Use of Humanised Rat Basophilic Leukaemia Cell Line RS-ATL8 for the Assessment of Allergenicity of Schistosoma mansoni Proteins

Background

Parasite-specific IgE is thought to correlate with protection against Schistosoma mansoni infection or re-infection. Only a few molecular targets of the IgE response in S. mansoni infection have been characterised. A better insight into the basic mechanisms of anti-parasite immunity could be gained from a genome-wide characterisation of such S. mansoni allergens. This would have repercussions on our understanding of allergy and the development of safe and efficacious vaccinations against helminthic parasites.

Methodology/Principal Findings

A complete medium- to high-throughput amenable workflow, including important quality controls, is described, which enables the rapid translation of S. mansoni proteins using wheat germ lysate and subsequent assessment of potential allergenicity with a humanised Rat Basophilic Leukemia (RBL) reporter cell line. Cell-free translation is completed within 90 minutes, generating sufficient amounts of parasitic protein for rapid screening of allergenicity without any need for purification. Antigenic integrity is demonstrated using Western Blotting. After overnight incubation with infected individuals'' serum, the RS-ATL8 reporter cell line is challenged with the complete wheat germ translation mixture and Luciferase activity measured, reporting cellular activation by the suspected allergen. The suitability of this system for characterization of novel S. mansoni allergens is demonstrated using well characterised plant and parasitic allergens such as Par j 2, SmTAL-1 and the IgE binding factor IPSE/alpha-1, expressed in wheat germ lysates and/or E. coli. SmTAL-1, but not SmTAL2 (used as a negative control), was able to activate the basophil reporter cell line.

Conclusion/Significance

This method offers an accessible way for assessment of potential allergenicity of anti-helminthic vaccine candidates and is suitable for medium- to high-throughput studies using infected individual sera. It is also suitable for the study of the basis of allergenicity of helminthic proteins.     

Osteoclast Derivation from Mouse Bone Marrow

Osteoclasts are highly specialized cells that are derived from the monocyte/macrophage lineage of the bone marrow. Their unique ability to resorb both the organic and inorganic matrices of bone means that they play a key role in regulating skeletal remodeling. Together, osteoblasts and osteoclasts are responsible for the dynamic coupling process that involves both bone resorption and bone formation acting together to maintain the normal skeleton during health and disease.As the principal bone-resorbing cell in the body, changes in osteoclast differentiation or function can result in profound effects in the body. Diseases associated with altered osteoclast function can range in severity from lethal neonatal disease due to failure to form a marrow space for hematopoiesis, to more commonly observed pathologies such as osteoporosis, in which excessive osteoclastic bone resorption predisposes to fracture formation.An ability to isolate osteoclasts in high numbers in vitro has allowed for significant advances in the understanding of the bone remodeling cycle and has paved the way for the discovery of novel therapeutic strategies that combat these diseases. Here, we describe a protocol to isolate and cultivate osteoclasts from mouse bone marrow that will yield large numbers of osteoclasts.     

Discrepant Expression of Cytokines in Inflammation- and Age-Related Cataract Patients

Purpose

Inflammatory cataracts secondary to Behcet''s disease (BD) or Vogt-Koyanagi-Harada disease (VKH) are thought to result from a pathological dysregulation of cytokines that is different from that of age-related (AR) cataracts. However, little is known about the function of cytokines in the development of inflammatory cataracts. The purpose of this study was to identify possible differences in cytokine expression in inflammation- and age-related cataract patients.

Methods

Analysis techniques involving the concomitant use of a cocktail of antibody-coated non-magnetic beads were used to determine the cytokine expression profiles of BD, VKH and AR cataract patients. Furthermore, anterior chamber aqueous flares and inflammatory cells were quantitatively measured with a laser flare cell meter (LFCM).

Results

The expressions of interleukin-2 (IL-2), IL-4, IL-6, IL-10, IL-17A, and interferon-γ (IFN-γ) were analyzed in aqueous humor (AqH), phytohemagglutinin (PHA)-stimulated and non-PHA-stimulated cultures of peripheral blood mononuclear cells (PBMCs) from the three types of cataract patients. IL-6 and IFN-γ were identified above the detection limits, but, among the BD and VKH cataract patients, only the levels of IL-6 were significantly higher in both the AqH and PBMC non-PHA cultures compared with the levels observed in the AR cataract patients. In contrast, IFN-γ was significantly elevated in the AqH of the BD cataract patients compared with the VKH and AR cataract patients. In the PHA-stimulated PBMC cultures, IL-2, IFN-γ, IL-6, and IL-17A were significantly increased, and the IL-6 level was significantly higher in the VKH patients than in the BD and AR cataract patients. The correlation analyses of the cytokines and inflammation indexes of the AqH obtained with the LFCM revealed that only IL-6 was significantly correlated with the inflammation index.

Conclusion

Distinct expression profiles of cytokines and the correlations of these profiles with in vivo inflammatory indexes for inflammatory and AR cataract patients were identified.     

Nanomanipulation of Single RNA Molecules by Optical Tweezers

A large portion of the human genome is transcribed but not translated. In this post genomic era, regulatory functions of RNA have been shown to be increasingly important. As RNA function often depends on its ability to adopt alternative structures, it is difficult to predict RNA three-dimensional structures directly from sequence. Single-molecule approaches show potentials to solve the problem of RNA structural polymorphism by monitoring molecular structures one molecule at a time. This work presents a method to precisely manipulate the folding and structure of single RNA molecules using optical tweezers. First, methods to synthesize molecules suitable for single-molecule mechanical work are described. Next, various calibration procedures to ensure the proper operations of the optical tweezers are discussed. Next, various experiments are explained. To demonstrate the utility of the technique, results of mechanically unfolding RNA hairpins and a single RNA kissing complex are used as evidence. In these examples, the nanomanipulation technique was used to study folding of each structural domain, including secondary and tertiary, independently. Lastly, the limitations and future applications of the method are discussed.     

SPAG5 upregulation predicts poor prognosis in cervical cancer patients and alters sensitivity to taxol treatment via the mTOR signaling pathway

Previously, we found that sperm-associated antigen 5 (SPAG5) was upregulated in pelvic lymph node metastasis–positive cervical cancer. The aim of this study is to examine the role of SPAG5 in the proliferation and tumorigenicity of cervical cancer and its clinical significance in tumor progression. In our study, SPAG5 expression in cervical cancer patients was detected using quantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry; cervical cancer cell function with downregulated SPAG5 in vitro was explored using tetrazolium assay, flow cytometry, and colony formation and Transwell assays. SPAG5 was upregulated in tumor tissue compared with paired adjacent noncancerous tissues; SPAG5 upregulation in tumor tissues indicated poor disease-free survival, which was also an independent prognostic indicator for cervical cancer patients. In vitro study demonstrated that SPAG5 downregulation inhibited cell proliferation and growth significantly by G2/M arrest and induction of apoptosis, and hindered cell migration and invasion. Under SPAG5 downregulation, the sensitivity of cervical cancer cells differed according to taxol dose, which correlated with mammalian target of rapamycin (mTOR) signaling pathway activity. In general, SPAG5 upregulation relates to poor prognosis in cervical cancer patients, and SPAG5 is a regulator of mTOR activity during taxol treatment in cervical cancer.     

氯苯胁迫对蚕豆幼苗生长和细胞分裂的影响

研究了1,2,4-三氯苯(TCB)对蚕豆幼苗生长、根尖细胞分裂及染色体畸变的影响．结果表明,随TCB浓度增加和处理时间延长,蚕豆幼苗根长的生长及根尖细胞有丝分裂指数降低甚至停止．TCB诱发蚕豆根尖细胞有丝分裂过程中染色体数目畸变和结构畸变．50-100μg.g^-1TCB胁迫12-24h,蚕豆根尖染色体的主要损伤形式为c-有丝分裂、染色体桥和不均匀排列,其出现百分率达1．0％--10.3％．300μg.g^-1TCB胁迫12-96h,蚕豆根尖细胞中染色体粘连(S)、S+染色体断裂(S+B)、S+染色体环(S+R)、S+染色体不均匀排列(S+A)及S+染色体桥(S+Be)出现的百分率达47．9％--88.9％,各种类型染色体断裂出现的百分率仅为18．1％--29．6％,说明蚕豆根尖细胞染色体畸变分析可作为TCB土壤污染监测的敏感生物监测指标．     

Trends in HSPB5 research: a 36-year bibliometric analysis

HSPB5 (heat shock protein B5), also known as αB-crystallin, is one of the most widespread and populous of the ten human small heat shock proteins (sHsps). Over the past decades, extensive research has been conducted on HSPB5. However, few studies have statistically analyzed these publications. Herein, we conducted a bibliometric analysis to track the global research trend and current development status of HSPB5 research from the Web of Science Core Collection (WoSCC) database between 1985 and 2020. Our results demonstrate that 1220 original articles cited 54,778 times in 391 scholarly journals were published. Visualization analyses reveal that the Journal of Biological Chemistry was the most influential journal with 85 articles. The USA dominated this field with 520 publications (42.62%), followed by Japan with 149 publications (12.21%), and Kato contributed the largest number of publications. Most related publications were published in journals focusing on biochemistry molecular biology, cell biology, neurosciences neurology, and ophthalmology. In addition, keyword co-occurrence analyses identify three predominant research topics: expression of HSPB5, chaperone studies for HSPB5, and pathological studies of HSPB5. This study provides valuable guidance for researchers and leads to collaborative opportunities between diverse research interests to be integrated for HSPB5 research.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01220-6.     

蝎毒对癫痫大鼠海马内强啡肽原mRNA表达的影响

目的和方法：本工作用红藻氨酸癫痫模型,通过对癫痫大鼠蝎毒处理后民内哟啡肽原ｍＲＮＡ（ＫＰＵＹＮｍＲＮＡ）表达的原位杂交观察,对国产蝎粗毒抗癫痫反复发作的细胞分子机制进行初步探讨。结果：原位杂交的实验显示,三周ＫＡ后,实验对照组与空白对照组相比,腹侧海马尤其是海马门区ＰＤＹＮｍＲＮＡ阳性数目明显减少（Ｐ〈０．０１）。实验给药组大鼠８例,其中有６例腹侧海马门区ＰＤＹＮｍＲＮＡ阳性神经元数目未见减少且有