Impaired expression of serine/arginine protein kinase 2 (SRPK2) affects melanoma progression.

Melanoma is one of the most aggressive tumors, and its lethality is associated with the ability of malignant cells to migrate and invade surrounding tissues to colonize distant organs and to generate widespread metastasis. The serine/arginine protein kinases 1 and 2 (SRPK1 and SRPK2) are classically related to the control of pre-mRNA splicing through SR protein phosphorylation and have been found overexpressed in many types of cancer, including melanoma. Previously, we have demonstrated that the pharmacological inhibition of SRPKs impairs pulmonary colonization of metastatic melanoma in mice. As the used compounds could target at least both SRPK1 and SRPK2, here we sought to obtain additional clues regarding the involvement of these paralogs in melanoma progression. We analyzed single-cell RNA sequencing data of melanoma patient cohorts and found that SRPK2 expression in melanoma cells is associated with poor prognosis. Consistently, CRISPR-Cas9 genome targeting of SRPK2, but not SRPK1, impaired actin polymerization dynamics as well as the proliferative and invasive capacity of B16F10 cells in vitro. In further in vivo experiments, genetic targeting of SRPK2, but not SRPK1, reduced tumor progression in both subcutaneous and caudal vein melanoma induction models. Taken together, these findings suggest different functional roles for SRPK1/2 in metastatic melanoma and highlight the relevance of pursuing selective pharmacological inhibitors of SRPK2.

Enhancing cell resistance for production of mixed microbiological reference materials with Salmonella and coliforms by freeze-drying.

The reference material (RM) is a technical requirement for the quality assurance of analytical results and proficiency tests or interlaboratory comparisons. Microbiological RMs are most available in the dehydrated form, mainly by freeze-drying, and maintaining bacterial survival after preparation is a challenge. Thus, obtaining the most resistant cells is essential. Considering that bacteria present cross-response to dehydration after being submitted to an array of stress conditions, this study aimed to evaluate the influence of growth conditions on enterobacteria for the production of mixed microbiological RMs by freeze-drying in skim milk powder. Salmonella enterica serovar Enteritidis, Cronobacter sakazakii, Escherichia coli, and Citrobacter freundii were grown in a minimal medium with 0.5 M NaCl and 0 to 5.0 mM of manganese sulfate (MnSO4) until stationary phase. Salmonella Enteritidis presented an increased resistance to dehydration in the presence of Mn, while C. sakazakii was the most resistant to freeze-drying and further storage for 90 days. Mixed microbiological RMs were produced by freeze-drying and containing Salmonella Enteritidis and coliforms in skim milk powder with 100 mM of trehalose and the Salmonella survival rate was 91.2 to 93.6%. The mixed RM was stable after 30 days at -20 °C, and Salmonella and coliforms were detected by different methods being, the Rambach Agar the best for the bacterial differentiation. The results showed that the culture conditions applied in this study resulted in bacterial cells being more resistant to dehydration, freeze-drying, and stabilization for the production of mixed microbiological RMs more stable and homogeneous.

Splicing Regulators and Their Roles in Cancer Biology and Therapy.

Alternative splicing allows cells to expand the encoding potential of their genomes. In this elegant mechanism, a single gene can yield protein isoforms with even antagonistic functions depending on the cellular physiological context. Alterations in splicing regulatory factors activity in cancer cells, however, can generate an abnormal protein expression pattern that promotes growth, survival, and other processes, which are relevant to tumor biology. In this review, we discuss dysregulated alternative splicing events and regulatory factors that impact pathways related to cancer. The SR proteins and their regulatory kinases SRPKs and CLKs have been frequently found altered in tumors and are examined in more detail. Finally, perspectives that support splicing machinery as target for the development of novel anticancer therapies are discussed.

Nucleotide polymorphism in the 5.8S nrDNA gene and internal transcribed spacers in Phakopsora pachyrhizi viewed from structural models.

The assessment of nucleotide polymorphisms in environmental samples of obligate pathogens requires DNA amplification through the polymerase chain reaction (PCR) and bacterial cloning of PCR products prior to sequencing. The drawback of this strategy is that it can give rise to false polymorphisms owing to DNA polymerase misincorporation during PCR or bacterial cloning. We investigated patterns of nucleotide polymorphism in the internal transcribed spacer (ITS) region for Phakopsora pachyrhizi, an obligate biotrophic fungus that causes the Asian soybean rust. Field-collected samples of P. pachyrhizi were obtained from all major soybean production areas worldwide, including Brazil and the United States. Bacterially-cloned, PCR products were obtained using a high fidelity DNA polymerase. A total of 370 ITS sequences that were subjected to an array of complementary sequence analyses, which included analyses of secondary structure stability, the pattern of nucleotide polymorphisms, GC content, and the presence of conserved motifs. The sequences exhibited features of functional rRNAs. Overall, polymorphisms took place within less conserved motives, such as loops and bulges; alternatively, they gave rise to non-canonical G-U pairs within conserved regions of double stranded helices. We discuss the usefulness of structural analyses to filter out putative 'suspicious' bacterially cloned ITS sequences, thus keeping artificially-induced sequence variation to a minimum.