Various chimeric lysins have been developed as efficacious antibiotics against multidrug-resistant bacteria, but direct comparisons of their antibacterial activities have been difficult due to the preparation of multiple recombinant chimeric lysins. Previously, we reported an Escherichia coli cell-free expression method to better screen chimeric lysins against Staphylococcus aureus, but we still needed to increase the amounts of expressed proteins enough to be able to detect them non-isotopically for quantity comparisons. In this study, we improved the previous cell-free expression system by adding a previously reported artificial T7 terminator and reversing the different nucleotides between the T7 promoter and start codon to those of the T7 phage. The new method increased the expressed amount of chimeric lysins enough for us to detect them using Western blotting. Therefore, the qualitative comparison of activity between different chimeric lysins has become possible via the adjustment of the number of variables between samples without protein purification. We applied this method to select more active chimeric lysins derived from our previously reported chimeric lysin (ALS2). Finally, we compared the antibacterial activities of our selected chimeric lysins with reported chimeric lysins (ClyC and ClyO) and lysostaphin and determined the rank orders of antibacterial activities on different Staphylococcus aureus strains in our experimental conditions.

The secretion of proteins that do not follow the well-characterized endoplasmic reticulum (ER)-Golgi apparatus pathway, known as unconventional protein secretion (UCPS), is gradually revealing its complexities. Our study has identified an ER-based tubulovesicular network, termed ER tubular body (ER-TB), as a central compartment in this process. We demonstrate that ER-TBs are formed by two reticulophagy receptors, ATL3 and RTN3L, under conditions of cellular stress. In addition to their role in stress-induced secretion, the activation of UCPS via ER-TBs facilitates cell surface trafficking of trafficking-deficient transmembrane proteins such as ΔF508-CFTR. Furthermore, their involvement in ER remodeling and vesicle trafficking suggests a potential role in viral replication, particularly in the formation of membrane compartments utilized by positive-strand RNA viruses. By uncovering ER-TBs as key cellular structures in stress-induced UCPS and demonstrating their regulation by autophagy-related factors, our findings offer valuable insights into protein homeostasis, viral pathogenesis, and potential therapeutic strategies for diseases linked to trafficking defects.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) cation channel is a bifunctional ion channel with intrinsic kinase activity and is ubiquitously expressed in the animal/human body. Accumulated knowledge of TRPM7 suggests that it plays an essential role in normal physiological processes, including the development, survival, proliferation, differentiation, and migration of cells. The aim of this study was to demonstrate the presence and expression patterns of TRPM7 in normal canine mammary glands using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. Normal mammary gland tissue samples were obtained from five female beagle dogs. RT-PCR and sequencing of the amplified PCR products demonstrated the presence of TRPM7 mRNA in normal mammary glands, and the presence of TRPM7 protein was confirmed by Western blotting. Immunohistochemical investigations demonstrated the expression of TRPM7 in the apical membrane of acinar and ductal epithelial cells in the canine mammary glands. These results provide the first evidence of the presence and distribution of TRPM7 in the canine mammary gland and could help explain the physiological and pathological roles of TRPM7 in the canine mammary gland; however, additional studies are required to elucidate these roles.