Biological Procedures Online最新文献

The emergence of antibiotic-resistant pathogens has threatened not only our ability to deal with common infectious diseases but also the management of life-threatening complications. Antimicrobial resistance (AMR) remains a significant threat in both industrialized and developing countries alike. In Africa, though, poor clinical care, indiscriminate antibiotic use, lack of robust AMR surveillance programs, lack of proper regulations and the burden of communicable diseases are factors aggravating the problem of AMR. In order to effectively address the challenge of AMR, antimicrobial stewardship programs, solid AMR surveillance systems to monitor the trend of resistance, as well as robust, affordable and rapid diagnostic tools which generate data that informs decision-making, have been demonstrated to be effective. However, we have identified a significant knowledge gap in the area of the application of fast and affordable diagnostic tools, surveillance, and stewardship programs in Africa. Therefore, we set out to provide up-to-date information in these areas. We discussed available hospital-based stewardship initiatives in addition to the role of governmental and non-governmental organizations. Finally, we have reviewed the application of various phenotypic and molecular AMR detection tools in both research and routine laboratory settings in Africa, deployment challenges and the efficiency of these methods.

Aims: We focused on the detailed functions of circ-ABCB10 in cervical cancer (CC) development and its mechanisms.

Background: The increasing findings have proposed the central roles of circular RNAs (circRNAs) in the tumorigenesis of various human cancers. Circ-ABCB10 displays promising oncogenic effect in several tumors.

Methods: Circ-ABCB10 and miR-128-3p production levels in CC tissues and cells were tested through RT-qPCR. The association of circ-ABCB10 expression with clinicopathologic parameters of CC patients was statistically analyzed. Cell proliferation, invasion, apoptosis, and epithelial-mesenchymal transition (EMT) were evaluated by MTT, transwell invasion assays, flow cytometry analyses, and western blot examination of EMT markers. The binding activity between miR-128-3p and circ-ABCB10 or zinc finger E-box binding homeobox 1 (ZEB1) was explored through pull-down assay or luciferase reporter assay. The influence of circ-ABCB10 on CC tumorigenesis was evaluated by in vivo xenograft experiments.

Results: The elevated circ-ABCB10 expression was determined in CC tissues and cells. Moreover, higher production level of circ-ABCB10 was close related to lymph-node metastasis, Federation of Gynecology and Obstetrics (FIGO) stage, and tumor size in CC patients. Loss of circ-ABCB10 weakened cell proliferative and invasive abilities, inhibited EMT, and induced apoptosis in CC. Loss of circ-ABCB10 inhibited ZEB1 expression by serving as a sponge of miR-128-3p in CC cells. Circ-ABCB10 sponged miR-128-3p to enhance cell proliferation, invasion, EMT and inhibit apoptosis in CC cells. Xenograft tumor assays confirmed that circ-ABCB10 knockdown inhibited CC tumor growth.

Conclusion: Our study suggests that circ-ABCB10 depletion inhibits proliferation, invasion and EMT and promotes apoptosis of cervical cancer cells through miR-128-3p/ZEB1 axis and represses CC tumor growth.

Background: Ocriplasmin (Jetrea) is using for the treatment of symptomatic vitreomacular adhesion. This enzyme undergoes rapid inactivation and limited activity duration as a result of its autolytic nature after injection within the eye. Moreover, the proteolytic activity can cause photoreceptor damage, which may result in visual impairment in more serious cases.

Results: The present research aimed to reduce the disadvantages of ocriplasmin using site-directed mutagenesis. To reduce the autolytic activity of ocriplasmin in the first variant, lysine 156 changed to glutamic acid and, in the second variant for the proteolytic activity reduction, alanine 59 mutated to threonine. The third variant contained both mutations. Expression of wild type and three mutant variants of ocriplasmin constructs were done in the Pichia pastoris expression system. The mutant variants were analyzed in silico and in vitro and compared to the wild type. The kinetic parameters of ocriplasmin variants showed both variants with K156E substitution were more resistant to autolytic degradation than wild-type. These variants also exhibited reduced K_cat and V_max values. An increase in their Km values, leading to a decreased catalytic efficiency (the K_cat/K_m ratio) of autolytic and mixed variants. Moreover, in the variant with A59T mutation, K_cat and V_max values have reduced compared to wild type. The mix variants showed the most increase in Km value (almost 2-fold) as well as reduced enzymatic affinity to the substrate. Thus, the results indicated that combined mutations at the ocriplasmin sequence were more effective compared with single mutations.

Conclusions: The results indicated such variants represent valuable tools for the investigation of therapeutic strategies aiming at the non-surgical resolution of vitreomacular adhesion.

An amendment to this paper has been published and can be accessed via the original article.

Background: DNA repair pathways, cell cycle arrest checkpoints, and cell death induction are present in cells to process DNA damage and prevent genomic instability caused by various extrinsic and intrinsic ionizing factors. Mutations in the genes involved in these pathways enhances the ionizing radiation sensitivity, reduces the individual's capacity to repair DNA damages, and subsequently increases susceptibility to tumorigenesis.

Body: BRCA1 and BRCA2 are two highly penetrant genes involved in the inherited breast cancer and contribute to different DNA damage pathways and cell cycle and apoptosis cascades. Mutations in these genes have been associated with hypersensitivity and genetic instability as well as manifesting severe radiotherapy complications in breast cancer patients. The genomic instability and DNA repair capacity of breast cancer patients with BRCA1/2 mutations have been analyzed in different studies using a variety of assays, including micronucleus assay, comet assay, chromosomal assay, colony-forming assay, γ -H2AX and 53BP1 biomarkers, and fluorescence in situ hybridization. The majority of studies confirmed the enhanced spontaneous & radiation-induced radiosensitivity of breast cancer patients compared to healthy controls. Using G2 micronucleus assay and G2 chromosomal assay, most studies have reported the lymphocyte of healthy carriers with BRCA1 mutation are hypersensitive to invitro ionizing radiation compared to non-carriers without a history of breast cancer. However, it seems this approach is not likely to be useful to distinguish the BRCA carriers from non-carrier with familial history of breast cancer.

Conclusion: In overall, breast cancer patients are more radiosensitive compared to healthy control; however, inconsistent results exist about the ability of current radiosensitive techniques in screening BRCA1/2 carriers or those susceptible to radiotherapy complications. Therefore, developing further radiosensitivity assay is still warranted to evaluate the DNA repair capacity of individuals with BRCA1/2 mutations and serve as a predictive factor for increased risk of cancer mainly in the relatives of breast cancer patients. Moreover, it can provide more evidence about who is susceptible to manifest severe complication after radiotherapy.

Interest in CRISPR technology, an instrumental component of prokaryotic adaptive immunity which enables prokaryotes to detect any foreign DNA and then destroy it, has gained popularity among members of the scientific community. This is due to CRISPR's remarkable gene editing and cleaving abilities. While the application of CRISPR in human genome editing and diagnosis needs to be researched more fully, and any potential side effects or ambiguities resolved, CRISPR has already shown its capacity in an astonishing variety of applications related to genome editing and genetic engineering. One of its most currently relevant applications is in diagnosis of infectious and non-infectious diseases. Since its initial discovery, 6 types and 22 subtypes of CRISPR systems have been discovered and explored. Diagnostic CRISPR systems are most often derived from types II, V, and VI. Different types of CRISPR-Cas systems which have been identified in different microorganisms can target DNA (e.g. Cas9 and Cas12 enzymes) or RNA (e.g. Cas13 enzyme). Viral, bacterial, and non-infectious diseases such as cancer can all be diagnosed using the cleavage activity of CRISPR enzymes from the aforementioned types. Diagnostic tests using Cas12 and Cas13 enzymes have already been developed for detection of the emerging SARS-CoV-2 virus. Additionally, CRISPR diagnostic tests can be performed using simple reagents and paper-based lateral flow assays, which can potentially reduce laboratory and patient costs significantly. In this review, the classification of CRISPR-Cas systems as well as the basis of the CRISPR/Cas mechanisms of action will be presented. The application of these systems in medical diagnostics with emphasis on the diagnosis of COVID-19 will be discussed.