-1 Genes in Qinghai-Tibet Plateau Bos grunniens and Lowland Bos taurus

  • Chen, Ya-bing;Fu, Mei;Lan, Dao-liang;Li, Jian
    • Asian-Australasian Journal of Animal Sciences
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    • 제28권1호
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    • pp.20-24
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    • 2015
  • Insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-1 (IGFBP-1) play a pivotal role in regulating cellular hypoxic response. In this study, we cloned and characterized the genes encoding IGF-1 and IGFBP-1 to improve the current knowledge on their roles in highland Bos grunniens (Yak). We also compared their expression levels in the liver and kidney tissues between yaks and lowland cattle. We obtained full-length 465 bp IGF-1 and 792 bp IGFBP-1, encoding 154 amino acids (AA) IGF-1, and 263 AA IGFBP-1 protein, respectively using reverse transcriptase-polyerase chain reaction (RT-PCR) technology. Analysis of their corresponding amino acid sequences showed a high identity between B. grunniens and lowland mammals. Moreover, the two genes were proved to be widely distributed in the examined tissues through expression pattern analysis. Real-time PCR results revealed that IGF-1 expression was higher in the liver and kidney tissues in B. grunniens than in Bos taurus (p<0.05). The IGFBP-1 gene was expressed at a higher level in the liver (p<0.05) of B. taurus than B. grunniens, but it has a similar expression level in the kidneys of the two species. These results indicated that upregulated IGF-1 and downregulated IGFBP-1 are associated with hypoxia adaptive response in B. grunniens.