The oncolytic peptide LTX-315 triggers necrotic cell death

The oncolytic peptide LTX-315 has been designed for killing human cancer cells and turned out to stimulate anti-cancer immune responses when locally injected into tumors established in immunocompetent mice. Here, we investigated the question whether LTX-315 induces apoptosis or necrosis. Transmission electron microscopy or morphometric analysis of chromatin-stained tumor cells revealed that LTX-315 failed to induce apoptotic nuclear condensation and rather induced a necrotic phenotype. Accordingly, LTX-315 failed to stimulate the activation of caspase-3, and inhibition of caspases by means of Z-VAD-fmk was unable to reduce cell killing by LTX-315. In addition, 2 prominent inhibitors of regulated necrosis (necroptosis), namely, necrostatin-1 and cycosporin A, failed to reduce LTX-315-induced cell death. In conclusion, it appears that LTX-315 triggers unregulated necrosis, which may contribute to its pro-inflammatory and pro-immune effects.     

Genome‐wide scans to detect positive selection in Large White and Tongcheng pigs

Due to the direction, intensity, duration and consistency of genetic selection, especially recent artificial selection, the production performance of domestic pigs has been greatly changed. Therefore, we reasoned that there must be footprints or selection signatures that had been left during domestication. In this study, with porcine 60K BeadChip genotyping data from both commercial Large White and local Chinese Tongcheng pigs, we calculated the extended haplotype homozygosity values of the two breeds using the long‐range haplotype method to detect selection signatures. We found 34 candidate regions, including 61 known genes, from Large White pigs and 25 regions comprising 57 known genes from Tongcheng pigs. Many selection signatures were found on SSC1, SSC4, SSC7 and SSC14 regions in both populations. According to quantitative trait loci and network pathway analyses, most of the regions and genes were linked to growth, reproduction and immune responses. In addition, the average genetic differentiation coefficient F_ST was 0.254, which means that there had already been a significant differentiation between the breeds. The findings from this study can contribute to further research on molecular mechanisms of pig evolution and domestication and also provide valuable references for improvement of their breeding and cultivation.     

Plasminogen Deficiency Causes Reduced Corticospinal Axonal Plasticity and Functional Recovery after Stroke in Mice

Tissue plasminogen activator (tPA) has been implicated in neurite outgrowth and neurological recovery post stroke. tPA converts the zymogen plasminogen (Plg) into plasmin. In this study, using plasminogen knockout (Plg^-/-) mice and their Plg-native littermates (Plg^+/+), we investigated the role of Plg in axonal remodeling and neurological recovery after stroke. Plg^+/+ and Plg^-/- mice (n = 10/group) were subjected to permanent intraluminal monofilament middle cerebral artery occlusion (MCAo). A foot-fault test and a single pellet reaching test were performed prior to and on day 3 after stroke, and weekly thereafter to monitor functional deficit and recovery. Biotinylated dextran amine (BDA) was injected into the left motor cortex to anterogradely label the corticospinal tract (CST). Animals were euthanized 4 weeks after stroke. Neurite outgrowth was also measured in primary cultured cortical neurons harvested from Plg^+/+ and Plg^-/- embryos. In Plg^+/+ mice, the motor functional deficiency after stroke progressively recovered with time. In contrast, recovery in Plg^-/- mice was significantly impaired compared to Plg^+/+ mice (p<0.01). BDA-positive axonal density of the CST originating from the contralesional cortex in the denervated side of the cervical gray matter was significantly reduced in Plg^-/- mice compared with Plg^+/+ mice (p<0.05). The behavioral outcome was highly correlated with the midline-crossing CST axonal density (R²>0.82, p<0.01). Plg^-/- neurons exhibited significantly reduced neurite outgrowth. Our data suggest that plasminogen-dependent proteolysis has a beneficial effect during neurological recovery after stroke, at least in part, by promoting axonal remodeling in the denervated spinal cord.     

Mammalian Fbh1 is important to restore normal mitotic progression following decatenation stress

We have addressed the role of the F-box helicase 1 (Fbh1) protein during genome maintenance in mammalian cells. For this, we generated two mouse embryonic stem cell lines deficient for Fbh1: one with a homozygous deletion of the N-terminal F-box domain (Fbh1^f/f), and the other with a homozygous disruption (Fbh1^?/?). Consistent with previous reports of Fbh1-deficiency in vertebrate cells, we found that Fbh1^?/? cells show a moderate increase in Rad51 localization to DNA damage, but no clear defect in chromosome break repair. In contrast, we found that Fbh1^f/f cells show a decrease in Rad51 localization to DNA damage and increased cytoplasmic localization of Rad51. However, these Fbh1^f/f cells show no clear defects in chromosome break repair. Since some Rad51 partners and F-box-associated proteins (Skp1-Cul1) have been implicated in progression through mitosis, we considered whether Fbh1 might play a role in this process. To test this hypothesis, we disrupted mitosis using catalytic topoisomerase II inhibitors (bisdioxopiperazines), which inhibit chromosome decatenation. We found that both Fbh1^f/f and Fbh1^?/? cells show hypersensitivity to topoisomerase II catalytic inhibitors, even though the degree of decatenation stress was not affected. Furthermore, following topoisomerase II catalytic inhibition, both Fbh1-deficient cell lines show substantial defects in anaphase separation of chromosomes. These results indicate that Fbh1 is important for restoration of normal mitotic progression following decatenation stress.     

Expression patterns and association analysis of the porcine DHX58 gene

RIG-1 signalling is responsible for the detection of cytoplasmic viral RNA molecules. DEXH (Asp-Glu-X-His) box polypeptide 58 (encoded by DHX58) is a negative regulator of the RIG-1 signalling pathway. In human, the DHX58 gene can be upregulated and can inhibit the RIG-1 signalling pathway during viral infection. In this study, porcine DHX58 gene expression patterns were studied. According to our results, the porcine DHX58 gene was upregulated not only by the stimulation of Poly I:C but also by the stimulation of 1ipopolysaccharides (LPS). One polymorphism (g.4919G>C), detected in the ninth intron, was significantly associated with some blood parameters including the red cell distribution width of 1-day-old pigs and white blood cell counts, lymphocyte absolute counts, and platelet distribution width of 17-day-old pigs (P < 0.05). Moreover, the individuals with the genotype GG have a significantly higher mean white blood cell count than individuals with genotype CC or GC (P < 0.05). Our study indicates that DHX58 is an important gene that is associated with the immune response in swine.     

Properties of herpes simplex virus type 1 and type 2 DNA polymerase

Herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) DNA polymerases were highly purified from infected HeLa BU cells by DEAE cellulose, phosphocellulose and DNA cellulose column chromatography. DNA exonuclease activity but not endonuclease activity was found associated with both types of DNA polymerase. Both DNA polymerase activities could be activated by salt in a similar fashion with the optimal activity in the range of ionic strength between 0.22 and 0.29 alpha. At an ionic strength of 0.14, spermidine and putrescine in the concentration range (0--5 mM) studied could mimic the action of KCI in stimulating DNA polymerase activity. Spermine, in the same concentration range, had a biphasic effect. At an ionic strength of 0.29 all three polyamines were inhibitory. HSV-1 and HSV-2 DNA polymerase are similar in their column chromatographic behavior, sedimentation rate in sucrose gradient centrifugation, and activation energy, but they differ in their heat stability at 45 degrees C with the HSV-2 enzyme more stable than the HSV-1 enzyme. Kinetic behavior of both enzymes is similar, with Km values for deoxyribonucleoside triphosphates in the range of 5 . 10(-7) to 1.8 . 10(-8) M. IdUTP and dUTP served as apparent competitive inhibitors with respect to dTTP, and AraATP acted as an apparent competitive inhibitor with respect to dATP. AraATP could not replace dATP in the DNA polymerization reaction; in contrast, IdUTP could replace TTP. Phosphonoformic acid behaved as an uncompetitive inhibitor with respect to DNA. The ID(50) value estimated was foind to be dependent on the purity of the DNA polymerase used and the ionic strength of the assay condition. Each DNA-polymerase associated DNA exonuclease had the same stability at 45 degrees C as its DNA polymerase. The associated DNAase activity was inhibited by phosphonoformic acid and high ionic strength of the assay condition.