Kounser Jan, Neelofar Hassan, Antonisamy James, Ishraq Hussain, S. Rashid
Tumors have posed significant threats to human health for over 250 years, emerging as the foremost cause of death. While chemotherapeutic drugs are effective in treating tumors, their side effects can sometimes be challenging to manage during therapy. Nonetheless, there is growing interest in exploring natural compounds as alternatives, which potentially achieve therapeutic outcomes comparable to conventional chemotherapeutics with fewer adverse effects. Paeoniflorin (PF), a monoterpene glycoside derived from the root of Paeonia lactiflora, has garnered significant attention lately due to its promising anti-cancer properties. This review offers an updated outline of the molecular mechanisms underlying PF’s anti-tumor function, with a focus on its modulation of various signaling pathways. PF exerts its anti-tumor activity by regulating crucial cellular processes including apoptosis, angiogenesis, proliferation, and metastasis. We explored the multifaceted impact of PF while modulating through signaling pathways, encompassing nuclear factor kappa B, NOTCH, caspase cascade, transforming growth factor-β, NEDD4, P53/14-3-3, STAT 3, MAPK, MMP-9, and SKP2 signaling pathways, highlighting its versatility in targeting diverse malignancies. Furthermore, we discuss future research directions aimed at exploring innovative and targeted cancer therapies facilitated by PF.
{"title":"Exploring molecular targets in cancer: Unveiling the anticancer potential of Paeoniflorin through a comprehensive analysis of diverse signaling pathways and recent advances","authors":"Kounser Jan, Neelofar Hassan, Antonisamy James, Ishraq Hussain, S. Rashid","doi":"10.14440/jbm.2024.0003","DOIUrl":"https://doi.org/10.14440/jbm.2024.0003","url":null,"abstract":"Tumors have posed significant threats to human health for over 250 years, emerging as the foremost cause of death. While chemotherapeutic drugs are effective in treating tumors, their side effects can sometimes be challenging to manage during therapy. Nonetheless, there is growing interest in exploring natural compounds as alternatives, which potentially achieve therapeutic outcomes comparable to conventional chemotherapeutics with fewer adverse effects. Paeoniflorin (PF), a monoterpene glycoside derived from the root of Paeonia lactiflora, has garnered significant attention lately due to its promising anti-cancer properties. This review offers an updated outline of the molecular mechanisms underlying PF’s anti-tumor function, with a focus on its modulation of various signaling pathways. PF exerts its anti-tumor activity by regulating crucial cellular processes including apoptosis, angiogenesis, proliferation, and metastasis. We explored the multifaceted impact of PF while modulating through signaling pathways, encompassing nuclear factor kappa B, NOTCH, caspase cascade, transforming growth factor-β, NEDD4, P53/14-3-3, STAT 3, MAPK, MMP-9, and SKP2 signaling pathways, highlighting its versatility in targeting diverse malignancies. Furthermore, we discuss future research directions aimed at exploring innovative and targeted cancer therapies facilitated by PF.\u0000","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"127 2","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141835042","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Protein A and Protein L affinity chromatographies are extensively used in mAb and bispecific antibody (bsAb) purification. In addition to product capture, they are both capable of separating certain product-related by-products and aggregates under appropriate conditions. For both types of chromatography, previous studies suggested that adding a salt additive to the mobile phase can significantly improve the resolution between product and by-products/aggregates. Nevertheless, the effects of different salt additives on antibody elution in Protein A and Protein L chromatography have not been compared. In the current study, we compared the effects of three salt additives, sodium chloride (NaCl), calcium chloride (CaCl2), and arginine hydrochloride (Arg·HCl), on antibody elution in Protein A and Protein L chromatography. Interestingly, while NaCl suppressed antibody elution in both types of chromatography, CaCl2, and Arg·HCl promoted antibody elution in Protein A chromatography but suppressed antibody elution in Protein L chromatography. In addition, we evaluated the effect of each salt gradient on aggregate removal by Protein L chromatography. The information provided by the current study should be useful to the selection of conditions/additives for improving by-product removal by Protein A and Protein L chromatography.
蛋白 A 和蛋白 L 亲和色谱广泛用于 mAb 和双特异性抗体（bsAb）的纯化。除了捕获产物外，它们还能在适当条件下分离某些与产物相关的副产物和聚集物。对于这两种色谱法，以往的研究表明，在流动相中添加盐添加剂可显著提高产物与副产物/聚集物之间的分辨力。然而，不同盐添加剂对蛋白质 A 和蛋白质 L 色谱中抗体洗脱的影响尚未进行过比较。在本研究中，我们比较了氯化钠（NaCl）、氯化钙（CaCl2）和盐酸精氨酸（Arg-HCl）这三种盐添加剂对蛋白 A 和蛋白 L 层析中抗体洗脱的影响。有趣的是，氯化钠抑制了抗体在两种层析中的洗脱，而氯化钙和盐酸精氨酸则促进了抗体在蛋白 A 层析中的洗脱，但抑制了抗体在蛋白 L 层析中的洗脱。此外，我们还评估了每种盐梯度对蛋白 L 层析去除聚集体的影响。本研究提供的信息将有助于选择条件/添加剂，以改善蛋白 A 和蛋白 L 色谱对副产物的去除。
{"title":"Calcium chloride and arginine show diametrically opposite effects on antibody elution in Protein A and Protein L chromatography","authors":"Ju Qu, Yan Wan, Yifeng Li","doi":"10.14440/jbm.2024.0006","DOIUrl":"https://doi.org/10.14440/jbm.2024.0006","url":null,"abstract":"Protein A and Protein L affinity chromatographies are extensively used in mAb and bispecific antibody (bsAb) purification. In addition to product capture, they are both capable of separating certain product-related by-products and aggregates under appropriate conditions. For both types of chromatography, previous studies suggested that adding a salt additive to the mobile phase can significantly improve the resolution between product and by-products/aggregates. Nevertheless, the effects of different salt additives on antibody elution in Protein A and Protein L chromatography have not been compared. In the current study, we compared the effects of three salt additives, sodium chloride (NaCl), calcium chloride (CaCl2), and arginine hydrochloride (Arg·HCl), on antibody elution in Protein A and Protein L chromatography. Interestingly, while NaCl suppressed antibody elution in both types of chromatography, CaCl2, and Arg·HCl promoted antibody elution in Protein A chromatography but suppressed antibody elution in Protein L chromatography. In addition, we evaluated the effect of each salt gradient on aggregate removal by Protein L chromatography. The information provided by the current study should be useful to the selection of conditions/additives for improving by-product removal by Protein A and Protein L chromatography.\u0000","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"33 S123","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141835328","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Spatiotemporal regulation of gene expression is essential for maintaining cellular homeostasis throughout kidney development and disease progression. Transcription factors (TFs) and epigenetic modifications play pivotal roles in controlling gene expression. Profiling chromatin modifications across the genome, along with the distribution and target regulation by TFs in specific kidney cell types, is crucial for understanding the dynamic changes in gene expression. Here, we presented a comprehensive workflow for epigenomic, cistromic, and transcriptomic analyses of primary kidney tubular cells. Specifically, our methodologies included the isolation of primary kidney tubular epithelial cells, RNA extraction, assay for transposase-accessible chromatin using sequencing, ultra-low-input micrococcal nuclease-based native chromatin immunoprecipitation, cleavage under targets and release using nuclease, and subsequent bioinformatic analysis. This protocol provides a methodological framework for investigating the roles of TFs and epigenetic modifications in kidney development and diseases.
{"title":"Epigenomic, cistromic, and transcriptomic profiling of primary kidney tubular cells","authors":"Zhiheng Liu, Lirong Zhang, Yupeng Chen","doi":"10.14440/jbm.2024.0009","DOIUrl":"https://doi.org/10.14440/jbm.2024.0009","url":null,"abstract":"Spatiotemporal regulation of gene expression is essential for maintaining cellular homeostasis throughout kidney development and disease progression. Transcription factors (TFs) and epigenetic modifications play pivotal roles in controlling gene expression. Profiling chromatin modifications across the genome, along with the distribution and target regulation by TFs in specific kidney cell types, is crucial for understanding the dynamic changes in gene expression. Here, we presented a comprehensive workflow for epigenomic, cistromic, and transcriptomic analyses of primary kidney tubular cells. Specifically, our methodologies included the isolation of primary kidney tubular epithelial cells, RNA extraction, assay for transposase-accessible chromatin using sequencing, ultra-low-input micrococcal nuclease-based native chromatin immunoprecipitation, cleavage under targets and release using nuclease, and subsequent bioinformatic analysis. This protocol provides a methodological framework for investigating the roles of TFs and epigenetic modifications in kidney development and diseases.\u0000","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"100 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141835607","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Riccardo Donelli, A. Gazzola, C. Mannu, Maryam Etebari, M. Navari, P. Piccaluga
Background: Clonality assessment is currently the major molecular analysis utilized to support the diagnosis of suspicious lymphoid malignancies. Clonal rearrangements of the V-J segments of T-cell receptor G chain locus (TCRγ or TRG) have been observed in almost all types of T neoplasms, such as T-cell-related non-Hodgkin lymphomas and leukemias. At present, the gold standard for clonality evaluation is multiplex polymerase chain reaction (PCR), plus subsequent capillary electrophoresis/heteroduplex analyses, and/or Sanger sequencing. This approach overcomes the problem with the conventional Southern blot hybridization and is more efficient, simple, fast, and reproducible. In the recent years, the new next-generation sequencing (NGS) technologies provided alternative techniques for the analysis of antigen receptors genes, which presented several advantages, such as increased efficiency, specificity (SP), sensitivity (ST), resolution, and objectivity of the results, leading to a better classification, stratification, and monitoring of lymphoid malignancies. Nonetheless, these technologies are still far from being the new gold standard since further studies are warranted to prove their utility. The present study aimed to assess the diagnostic accuracy of these two methods by comparing a commercial NGS-based assay for the evaluation of TRG locus with the gold standard PCR-based one, to fulfill the requirements of a phase 3 diagnostic accuracy study. Methods: We assessed the TRG gene rearrangements in 72 cases using the conventional and highly-validated PCR-based assay proposed by EuroClonality consortium, an alternative commercial PCR-based assay, namely, IdentiClone® TCR Gamma Gene Rearrangement Assay 2.0, and a commercial NGS-based assay, that is, Invivoscribe LymphoTrack® Dx MiSeq® (both by Invivoscribe Technologies Inc., San Diego, CA, USA), to determine the diagnostic accuracy of the latter, and compare them with reference diagnoses made based on observation of clinical manifestations, cytohistological, and immunohistochemical analyses. Statistical values were calculated using the Oxford CATmaker software package. Results: Using standardized criteria of interpretation, the obtained results showed a diagnostic accuracy of 90.3% (correspondence in 65 out of 72 cases) of the test under investigation, with a ST of 86%, a SP of 95%, a positive predicting value of 94%, and a negative predicting value of 88%, demonstrating that Invivoscribe LymphoTrack® Dx MiSeq® assay had high efficiency and reliability in detecting clonal TRG gene rearrangements in T-cell non-Hodgkin lymphomas. Conclusions: This diagnostic accuracy study yielded comparable results using a validated PCR-based approach and a new NGS-based one. Subsequent studies and cost-effectiveness evaluation are needed to put the NGS-based clonality assessment into routine diagnostic practice.
背景：克隆性评估是目前用于支持诊断可疑淋巴恶性肿瘤的主要分子分析方法。在几乎所有类型的 T 细胞肿瘤（如 T 细胞相关的非霍奇金淋巴瘤和白血病）中都可观察到 T 细胞受体 G 链基因座（TCRγ 或 TRG）V-J 段的克隆重排。目前，克隆性评估的黄金标准是多重聚合酶链反应（PCR），以及随后的毛细管电泳/双链分析和/或桑格测序。这种方法克服了传统 Southern 印迹杂交的问题，更高效、简单、快速、可重复。近年来，新的新一代测序（NGS）技术为抗原受体基因分析提供了替代技术，具有效率高、特异性（SP）强、灵敏度（ST）高、分辨率高、结果客观等优点，可更好地对淋巴恶性肿瘤进行分类、分层和监测。尽管如此，这些技术仍远未成为新的金标准，因为还需要进一步的研究来证明它们的实用性。本研究旨在评估这两种方法的诊断准确性，将基于 NGS 的商用检测方法与基于 PCR 的金标准检测方法进行比较，以评估 TRG 基因座的诊断准确性，从而满足 3 期诊断准确性研究的要求。方法：我们评估了 72 个病例的 TRG 基因重排情况，分别使用了欧洲克隆联盟（EuroClonality consortium）提出的经过高度验证的传统 PCR 检测方法、另一种基于 PCR 的商业检测方法 IdentiClone® TCR Gamma 基因重排检测 2.0 和基于 NGS 的商业检测方法 Invivoscribe LymphoTrack® Dx MiSeq®（均由 Invivoscribe Technologies Inc、美国加利福尼亚州圣迭戈市），以确定后者的诊断准确性，并将其与根据临床表现观察、细胞组织学和免疫组化分析做出的参考诊断进行比较。统计值使用牛津 CATmaker 软件包进行计算。结果：使用标准化解释标准得出的结果显示，该检测方法的诊断准确率为 90.3%（72 个病例中有 65 个对应），ST 为 86%，SP 为 95%，阳性预测值为 94%，阴性预测值为 88%，表明 Invivoscribe LymphoTrack® Dx MiSeq® 检测方法在检测 T 细胞非霍奇金淋巴瘤的克隆 TRG 基因重排方面具有很高的效率和可靠性。结论：这项诊断准确性研究使用基于 PCR 的有效方法和基于 NGS 的新方法得出了相似的结果。要将基于 NGS 的克隆性评估应用到常规诊断实践中，还需要进行后续研究和成本效益评估。
{"title":"Conventional PCR-based versus next-generation sequencing-based approach for T-cell receptor γ gene clonality assessment in mature T-cell lymphomas: A phase 3 diagnostic accuracy study","authors":"Riccardo Donelli, A. Gazzola, C. Mannu, Maryam Etebari, M. Navari, P. Piccaluga","doi":"10.14440/jbm.2024.0002","DOIUrl":"https://doi.org/10.14440/jbm.2024.0002","url":null,"abstract":"Background: Clonality assessment is currently the major molecular analysis utilized to support the diagnosis of suspicious lymphoid malignancies. Clonal rearrangements of the V-J segments of T-cell receptor G chain locus (TCRγ or TRG) have been observed in almost all types of T neoplasms, such as T-cell-related non-Hodgkin lymphomas and leukemias. At present, the gold standard for clonality evaluation is multiplex polymerase chain reaction (PCR), plus subsequent capillary electrophoresis/heteroduplex analyses, and/or Sanger sequencing. This approach overcomes the problem with the conventional Southern blot hybridization and is more efficient, simple, fast, and reproducible. In the recent years, the new next-generation sequencing (NGS) technologies provided alternative techniques for the analysis of antigen receptors genes, which presented several advantages, such as increased efficiency, specificity (SP), sensitivity (ST), resolution, and objectivity of the results, leading to a better classification, stratification, and monitoring of lymphoid malignancies. Nonetheless, these technologies are still far from being the new gold standard since further studies are warranted to prove their utility. The present study aimed to assess the diagnostic accuracy of these two methods by comparing a commercial NGS-based assay for the evaluation of TRG locus with the gold standard PCR-based one, to fulfill the requirements of a phase 3 diagnostic accuracy study. Methods: We assessed the TRG gene rearrangements in 72 cases using the conventional and highly-validated PCR-based assay proposed by EuroClonality consortium, an alternative commercial PCR-based assay, namely, IdentiClone® TCR Gamma Gene Rearrangement Assay 2.0, and a commercial NGS-based assay, that is, Invivoscribe LymphoTrack® Dx MiSeq® (both by Invivoscribe Technologies Inc., San Diego, CA, USA), to determine the diagnostic accuracy of the latter, and compare them with reference diagnoses made based on observation of clinical manifestations, cytohistological, and immunohistochemical analyses. Statistical values were calculated using the Oxford CATmaker software package. Results: Using standardized criteria of interpretation, the obtained results showed a diagnostic accuracy of 90.3% (correspondence in 65 out of 72 cases) of the test under investigation, with a ST of 86%, a SP of 95%, a positive predicting value of 94%, and a negative predicting value of 88%, demonstrating that Invivoscribe LymphoTrack® Dx MiSeq® assay had high efficiency and reliability in detecting clonal TRG gene rearrangements in T-cell non-Hodgkin lymphomas. Conclusions: This diagnostic accuracy study yielded comparable results using a validated PCR-based approach and a new NGS-based one. Subsequent studies and cost-effectiveness evaluation are needed to put the NGS-based clonality assessment into routine diagnostic practice.\u0000","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"54 1","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141835717","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
R. K. Jain, Archa Sharma, J. Lalwani, D. Chaurasia, Nagaraj Perumal
This study investigated the influence of relative humidity (RH) on the efficiency of SARS-CoV-2 RNA extraction using the Nextractor automated system. Experiments employing clinical samples demonstrated satisfactory sensitivity and reproducibility for RNA extraction at low humidity (below 50% RH). Conversely, extractions at high humidity (above 70% RH) resulted in complete failure of reverse transcription-polymerase chain reaction assays, with neither SARS-CoV-2 RNA nor the human RNase P gene (internal control) detected. Analysis suggested that residual ethanol, incompletely evaporating due to high humidity, acted as a potent polymerase chain reaction inhibitor in these samples. These findings highlighted the importance of maintaining optimal laboratory humidity (<50% RH) for reliable SARS-CoV-2 RNA extraction using the Nextractor system. Furthermore, laboratories should implement strategies such as regular humidity monitoring, staff training on humidity’s impact, and system validation under specific humidity conditions to ensure accurate molecular diagnostic workflows for COVID-19 testing.
{"title":"Impact of relative humidity on SARS-CoV-2 RNA extraction using Nextractor automated extraction system","authors":"R. K. Jain, Archa Sharma, J. Lalwani, D. Chaurasia, Nagaraj Perumal","doi":"10.14440/jbm.2024.0001","DOIUrl":"https://doi.org/10.14440/jbm.2024.0001","url":null,"abstract":"This study investigated the influence of relative humidity (RH) on the efficiency of SARS-CoV-2 RNA extraction using the Nextractor automated system. Experiments employing clinical samples demonstrated satisfactory sensitivity and reproducibility for RNA extraction at low humidity (below 50% RH). Conversely, extractions at high humidity (above 70% RH) resulted in complete failure of reverse transcription-polymerase chain reaction assays, with neither SARS-CoV-2 RNA nor the human RNase P gene (internal control) detected. Analysis suggested that residual ethanol, incompletely evaporating due to high humidity, acted as a potent polymerase chain reaction inhibitor in these samples. These findings highlighted the importance of maintaining optimal laboratory humidity (<50% RH) for reliable SARS-CoV-2 RNA extraction using the Nextractor system. Furthermore, laboratories should implement strategies such as regular humidity monitoring, staff training on humidity’s impact, and system validation under specific humidity conditions to ensure accurate molecular diagnostic workflows for COVID-19 testing.\u0000","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"65 9","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-07-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141837751","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yan Mu, Ke-Xin Chang, Yu-Feng Chen, Ke Yan, Chun-Xiang Wang, Qian Hua
Alzheimer’s disease (AD) is a serious dementia afflicting aging population and is characterized by cognitive decline, amyloid-β plaques, and neurofibrillary tangles. AD substantially impairs the life quality of the victims and poses a heavy burden on the society at large. The number of people with dementia due to AD, prodromal AD, and preclinical AD is estimated to stand at roughly 3.2, 69, and 315 million worldwide, respectively. Current clinical diagnosis is based on clinical symptoms, and clinical research demonstrated that positron emission tomography (PET) and cerebrospinal fluid (CSF) biomarkers had excellent diagnostic performance. However, the application of CSF biomarker tests and PET are restricted by the invasiveness and high cost. The presence of clinical symptoms means that AD pathology has been progressing for many years, and only a few drugs have been approved for the traetemnt of AD. Therefore, early diagnosis is extremely important for controlling the outcomes caused by AD. In this review, we provided an overview of developing clinical diagnostic criteria, diagnostic strategies under clinical research, developing blood based-biomarker assays, and promising nanotechnologically-based assays.
阿尔茨海默病（AD）是一种严重困扰老年人群的痴呆症，以认知能力下降、淀粉样β斑块和神经纤维缠结为特征。老年痴呆症严重影响患者的生活质量，并给整个社会带来沉重负担。据估计，全球因渐变性痴呆、渐变性痴呆前兆和临床前渐变性痴呆导致的痴呆患者人数分别约为 320 万、6 900 万和 3.15 亿。目前的临床诊断以临床症状为基础，临床研究表明正电子发射断层扫描（PET）和脑脊液（CSF）生物标志物具有良好的诊断性能。然而，脑脊液生物标志物检测和正电子发射计算机断层扫描因其侵入性和高成本而限制了其应用。临床症状的出现意味着AD病理已发展多年，而目前仅有少数药物被批准用于AD的治疗。因此，早期诊断对于控制 AD 所造成的后果极其重要。在这篇综述中，我们概述了正在制定的临床诊断标准、临床研究中的诊断策略、正在开发的基于血液的生物标志物检测方法以及前景看好的基于纳米技术的检测方法。
{"title":"Diagnosis of Alzheimer's disease: Towards accuracy and accessibility","authors":"Yan Mu, Ke-Xin Chang, Yu-Feng Chen, Ke Yan, Chun-Xiang Wang, Qian Hua","doi":"10.14440/jbm.2024.412","DOIUrl":"https://doi.org/10.14440/jbm.2024.412","url":null,"abstract":"Alzheimer’s disease (AD) is a serious dementia afflicting aging population and is characterized by cognitive decline, amyloid-β plaques, and neurofibrillary tangles. AD substantially impairs the life quality of the victims and poses a heavy burden on the society at large. The number of people with dementia due to AD, prodromal AD, and preclinical AD is estimated to stand at roughly 3.2, 69, and 315 million worldwide, respectively. Current clinical diagnosis is based on clinical symptoms, and clinical research demonstrated that positron emission tomography (PET) and cerebrospinal fluid (CSF) biomarkers had excellent diagnostic performance. However, the application of CSF biomarker tests and PET are restricted by the invasiveness and high cost. The presence of clinical symptoms means that AD pathology has been progressing for many years, and only a few drugs have been approved for the traetemnt of AD. Therefore, early diagnosis is extremely important for controlling the outcomes caused by AD. In this review, we provided an overview of developing clinical diagnostic criteria, diagnostic strategies under clinical research, developing blood based-biomarker assays, and promising nanotechnologically-based assays.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":" 18","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-03-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140390585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Akabane virus (AKAV) is an arbovirus belonging to the family Bunyaviridae, genus Orthobunyavirus. AKAV consists of three-segment (L, M, and S RNA segments), negative single-stranded RNA. The aim of this study was to investigate an in situ hybridization method (ISH) in a Vero E6 cell line infected with Akabane virus. The 320 base pair amplicon was obtained by RT-PCR with a primer pair and labeled with digoxigenin. Akabane virus RNAs were seen as a granular pattern in the cytoplasm of infected cells. As a result, the expression of the particular Akabane virus gene area was successfully disclosed in the current investigation using the ISH method with a digoxigenin-labeled probe.
赤羽病毒（AKAV）是一种虫媒病毒，属于布尼亚病毒科、正布尼亚病毒属。AKAV 由三段（L、M 和 S RNA 段）阴性单链 RNA 组成。本研究的目的是在感染赤羽病毒的 Vero E6 细胞系中研究一种原位杂交方法（ISH）。使用引物对通过 RT-PCR 获得 320 碱基对扩增片段，并用地高辛标记。在感染细胞的细胞质中，可以看到赤羽病毒 RNA 呈颗粒状。因此，在本次研究中，使用地高辛标记探针的 ISH 方法成功地揭示了赤羽病毒特定基因区域的表达。
{"title":"Development of an In Situ Hybridization Method for Detection of Akabane Virus","authors":"Nihat Toplu, T. Ç. Oğuzoğlu, A. Akkoç","doi":"10.14440/jbm.2024.413","DOIUrl":"https://doi.org/10.14440/jbm.2024.413","url":null,"abstract":"Akabane virus (AKAV) is an arbovirus belonging to the family Bunyaviridae, genus Orthobunyavirus. AKAV consists of three-segment (L, M, and S RNA segments), negative single-stranded RNA. The aim of this study was to investigate an in situ hybridization method (ISH) in a Vero E6 cell line infected with Akabane virus. The 320 base pair amplicon was obtained by RT-PCR with a primer pair and labeled with digoxigenin. Akabane virus RNAs were seen as a granular pattern in the cytoplasm of infected cells. As a result, the expression of the particular Akabane virus gene area was successfully disclosed in the current investigation using the ISH method with a digoxigenin-labeled probe.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"17 3","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-02-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140437393","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Minghang Yu, Yang Xiong, Pu Liang, Danying Chen, Yuting Zhang, Huan Liu, Yuanyuan Zhang, Xuesen Zhao, Ronghua Jin, Xi Wang
The rapid identification SARS-CoV-2 virus has become the basis for the control of the COVID-19 outbreak. The rapid antigen tests for SARS-CoV-2 are quick, widely available, and inexpensive. Rapid antigen tests have gradually replaced the time-consuming and costly RT-PCR. Currently, although several RAT kits have been extensively used for the diagnosis of COVID-19, validity data are limited due to the inconsistent sensitivity and poor reproducibility. Meanwhile, WHO does not recommend specific commercial RAT kits. Therefore, it is crucial to establish a method to evaluate the effectiveness of different rapid antigen tests kits. This study aimed to develop an evaluation system for rapid antigen tests to provide an efficient and accurate technique for screening SARS-CoV-2 antigen detection kits. Given large number of rapid antigen tests kits available, this study only focused on those that are representative and commonely used in China. By minimzing biases through randomization, concealment, and blinding, we eventually found that the Test 1 had the lowest sensitivity and the Test VI had the highest sensitivity. This study provided an evaluation platform that can potentially serve as a reference for COVID-19 diagnostic strategies.
快速鉴定 SARS-CoV-2 病毒已成为控制 COVID-19 爆发的基础。SARS-CoV-2 的快速抗原检测快速、广泛、廉价。快速抗原检测已逐渐取代耗时费钱的 RT-PCR。目前，虽然有几种快速抗原检测试剂盒已被广泛用于诊断 COVID-19，但由于灵敏度不一致和重现性差，有效数据有限。同时，世卫组织并未推荐特定的商业 RAT 试剂盒。因此，建立一套评估不同快速抗原检测试剂盒有效性的方法至关重要。本研究旨在开发一套快速抗原检测评估系统，为筛选 SARS-CoV-2 抗原检测试剂盒提供一种高效、准确的技术。鉴于快速抗原检测试剂盒种类繁多，本研究只关注在中国具有代表性且普遍使用的试剂盒。通过随机、隐蔽和盲法将偏倚最小化，我们最终发现检测 1 的灵敏度最低，而检测 VI 的灵敏度最高。这项研究提供了一个评估平台，有可能作为 COVID-19 诊断策略的参考。
{"title":"Validation of 12 Rapid Antigen Tests for the Detection of SARS-CoV-2","authors":"Minghang Yu, Yang Xiong, Pu Liang, Danying Chen, Yuting Zhang, Huan Liu, Yuanyuan Zhang, Xuesen Zhao, Ronghua Jin, Xi Wang","doi":"10.14440/jbm.2024.409","DOIUrl":"https://doi.org/10.14440/jbm.2024.409","url":null,"abstract":"The rapid identification SARS-CoV-2 virus has become the basis for the control of the COVID-19 outbreak. The rapid antigen tests for SARS-CoV-2 are quick, widely available, and inexpensive. Rapid antigen tests have gradually replaced the time-consuming and costly RT-PCR. Currently, although several RAT kits have been extensively used for the diagnosis of COVID-19, validity data are limited due to the inconsistent sensitivity and poor reproducibility. Meanwhile, WHO does not recommend specific commercial RAT kits. Therefore, it is crucial to establish a method to evaluate the effectiveness of different rapid antigen tests kits. This study aimed to develop an evaluation system for rapid antigen tests to provide an efficient and accurate technique for screening SARS-CoV-2 antigen detection kits. Given large number of rapid antigen tests kits available, this study only focused on those that are representative and commonely used in China. By minimzing biases through randomization, concealment, and blinding, we eventually found that the Test 1 had the lowest sensitivity and the Test VI had the highest sensitivity. This study provided an evaluation platform that can potentially serve as a reference for COVID-19 diagnostic strategies.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"12 12","pages":""},"PeriodicalIF":0.0,"publicationDate":"2024-01-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"139532789","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2023-11-20eCollection Date: 2023-01-01DOI: 10.14440/jbm.2023.408
Rachel Twomey, Sean Graham, Joseph S Spina, Xiaoming Wu, Philip E Dubé, Courtney Ferrebee, William Housley
Mouse models with humanized immune systems are becoming increasingly prevalent in pharmaceutical research as a platform for preclinical testing with potential for greater translatability to clinical applications. However, the presence of both mouse and human cells that respond to TLR ligands poses a challenge for investigating therapeutic modalities targeting TLR signaling. AZ617 is a human TLR4 agonist, which has been shown in vitro to preferentially induce human cytokines via the TLR4 signaling pathway. We sought to examine the ability of AZ617 to preferentially induce human cytokines in CD34+ stem cell-engrafted NOG-EXL mice (huNOG-EXL), to determine its suitability as an in vivo human functional readout. AZ617 elicited a strong human TNFα and IL-6 response in vivo that demonstrated a 10- and 5-fold preference, respectively, over the mouse TNFα and IL-6. To assess efficacy of inhibiting a key protein in the TLR4 signaling pathway, PF-06650833, a small molecule inhibitor of IRAK4, was used as a tool molecule. PF-0660833 was found to effectively inhibit AZ617-induced human TNFα release in vitro. Likewise, PF-06650833 reduced AZ617-induced human TNFα in the huNOG-EXL mouse model, with a weaker effect on human IL-6. A longitudinal study tracking functionality of monocytes revealed that the ability of monocytes to respond to ex vivo stimuli was increased by 21 weeks after engraftment. Taken together, our data suggests that human selective TLR ligands could preferentially drive cytokine production from human cells in huNOG-EXL mice. This model will allow for investigation of pharmacological inhibition of human TLR signaling pathways in an in vivo model system.
{"title":"Utilizing a human TLR selective ligand in a humanized immune system mouse model to investigate human TLR4 signaling.","authors":"Rachel Twomey, Sean Graham, Joseph S Spina, Xiaoming Wu, Philip E Dubé, Courtney Ferrebee, William Housley","doi":"10.14440/jbm.2023.408","DOIUrl":"10.14440/jbm.2023.408","url":null,"abstract":"<p><p>Mouse models with humanized immune systems are becoming increasingly prevalent in pharmaceutical research as a platform for preclinical testing with potential for greater translatability to clinical applications. However, the presence of both mouse and human cells that respond to TLR ligands poses a challenge for investigating therapeutic modalities targeting TLR signaling. AZ617 is a human TLR4 agonist, which has been shown <i>in vitro</i> to preferentially induce human cytokines via the TLR4 signaling pathway. We sought to examine the ability of AZ617 to preferentially induce human cytokines in CD34+ stem cell-engrafted NOG-EXL mice (huNOG-EXL), to determine its suitability as an <i>in vivo</i> human functional readout. AZ617 elicited a strong human TNFα and IL-6 response <i>in vivo</i> that demonstrated a 10- and 5-fold preference, respectively, over the mouse TNFα and IL-6. To assess efficacy of inhibiting a key protein in the TLR4 signaling pathway, PF-06650833, a small molecule inhibitor of IRAK4, was used as a tool molecule. PF-0660833 was found to effectively inhibit AZ617-induced human TNFα release <i>in vitro</i>. Likewise, PF-06650833 reduced AZ617-induced human TNFα in the huNOG-EXL mouse model, with a weaker effect on human IL-6. A longitudinal study tracking functionality of monocytes revealed that the ability of monocytes to respond to <i>ex vivo</i> stimuli was increased by 21 weeks after engraftment. Taken together, our data suggests that human selective TLR ligands could preferentially drive cytokine production from human cells in huNOG-EXL mice. This model will allow for investigation of pharmacological inhibition of human TLR signaling pathways in an <i>in vivo</i> model system.</p>","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"10 ","pages":""},"PeriodicalIF":0.0,"publicationDate":"2023-11-20","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10691501/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138479727","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sonia Del Prete, Marta Gogliettino, Gianna Palmieri, Ennio Cocca
Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation.We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5’ end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.
{"title":"A strategy to recover a poor-quality ligase product","authors":"Sonia Del Prete, Marta Gogliettino, Gianna Palmieri, Ennio Cocca","doi":"10.14440/jbm.2023.411","DOIUrl":"https://doi.org/10.14440/jbm.2023.411","url":null,"abstract":"Over the last decades, PCR and molecular cloning have profoundly impacted various biological areas, from basic to pharmaceutical sciences. Presented in this study is a simple and step-by-step protocol that uses PCR to recover a poor-quality ligase product. In fact, a classic step that can be problematic in typical recombinant DNA manipulations can be the recovery of a product from a T4 DNA ligase reaction between two or more suitably prepared DNA fragments (sticky ends, blunt ends, TA cloning, etc.). This reaction can result in poor yields of the ligation product, due to various causes, mainly the preparation of the DNA fragments, and the poor yield can severely invalidate all subsequent steps. To overcome this problem, we designed a pair of PCR primers to amplify the entire ligase product into satisfactory amount. Of course, high-fidelity DNA polymerase must be used to obtain a faithful copy of the DNA of interest. The fragment thus amplified can then be inserted into a suitable vector and propagated by bacterial transformation.We applied this procedure to modify a synthetic gene by adding a His-Tag to its 5’ end, and to insert this new construct into an expression cassette. This last step was achieved by employing a PCR cloning system. In our practical example, comprehensive PCR-based protocol with important tips were introduced. This methodological paper can serve as a roadmap for biologists who want to quickly/fully exploit the potential of the PCR-cloning to get desired constructs.","PeriodicalId":73618,"journal":{"name":"Journal of biological methods","volume":"98 8","pages":"0"},"PeriodicalIF":0.0,"publicationDate":"2023-11-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"135091708","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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