The DNA polymerase (POL) of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral DNA replication and, thus, may be considered as a viable target for anti-KSHV therapeutics. To produce large quantities of homogeneous and pure POL required for high-throughput screening (HTS) for inhibitors, we generated a recombinant baculovirus vector encoding a hexahistidine (His6)-tagged POL and infected Spodoptera frugiperda Sf-9 insect cells. High expression of recombinant POL (rPOL) was achieved for up to 72h post-infection. The rPOL was solubilized in lysis buffer containing 0.3% Cymal-5 detergent, purified by metal-chelating and dsDNA-cellulose affinity chromatography, and analyzed by anti-His antibody Western blot and mass spectrometry. The functionality of rPOL was confirmed by its DNA synthesis activity in vitro, which was effectively blocked by the anti-herpetic DNA polymerase inhibitors, foscarnet and cidofovir diphosphate, in a dose-dependent manner. The POL expressed and purified from the recombinant baculovirus-infected insect cells may be useful toward the development of HTS of large chemical libraries to identify novel KSHV DNA polymerase inhibitors.
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http://dx.doi.org/10.1016/s1046-5928(03)00017-2 | DOI Listing |
BMC Infect Dis
January 2025
Diagnostic Systems Division, United States Army Medical Research Institute of Infectious Diseases, Fort Detrick, Maryland, 21702, United States of America.
Background: Point of need diagnostics provide efficient testing capability for remote or austere locations, decreasing the time to answer by minimizing travel or sample transport requirements. Loop-mediated isothermal amplification (LAMP) is an appealing technology for point-of-need diagnostics due to its rapid analysis time and minimal instrumentation requirements.
Methods: Here, we designed and optimized nine LAMP assays that are sensitive and specific to targeted bacterial select agents including Bacillus anthracis, Francisella tularensis, Yersinia pestis, and Brucella spp.
Medicine (Baltimore)
November 2024
Department of Neurosurgery, Wuhan Hanyang Hospital, Wuhan, Hubei, China.
This study aimed to investigate the polymorphisms of circadian clock genes and the association of shift work and gene polymorphisms with hypertension in healthcare workers. This study recruited 222 healthcare workers, of whom 76 had primary hypertension (Hyp group) and 146 served as controls (Control group). General information and working hours were collected through questionnaires.
View Article and Find Full Text PDFKlin Mikrobiol Infekc Lek
March 2024
Department of Microbiology, Olomouc University Hospital, Czech Republic, e-mail:
Objectives: Staphylococcus aureus is part of the human microbiota, but at the same time, it is capable of causing a wide range of diseases. Due to the ever-increasing resistance to antimicrobial agents and the existence of methicillin-resistant S. aureus (MRSA) strains, there is a real possibility of carrying even this resistant bacterium, which can subsequently cause a severe infection.
View Article and Find Full Text PDFBackground: This study aimed to evaluate the efficacy of third-generation sequencing (TGS) and a thalassemia (Thal) gene diagnostic kit in identifying Thal gene mutations.
Methods: Blood samples (n = 119) with positive hematology screening results were tested using polymerase chain reaction (PCR)-based methods and TGS on the PacBio-Sequel-II-platform, respectively.
Results: Out of the 119 cases, 106 cases showed fully consistent results between the two methods, with TGS identified HBA1/2 and HBB gene mutations in 82 individuals.
Background: Colorectal cancer (CRC) claims 900,000 lives per year. Colonoscopy offers reliable detection, but with low patient adherence rates. To significantly reduce CRC incidence and mortality, a more convenient screening measure for advanced precancerous lesions (APL) and CRC is urgently needed.
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