Search Results (9)

Cancer involves uncontrolled cell growth, leading to tumor formation, and remains a major cause of mortality worldwide. Colorectal cancer (CRC) arises from abnormal proliferation of colon glandular epithelial cells. We assessed the cytotoxic and molecular effects of lithium carbonate (Li₂CO₃) and lithium chloride (LiCl) in two CRC cell lines (HCT-116 and SW-620) and a non-tumorigenic line (CRL-1790). Viability assays revealed dose-dependent cytotoxicity, with HCT-116 being the most sensitive cell line (IC₅₀: 8.14 mM for Li₂CO₃). Notably, long-term lithium exposure reduced proliferation, lowering colony-forming efficiency (CFE) and a phenotypic shift from holoclones to meroclones and paraclones, indicating diminished self-renewal capacity. Minimal membrane damage was observed (LDH assay), suggesting non-lytic mechanisms consistent with apoptosis. TUNEL and Annexin-V/IP assays confirmed apoptosis in >40% of cells, without caspase-3 cleavage, suggesting a caspase-independent pathway. PARP-1 cleavage occurred only in SW-620 cells. Western blotting exposed cell-specific modulation of GSK-3β: increased inactive form (p-Ser9) in CRC cells and decreased in CRL-1790 cells, implying differential disruption of Wnt/β-catenin signaling. c-Myc levels remained unchanged, suggesting possible post-translational regulatory effects. Overall, these findings indicate that lithium salts selectively reduce CRC cell viability, impair stem-like characteristics, and induced caspase-independent apoptosis. Therefore, we expand the proof of concept of the potential of lithium-based compounds as low-toxicity adjuvant agents in colorectal cancer therapy. Full article

Background: Liuwei Dihuang Pills, a classic traditional Chinese medicine formula, has been widely used in clinical practice for its multiple pharmacological effects. However, the systematic characterization and identification of its chemical constituents, especially the aqueous decoction, remain insufficient, which hinders in-depth research on its pharmacodynamic material basis. Thus, there is an urgent need for a comprehensive analysis of its chemical components using advanced analytical techniques. Methods: After screening chromatographic columns, the ACQUITY UPLC™ HSS T3 column (100 mm × 2.1 mm, 1.8 μm) was selected. The column temperature was set to 40 °C, and the mobile phase consisted of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B). A gradient elution program was adopted, and the separation was completed within 20 min. Ultra-high performance liquid chromatography–Orbitrap mass spectrometry (UPLC-Orbitrap-MS) combined with a self-established information database was used for the analysis. Results: A total of 80 compounds were tentatively identified, including 13 monoterpenoids, 6 phenolic acids, 16 iridoids, 11 flavonoids, 25 triterpenoids, and 9 other types. Triterpenoids are mainly derived from Poria cocos and Alisma orientale; iridoids are mainly from Rehmannia glutinosa; monoterpenoids are mainly from Moutan Cortex; and flavonoids are mainly from Dioscorea opposita. Among them, monoterpenoids, iridoids, and triterpenoids are important pharmacodynamic components. The cleavage pathways of typical compounds (such as pachymic acid, catalpol, oxidized paeoniflorin, and puerarin) are clear, and their mass spectral fragment characteristics are consistent with the literature reports. Conclusions: Through UPLC-Orbitrap-MS technology and systematic optimization of conditions, this study significantly improved the coverage of chemical component identification in Liuwei Dihuang Pills, providing a comprehensive reference for the research on its pharmacodynamic substances. However, challenges remain in the identification of trace components and isomers. In the future, analytical methods will be further improved by combining technologies such as ion mobility mass spectrometry or multi-dimensional liquid chromatography. Full article

The methylotrophic yeast Komagataella kurtzmanii belongs to the group of homothallic fungi that are able to spontaneously change their mating type by inversion of chromosomal DNA in the MAT locus region. As a result, natural and genetically engineered cultures of these yeasts typically contain a mixture of sexually dimorphic cells that are prone to self-diploidisation and spore formation accompanied by genetic rearrangements. These characteristics pose a significant challenge to the development of genetically stable producers for industrial use. In the present study, we constructed heterothallic strains of K. kurtzmanii, ensuring a constant mating type by unifying the genetic sequences in the active and silent MAT loci. To obtain such strains, we performed site-directed inactivation of one of the two yeast MAT loci, replacing its sequence with a selective HIS4 gene surrounded by I-SceI meganuclease recognition sites. We then used transient expression of the SCE1 gene, encoding a recombinant I-SceI meganuclease, to induce site-specific cleavage of HIS4, followed by damage repair by homologous recombination in mutant cells. As a result, heterothallic strains designated ‘Y-727-2(alpha)’ and ‘Y-727-9(a)’, which correspond to the α and a mating type, respectively, were obtained. The strains demonstrated a loss of the ability to self-diploidize. The results of PCR and whole genome analysis confirmed the identity of the contents of the MAT loci. Analysis of the genomes of the final strains, however, revealed a fusion of chromosome 3 and chromosome 4 in strain Y-727-2(alpha)-1. This finding was subsequently confirmed by pulsed-field gel electrophoresis of yeast chromosomes. However, the ability of the Y-727-2(alpha)-derived producers to efficiently secrete recombinant β-galactosidase was unaffected by this genomic rearrangement. Full article

Laodelphax striatellus permutotetra-like virus (LsPLV) is a novel insect virus identified via small RNA deep sequencing. At present, there is a lack of awareness of LsPLV, restricting research on its utilization in biocontrol. In this paper, the full-length genome of LsPLV was cloned and analyzed, then viral capsid protein (CP) was expressed and prepared as an antibody, and CP property was tested. It was found that the LsPLV genome was 4667 nt in length, encoding two proteins, RNA-dependent RNA polymerase (RdRP) and CP, and the palm subdomain conserved region in RdRp was arranged in a “C–A–B” permutation pattern, exhibiting the typical characteristics of permutotetra-like viruses. Phylogenetic analysis suggested that LsPLV shared the highest homology (excluding LsPLV1) with a Nodaviridae virus (QLI47702.1), and their nucleotide identities of RdRP and CP were 55.4% and 59.2%, respectively. After expression, purified CP exhibited two bands of 60 kDa and 47 kDa, suggesting a potential cleavage in the protein. LsPLV CP in L. striatellus was detected by Western blot, and except for the complete CP band, the specific bands with molecular weights lower than CP were also detected, indicating that CP underwent cleavage. Detection of purified CP in vitro showed that the cleavage could occur independent of any protease, confirming that CP has self-cleavage characteristics. Full article

N-terminal nucleophile (Ntn)-hydrolases catalyze the cleavage of amide bonds in a variety of macromolecules, including the peptide bond in proteins, the amide bond in N-linked protein glycosylation, and the amide bond linking a fatty acid to sphingosine in complex sphingolipids. Ntn-hydrolases are all sharing two common hallmarks: Firstly, the enzymes are synthesized as inactive precursors that undergo auto-proteolytic self-activation, which, as a consequence, reveals the active site nucleophile at the newly formed N-terminus. Secondly, all Ntn-hydrolases share a structural consistent αββα-fold, notwithstanding the total lack of amino acid sequence homology. In humans, five subclasses of the Ntn-superfamily have been identified so far, comprising relevant members such as the catalytic active subunits of the proteasome or a number of lysosomal hydrolases, which are often associated with lysosomal storage diseases. This review gives an updated overview on the structural, functional, and (patho-)physiological characteristics of human Ntn-hydrolases, in particular. Full article

Newcastle disease virus (NDV) strain R2B, with an altered fusion protein cleavage site, was used as a viral vector to deliver the immunogenic genes VP2 and VP1 of chicken infectious anaemia virus (CIAV) to generate a bivalent vaccine candidate against these diseases in chickens. The immunogenic genes of CIAV were expressed as a single transcriptional unit from the NDV backbone and the two CIA viral proteins were obtained as separate entities using a self-cleaving foot-and-mouth disease virus 2A protease sequence between them. The recombinant virus (rR2B-FPCS-CAV) had similar growth kinetics as that of the parent recombinant virus (rR2B-FPCS) in vitro with similar pathogenicity characteristics. The bivalent vaccine candidate when given in specific pathogen-free chickens as primary and booster doses was able to elicit robust humoral and cell-mediated immune (CMI) responses obtained in a vaccination study that was conducted over a period of 15 weeks. In an NDV and CIAV ELISA trial, there was a significant difference in the titres of antibody between vaccinated and control groups which showed slight reduction in antibody titre by 56 days of age. Hence, a second booster was administered and the antibody titres were maintained until 84 days of age. Similar trends were noticed in CMI response carried out by lymphocyte transformation test, CD4⁺ and CD8⁺ response by flow cytometry analysis and response of real time PCR analysis of cytokine genes. Birds were challenged with virulent NDV and CIAV at 84 days and there was significant reduction in the NDV shed on the 2nd and 4th days post challenge in vaccinated birds as compared to unvaccinated controls. Haematological parameters comprising PCV, TLC, PLC and PHC were estimated in birds that were challenged with CIAV that indicated a significant reduction in the blood parameters of controls. Our findings support the development and assessment of a bivalent vaccine candidate against NDV and CIAV in chickens. Full article

Radiation therapy is an effective cancer therapy, but damage to normal tissues surrounding the tumor due to radiotherapy causes severe complications. The importance of the therapeutic area between tumor suppression and normal tissue injury has long been highlighted in radiation therapy. Recent advances in stem cell biology have shown that stem cell (SC) responses to genotoxic stresses of ionizing radiation can improve the therapeutic effect of radiation by repairing damaged cells. In contrast, cancer stem cells (CSCs), a small subpopulation of cells within tumors, are generally resistant to chemotherapy and radiotherapy and cause tumor recurrence. Although the underlying mechanisms are not clearly understood in detail, efforts are still underway to identify SC treatment or CSC resistant pathogenesis of DNA damage agents such as radiation therapy. In response to radiation, CSCs differ from normal SCs in their biological properties due to severe deregulation of the self-renewal ability in CSCs. Differences of cleavage mode, cell cycle characteristics, replication potential, and activation/inactivation of DNA damage treatment and cancer-specific molecular pathways between normal SCs and CSCs confer a malignant phenotype upon CSCs. However, further studies are needed to identify normal SC and CSC-specific targets. In this review, we summarize the current advances in research regarding how normal SCs and CSCs respond to ionizing radiation, with a special emphasis on cell toxicity, radiosensitivity, signaling networks, DNA damage response (DDR) and DNA repair. In addition, we discuss strategies to develop new diagnostic and therapeutic techniques for predicting responses to cancer treatment and overcoming radiation-related toxicity. Full article

Given the existing divide related to Internet skills and types of Internet use, it is safe to assume that a large proportion of the population uses the Internet for health purposes in a partially productive fashion. We suggest that in addition to user characteristics, another factor that inhibits productive Internet use, and thus contributes to the existing gap, is related to the ways in which the technology is configured. The goal of this study was to explore the processes that webmasters and content managers use for constructing and producing, or selecting content, for health websites. Interviews conducted with 23 website builders and managers of websites that represent public and non-public health organizations revealed that they do not plan or conduct activities for content needs elicitation, either in the design stage or on an ongoing basis. Rather, these professionals rely on a “self-embodiment” standard, whereby their and their cohorts’ expectations determine the quality and functionality of the websites’ structure and content. Hence, target groups beyond their social sphere are disregarded, and instead of new opportunities, new cleavages are created. We recommended that government, public and non-public stakeholders work to establish construction standards, to ensure that health websites meet the needs of varied end-user populations. Full article

Viroids are small pathogenic circular single-stranded RNAs, present in two complementary sequences, named plus and minus, in infected plant cells. A high degree of complementarities between different regions of the RNAs allows them to adopt complex structures. Since viroids are naked non-coding RNAs, interactions with host factors appear to be closely related to their structural and catalytic characteristics. Avocado sunblotch viroid (ASBVd), a member of the family Avsunviroidae, replicates via a symmetric RNA-dependant rolling-circle process, involving self-cleavage via hammerhead ribozymes. Consequently, it is assumed that ASBVd plus and minus strands adopt similar structures. Moreover, by computer analyses, a quasi-rod-like secondary structure has been predicted. Nevertheless, secondary and tertiary structures of both polarities of ASBVd remain unsolved. In this study, we analyzed the characteristic of each strand of ASBVd through biophysical analyses. We report that ASBVd transcripts of plus and minus polarities exhibit differences in electrophoretic mobility under native conditions and in thermal denaturation profiles. Subsequently, the secondary structures of plus and minus polarities of ASBVd were probed using the RNA-selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE) method. The models obtained show that both polarities fold into different structures. Moreover, our results suggest the existence of a kissing-loop interaction within the minus strand that may play a role in in vivo viroid life cycle. Full article