Downregulation of Rad51 Expression and Activity Potentiates the Cytotoxic Effect of Osimertinib in Human Non-Small Cell Lung Cancer Cells.

INTRODUCTION: Osimertinib (AZD9291) is a third-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor that has shown significant clinical benefits in patients with EGFR-sensitizing mutations or the EGFR T790M mutation. The homologous recombination (HR) pathway is crucial for repairing DNA double-strand breaks (DSBs). Rad51 plays a central role in HR, facilitating the search for homology and promoting DNA strand exchange between homologous DNA molecules. Rad51 is overexpressed in numerous types of cancer cells. B02, a specific small molecule inhibitor of Rad51, inhibits the DNA strand exchange activity of Rad51. Previous studies have indicated that B02 disrupted Rad51 foci formation in response to DNA damage and inhibited DSBs repair in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. However, the potential therapeutic effects of combining osimertinib with a Rad51 inhibitor are not well understood. The aim of this study was to elucidate whether the downregulation of Rad51 expression and activity can enhance the osimertinib-induced cytotoxicity in non-small cell lung cancer (NSCLC) cells. METHODS: We used the MTS, trypan blue dye exclusion and colony-formation ability assay to determine whether osimertinib alone or in combination with B02 had cytotoxic effects on NSCLC cell lines. Real-time polymerase chain reaction was conducted to measure the amounts of Rad51 mRNA. The protein levels of phosphorylated AKT and Rad51 were determined by Western blot analysis. RESULTS: We found that osimertinib reduced Rad51 expression by inactivating AKT activity. Rad51 knockdown using small interfering RNA or AKT inactivation through the phosphatidylinositol 3-kinase inhibitor LY294002 or si-AKT RNA transfection enhanced the cytotoxic and growth inhibitory effects of osimertinib. In contrast, AKT-CA (a constitutively active form of AKT) vector-enforced expression could mitigate the cytotoxic and cell growth inhibitory effects of osimertinib. Furthermore, B02 significantly enhanced the cytotoxic and cell growth inhibitory effects of osimertinib in NSCLC cells. Compared to parental cells, the activation of AKT and Rad51 expression in osimertinib-resistant cells could not be significantly inhibited by osimertinib treatment. Moreover, the increased expression of Rad51 is associated with the resistance mechanism in osimertinib-resistant H1975 and A549 cells. CONCLUSION: Collectively, the downregulation of Rad51 expression and activity enhances the cytotoxic effect of osimertinib in human NSCLC cells.

Identification of Nonsynonymous SNPs in Immune-Related Genes Associated with Pneumonia Severity in Pigs.

We previously showed that several polymorphisms in genes encoding pattern recognition receptors that cause amino acid substitutions alter pathogen recognition ability and disease susceptibility in pigs. In this study, we expanded our analysis to a wide range of immune-related genes and investigated polymorphism distribution and its influence on pneumonia in multiple commercial pig populations. Among the polymorphisms in 42 genes causing 634 amino acid substitutions extracted from the swine genome database, 80 in 24 genes were found to have a minor allele frequency of at least 10% in Japanese breeding stock pigs via targeted resequencing. Of these, 62 single nucleotide polymorphisms (SNPs) in 23 genes were successfully genotyped in 862 pigs belonging to four populations with data on pneumonia severity. Association analysis using a generalized linear mixed model revealed that 12 SNPs in nine genes were associated with pneumonia severity. In particular, SNPs in the cellular receptor for immunoglobulin G FCGR2B and the intracellular nucleic acid sensors IFI16 and LRRFIP1 were found to be associated with mycoplasmal pneumonia of swine or porcine pleuropneumonia in multiple populations and may therefore have wide applications in the improvement of disease resistance in pigs. Functional analyses at the cellular and animal levels are required to clarify the mechanisms underlying the effects of these SNPs on disease susceptibility.

Arf GTPase-Activating proteins ADAP1 and ARAP1 regulate incorporation of CD63 in multivesicular bodies.

Arf GTPase-activating proteins (ArfGAPs) mediate the hydrolysis of GTP bound to ADP-ribosylation factors. ArfGAPs are critical for cargo sorting in the Golgi-to-ER traffic. However, the role of ArfGAPs in sorting into intralumenal vesicles (ILVs) in multivesicular bodies (MVBs) in post-Golgi traffic remains unclear. Exosomes are extracellular vesicles (EVs) of endosomal origin. CD63 is an EV marker. CD63 is enriched ILVs in MVBs of cells. However, the secretion of CD63 positive EVs has not been consistent with the data on CD63 localization in MVBs, and how CD63-containing EVs are formed is yet to be understood. To elucidate the mechanism of CD63 transport to ILVs, we focused on CD63 localization in MVBs and searched for the ArfGAPs involved in CD63 localization. We observed that ADAP1 and ARAP1 depletion inhibited CD63 localization to enlarged endosomes after Rab5Q79L overexpression. We tested epidermal growth factor (EGF) and CD9 localization in MVBs. We observed that ADAP1 and ARAP1 depletion inhibited CD9 localization in enlarged endosomes but not EGF. Our results indicate ADAP1 and ARAP1, regulate incorporation of CD63 and CD9, but not EGF, in overlapped and different MVBs. Our work will contribute to distinguish heterogenous ILVs and exosomes by ArfGAPs.

Polymorphisms in Pattern Recognition Receptor Genes Are Associated with Respiratory Disease Severity in Pig Farms.

Reduced productivity caused by infections, particularly respiratory diseases, is a serious problem in pig farming. We have previously reported polymorphisms in porcine pattern recognition receptor genes affecting molecular functions and demonstrated that the 2197A/C polymorphism in the nucleotide-binding oligomerization domain containing 2 (NOD2) gene influences porcine circovirus 2-induced mortality. Here, we investigated how these polymorphisms affect respiratory disease-induced lesions, using samples from a slaughterhouse dealing with pigs from two farms. Lung lesions were evaluated using two scoring systems, Goodwin (GW) and slaughterhouse pleuritis evaluation system (SPES), to determine the influence of Mycoplasma hyopneumoniae (Mhp) and Actinobacillus pleuropneumoniae (App), respectively. SPES scores were significantly higher when the 1205T allele of Toll-like receptor 5 (TLR5-1205T), rather than TLR5-1205C, was present. On the farm with more severe Mhp invasion, lower GW lesion scores were significantly associated with the presence of the NOD-like receptor family pyrin domain containing 3 (NLRP3)-2906G allele; where App invasion was worse, lower SPES scores were significantly associated with the presence of the NOD2-2197C allele. Combinations of polymorphisms in pattern recognition receptor genes can therefore be utilized for breeding for resistance against respiratory diseases in pigs. DNA markers of these polymorphisms can thus be used to improve productivity by reducing respiratory diseases due to bacterial pathogens in pig livestock.

NOD2 Genotypes Affect the Symptoms and Mortality in the Porcine Circovirus 2-Spreading Pig Population.

The nucleotide oligomerization domain (NOD)-like receptor 2 (NOD2) is an intracellular pattern recognition receptor that detects components of peptidoglycans from bacterial cell walls. NOD2 regulates bowel microorganisms, provides resistance against infections such as diarrhea, and reduces the risk of inflammatory bowel diseases in humans and mice. We previously demonstrated that a specific porcine NOD2 polymorphism (NOD2-2197A > C) augments the recognition of peptidoglycan components. In this study, the relationships between porcine NOD2-2197A/C genotypes affecting molecular functions and symptoms in a porcine circovirus 2b (PCV2b)-spreading Duroc pig population were investigated. The NOD2 allele (NOD2-2197A) with reduced recognition of the peptidoglycan components augmented the mortality of pigs at the growing stage in the PCV2b-spreading population. Comparison of NOD2 allele frequencies in the piglets before and after invasion of PCV2b indicated that the ratio of NOD2-2197A decreased in the population after the PCV2b epidemic. This data indicated that functional differences caused by NOD2-2197 polymorphisms have a marked impact on pig health and livestock productivity. We suggest that NOD2-2197CC is a PCV2 disease resistant polymorphism, which is useful for selective breeding by reducing mortality and increasing productivity.

Persistence of extended-spectrum ß-lactamase plasmids among Enterobacteriaceae in commercial broiler farms.

To clarify the persistence of extended-spectrum ß-lactamase (ESBL) producers, 13 plasmids from two broiler farms were analyzed. On the farm not using antimicrobials, one plasmid from Klebsiella pneumoniae isolated from a day-old chick was similar to that from Escherichia coli isolated a year later, with the deletion of two transposons. On the farm using antimicrobials, most circulating plasmids (eight out of nine) in a flock of 40-days-old chicks were identical, although one from K. pneumoniae had a deletion of a transposon carrying a class 1 integron containing aadA2 and dfrA12. Thus, ESBL plasmids persisted in the farms with or without antimicrobial agent use.

Mastication of Hard Gumi Decreases the Gustatory Threshold for Sodium Chloride.

In recent years, there has been an increase in the number of hypertensive diseases and the various diseases associated with them. A major cause of these is excessive salt intake. The purpose of the present study was to examine whether chewing hard foods lowers the saltiness threshold. Fifteen subjects (fourteen women and one man) participated in the present study. Two types of gummies are available as ingredients: hard and soft gummies. The saltiness thresholds before and after chewing of each gummi were studied using 11 different NaCl solutions. Then, points of subjective equality (PSEs) were calculated to detect changes in the saltiness for each subject. In the soft Gumi condition, there was no significant difference in PSE for the saltiness between before and after ingesting Gumi (p>0.05), while in the hard Gumi condition, the PSE for the saltiness significantly decreased after ingesting Gumi compared with the value of before ingesting Gumi (p=0.001). From these results, we concluded that sensitivity to saltiness would increase after mastication of hard foods such as hard Gumi.

Effects of Antimicrobial Administration on the Prevalence of Antimicrobial-Resistant Escherichia coli in Broiler Flocks.

The increase in antimicrobial-resistant (AMR) bacteria caused by antimicrobial usage is a public health problem. We investigated the proportion of cephalexin (LEX)-resistant bacteria in fresh feces obtained from antimicrobial-free broilers in three flocks at ＜15, 15-40, and＞ 40 days old. DHL agar plates containing 25 µg/mL LEX (DHL-L) showed LEX-resistant bacteria in all flocks at ＜15 days old and in one flock at ＞ 40 days old. The bacterial counts on DHL and DHL-L were 105-108 colony forming units (CFU)/g feces and ＜102-105 CFU/g feces, respectively. We also assessed the proportion of AMR bacteria in feces collected at 5, 12, 19, 26, 33, and 40 days old from two flocks treated with amoxicillin at 5-7 days old and co-trimoxazole at 24-26 days old. The proportion of ampicillin (AMP)-resistant bacteria was elevated at 12 and 26-33 days old on DHL containing 50 µg/mL AMP, while no increase in LEX-resistant bacteria was observed on DHL-L. All isolates tested exhibited AMP resistance at 12 days old, while most exhibited resistance to both AMP and trimethoprim/sulfamethoxazole at 26-33 days old. Our results suggest that antimicrobial administration influenced the selection of AMR bacteria with cross- and coresistance.

The Response Time of the Stroop Test Is Delayed during Lemon-Flavored Gum Chewing.

In the present study, we examined the cognitive function during mastication of lemon-flavored gum, which is said to enhance cerebral blood flow. Nine healthy subjects (8 female and 1 male) participated in this study. Subjects chewed the gum for 3 min after fasting for 2 h and conducted a Stroop test while continuing to chew. At the end of all answers, gum chewing ended. The response time in the Stroop test was used as an indicator of cognitive function. We set the three conditions (lemon-flavored gum, mint-flavored gum, no gum chewing). There was no significant difference in reaction time between chewing mint-flavored gum and not chewing (p>0.05). However, the response time during chewing gum with a lemon flavor was significant slower than the conditions with mint-flavored gum and without gum chewing (p<0.05, in both). From the results of the present study, it was suggested that the response time delay of the Stroop test observed during the chewing of lemon-flavored gum revealed temporary decay of cognitive function during lemon-flavored gum chewing. It is suggested that lemon-flavored chewing gum forces a brain overload, resulting in a temporary decrease in cognitive function.

Effect of Gum Chewing Frequency on Oxygenation of the Prefrontal Cortex.

Since increased cerebral oxygenation reflects cerebral activation, this study investigated the effect of mastication frequency on prefrontal cortex oxygenation. Eleven young volunteers (nine women, two men; M age = 20.9 years, SD = 0.9) carried out three trials in which they were asked to chew a tasteless gum for 3 min at varying (rates of mastication frequency: 30, 70, and 110). Breaks of 2 min each were interleaved between trials. The oxygenation of the left prefrontal cortex was monitored by near-infrared spectroscopy. We found a significant increase in cortical oxygenation during gum chewing in all three conditions ( p < .05), compared with a resting level; we also found a significant difference between the Fast and Slow chewing conditions, and between the Fast and Normal (70 rpm) conditions, both findings seemingly related to activation of a motor command in frontal brain regions. To our knowledge, this is the first report on the effect of mastication frequency on cerebral oxygenation. Possible implications of this finding are discussed.

Interferon-alpha sensitizes human hepatoma cells to TRAIL-induced apoptosis through DR5 upregulation and NF-kappa B inactivation.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces apoptosis in a variety of cancer cells with little or no effect on normal cells. Human hepatoma cells, however, are resistant to TRAIL-induced apoptosis. Since interferon-alpha (IFN-alpha) is capable of enhancing TNF-alpha-induced apoptosis in certain cancer cells, we evaluated the effect of IFN-alpha on TRAIL-induced apoptosis of human hepatoma cells. IFN-alpha pretreatment enhanced TRAIL-induced apoptosis of HuH-7 and Hep3B cells, in which IFN-alpha upregulated the expression of DR5, a death receptor of TRAIL, and downregulated the expression of survivin, which has an antiapoptotic function. In contrast, IFN-alpha did not enhance TRAIL-induced apoptosis of HepG2 cells, in which expression of DR5 and survivin was not affected by IFN-alpha. On the other hand, TRAIL activated NF-kappa B composed of RelA-p50 heterodimer, a key transcription factor regulating cell survival, in HuH-7 and HepG2 cells. However, IFN-alpha pretreatment repressed the TRAIL-mediated activation of NF-kappaB and decreased its transcriptional activity in HuH-7 but not in HepG2 cells. Moreover, IFN-alpha pretreatment clearly augmented TRAIL-mediated caspase-8 activation in HuH-7 cells. Our results suggest that IFN-alpha could sensitize certain human hepatoma cells to TRAIL-induced apoptosis by stimulating its death signaling and by repressing the survival function in these cells.