Analysis of CYP1B1 Polymorphisms in Lung Cancer Patients Using Novel, Quick and Easy Methods Based on CAPS and ACRS-PCR Techniques.

Within the sequence of the CYP1B1 gene, more than 50 polymorphisms, resulting from single-nucleotide polymorphisms (SNPs), have been described. Some of them play an important role as specific genetic markers in the process of carcinogenesis and for therapeutic purposes. In this publication, we present methods we have developed that enable the specific and unambiguous identification of four polymorphisms that result in amino acid changes: c. 142C > G, c. 355G > T, c. 1294C > G, and c. 1358A > G. Our studies are based on cleaved amplified polymorphic sequences (CAPSs) and artificially created restriction site (ACRS) PCR techniques; therefore, they require only basic laboratory equipment and low financial outlays. Utilizing the described methods allows for the reduction of research time and cost, and the minimization of errors. Their effectiveness and efficiency depend on the careful design of appropriate primers and the precise selection of suitable restriction enzymes. As a result, further confirmation by sequencing is not necessary. Using the developed method, we examined 63 patients diagnosed with lung cancer and observed a 1.5 to 2.1 times higher frequency of the analyzed single-nucleotide polymorphisms compared to the frequency in the European population.

3-bromopyruvate induces morphological alteration and may initiate programmed cell death in Cryptococcus neoformans cells.

3-Bromopyruvate (3BP), known for its potent anticancer properties, also exhibits remarkable efficacy against the pathogenic fungus Cryptococcus neoformans. So far it has been proven that the main fungicidal activity of 3BP is based on ATP depletion and a reduction of intracellular level of glutathione. The presented study includes a broad range of methods to further investigate the mechanistic effects of 3BP on C. neoformans cells. The use of flow cytometry allowed a thorough examination of their survival during 3BP treatment, while observations using electron microscopy made it possible to note the changes in cellular morphology. Utilizing ruthenium red, the study suggests a mitochondrial pathway may initiate programmed cell death in response to 3BP. Analysis of free radical generation and gene expression changes supports this hypothesis. These findings enhance comprehension of 3BP's mechanisms in fungal cells, paving the way for its potential application as a therapeutic agent against cryptococcosis.

Plant-Fungal Interactions: A Case Study of Epicoccoum nigrum Link.

Epicoccum nigrum Link is a cosmopolitan species, and it has been described as both an in vitro and in vivo antagonist of many fungal pathogens of plants. However, there are no clear reports about the interactions between E. nigrum and various plant species, and about the effects of culture filtrates produced by this fungus on plants. Therefore, we assessed the interactions between E. nigrum and different plant species, such as sugar beet (Beta vulgaris L. ssp. vulgaris), spring wheat (Triticum aestivum L.), red clover (Trifolium pratense L.), and winter oilseed rape (Brassica napus L.). Additionally, we evaluated the effect of E. nigrum culture filtrates on garden cress (Lepidium sativum L.). Our study showed that the E. nigrum strains varied in terms of the color of excreted culture filtrates and showed different interactions with garden cress. Overall, fungal strains only affected adversely the sprout length in a significant way and, partially, the growth of the tested plant. In addition, we confirmed the suitability of the garden cress as a test plant in in vitro toxicological tests. Most strains of E. nigrum (61.1%) secreted enzymes expected to participate mainly in the later stages of the infection (amylases and proteases) and not those expected to operate in the early phases of host penetration (cellulases and pectinases) that were secreted by 33.3% of fungal strains. The group of pectinolytic enzymes represented the catalysts with the highest activity. Host specialization tests showed that E. nigrum was mainly re-isolated from the plant surface and the number of infected seedlings as well as the disease index depended on a studied plant species, with sugar beet and red clover being most sensitive to infection. In turn, the lowest value of the disease index caused by E. nigrum strains was recorded for spring wheat and winter oilseed rape. Overall, statistically significant differences in the growth of plant seedlings during the host specialization test were noted only for sugar beet and red clover seedlings. The seedlings of plants in the control group (without fungal inoculum) exhibited an increased length compared to those treated with E. nigrum inoculum. Our studies also showed that E. nigrum is probably a facultative saprotroph of plants and it may winter on red clover, which is presumably its main reservoirs, among the species considered.