Isolation and evaluation of the pathogenicity of a hybrid shiga toxin-producing and Enterotoxigenic Escherichia coli in pigs.

BACKGROUND: Porcine pathogenic Escherichia coli (E. coli), the globally recognized important pathogen, causes significant economic loss in the field. Enterotoxigenic E. coli (ETEC) causes porcine neonatal and post-weaning diarrhea (PWD), frequently carrying F4 adhesin, F18 adhesin, Heat-Stable toxin (ST), and Heat-Labile toxin (LT). Shiga Toxin-Producing E. coli (STEC) produces F18 adhesin and Shiga toxin type 2e (stx2e), majorly leading to systemic endothelial cell damage and edema disease. In this study, hemolytic pathogenic hybrid STEC/ETEC strains carrying ST and LT genes of ETEC and the Stx2e gene of STEC isolated from pigs with PWD in Taiwan were identified. The pathogenicity of a Taiwan hybrid STEC/ETEC strain was evaluated by oral inoculation in post-weaning pigs. RESULTS: Next generation sequencing and multilocus sequence typing of two hybrid Taiwan porcine STEC/ETEC isolates indicated that these two isolates were closely related to the ST88 porcine hybrid STEC/ETEC isolated from pigs with watery diarrhea. Furthermore, the two hybrid Taiwan porcine STEC/ETEC isolates also displayed combinations of multiple resistance genes encoding mechanisms for target modification and antibiotic inactivation. Animal experiments confirmed that the Taiwan hybrid STEC/ETEC could cause watery diarrhea in post-weaning pigs with no signs of edema disease and minimal histopathological lesions. CONCLUSION: To the best of the authors' knowledge, the present study is the first study demonstrating intestinal pathogenicity of the hybrid STEC/ETEC in pigs. The result suggests that the hybrid STEC/ETEC should be considered as a new emerging pathogen and a new target for vaccine development.

Insulinoma-associated protein 1 (INSM1) immunohistochemical expression in normal, hyperplastic, and neoplastic canine neuroendocrine tissues.

Insulinoma-associated protein 1 (INSM1), a recently identified neuroendocrine marker, is a transcriptional regulator with highly conserved INSM1 homologues in various species. This study investigated the immunohistochemical reactivity of the INSM1 antibody in 20 normal canine neuroendocrine tissues from various anatomical locations, 87 hyperplastic or neoplastic tissues of neuroendocrine origin, and 62 non-neuroendocrine neoplasms and compared the results with those of chromogranin A and synaptophysin in neuroendocrine neoplasms. Western blot was performed on fresh canine pituitary glands and canine parathyroid glands to confirm the specificity of the anti-INSM1 antibody. The results showed that the anti-INSM1 antibody could detect nuclear expression in normal canine neuroendocrine tissues, except for the parathyroid glands. INSM1 was detectable in 79/87 (91%) of the hyperplastic and neoplastic neuroendocrine lesions, but all parathyroid carcinomas and parathyroid adenomas (three samples each) were negative for INSM1. In contrast, INSM1 was detected in only one of 62 (2%) non-neuroendocrine neoplasms. The overall percentage of neuroendocrine neoplasms that immunolabeled positively for all three markers was 89%. In addition, the nuclear expression of INSM1 was easier to interpret than that of chromogranin A or synaptophysin. These findings confirm that INSM1 is a useful immunohistochemical marker for diagnosing canine neuroendocrine neoplasms, except for parathyroid neoplasms, and should be considered as part of immunohistochemistry panels to improve diagnostic capability.

Lower Repetition Induces Similar Postactivation Performance Enhancement to Repetition Maximum After a Single Set of Heavy-Resistance Exercise.

ABSTRACT: Chen, C-F, Chuang, C-Y, Wang, C-C, Liu, S-A, Chang, H-W, and Chan, K-H. Lower repetition induces similar postactivation performance enhancement to repetition maximum after a single set of heavy-resistance exercise. J Strength Cond Res XX(X): 000-000, 2023-The study was divided into 2 parts to investigate the acute postactivation performance enhancement (PAPE) responses to lower repetitions at the same load of 87% 1 repetition maximum (1RM) in the upper and lower body. In part 1, 14 athletes performed plyometric push-up (PPU) after the conditioning activity (CA) of bench press (BP). In part 2, 13 athletes performed a countermovement jump (CMJ) after the CA of parallel squat (PS). Subjects completed 3, 4, or 5 repetitions (trials CA-3, CA-4, or CA-5) of BP or PS in randomized and counterbalanced order. The velocity of each movement of the trial was recorded. The PPU or CMJ was tested every 2 minutes after the trial up to 12 minutes to assess the Post-Max and optimal individual PAPE time. The mean velocity of the last movement of BP in CA-5 was significantly lower than that in CA-3 (0.23 ± 0.06 vs. 0.28 ± 0.06 m·second -1 , p < 0.05), and the velocity of PS in CA-4 or CA-5 was significantly lower than that in CA-3 (0.53 ± 0.07 and 0.50 ± 0.05 vs. 0.57 ± 0.07 m·second -1 , p < 0.05). The peak force of PPU and jump height of CMJ at Post-Max in the 3 trials were significantly greater than those at Pre ( p < 0.05). There were no significant differences among trials in the optimal individual PAPE times in either part of the study. A single set of 87% 1RM resistance exercises with 3 or 4 repetitions in both the upper body and the lower body induces similar PAPE to repetition maximum.

Effects of selective cyclooxygenase-2 inhibitor robenacoxib on primary cells derived from feline injection-site sarcoma.

Feline injection-site sarcomas (FISSs) are highly invasive malignant mesenchymal neoplasms that arise from injection sites in cats. Although the tumorigenesis of FISSs is still uncertain, there is a consensus that FISS is associated with chronic inflammation caused by irritation of injection-related trauma and foreign chemical substances. Chronic inflammation can provide a proper microenvironment for tumour development, which has been known as one of the risk factors of tumorigenesis in many tumours. To investigate the tumorigenesis of FISS and screen for its potential therapeutic targets, cyclooxygenase-2 (COX-2), an inflammation-enhancing enzyme, was selected as a target for this study. In vitro experiments using FISS- and normal tissue-derived primary cells and robenacoxib, a highly selective COX-2 inhibitor, were performed. The results demonstrated that expression of COX-2 could be detected in formalin-fixed and paraffin-embedded FISS tissues and FISS-derived primary cells. Cell viability, migration and colony formation of FISS-derived primary cells were inhibited, and cell apoptosis was enhanced by robenacoxib in a dose-dependent manner. However, susceptibility to robenacoxib varied in different lines of FISS primary cells and was not completely correlated with COX-2 expression. Our results suggest that COX-2 inhibitors could be potential adjuvant therapeutics against FISSs.

Cryo-EM analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans.

Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3.3-Å cryoelectron microscopy (cryo-EM) structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type N-glycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.

Identification and genomic characterization of Baculovirus penaei in Litopenaeus vannamei in Taiwan.

Baculovirus penaei (BP), the causative agent of tetrahedral baculovirosis, causes the death of penaeid genera at the larval and post-larval stages. BP has been reported in the Western Pacific, South-East Atlantic, and the State of Hawaii, but never in Asia. The clinical features of BP infection are non-specific, and diagnosis relies on histological and molecular methods. In the present study, we report the first identification of BP infection in a shrimp farm in Northern Taiwan in 2022. Histopathologically, several tetrahedral eosinophilic intranuclear occlusion bodies were observed in or budding out of the nuclei of the degenerative hepatopancreatic cells. In situ hybridization and polymerase chain reaction confirmed tetrahedral baculovirosis infection caused by BP. Sequence alignment of the TW BP-1 with the USA BP strain reported in 1995 revealed 94.81% identity in the partial gene. The possibility of the emergence of USA-like BP in Taiwan highlights the importance of further epidemiological investigations on the prevalence and impact of BP in Asia.

Concurrent infection of a novel genotype of hepatopancreatic parvovirus and Enterocytozoon hepatopenaei in Penaeus vannamei in Taiwan.

Hepatopancreatic parvovirus (HPV) and Enterocytozoon hepatopenaei (EHP) are emerging and reemerging pathogens in shrimps. In the present study, a novel genotype of HPV concurrently infected with EHP in Penaeus vannamei in Taiwan leading to severe atrophy and damage of hepatopancreas were confirmed by histopathology, in situ hybridization, and PCR. The novel genotype of HPV exhibited 66%-69.5% sequence identities with all known HPVs and carried unique amino acid deletions and insertions in the VP gene. According to phylogenetic analysis, the Taiwan HPV isolates were classified as the genotype IV. The present study not only provided the histopathological and molecular proof of HPV and EHP co-infection in Taiwan, but also revealed the importance of investigating the geographical expansion of novel HPV genotypes.

The Use of Distinctive Monoclonal Antibodies in FMD VLP- and P1-Based Blocking ELISA for the Seromonitoring of Vaccinated Swine.

The serum neutralization (SN) test has been regarded as the "gold standard" for seroconversion following foot-and-mouth disease virus (FMDV) vaccination, although a high-level biosafety laboratory is necessary. ELISA is one alternative, and its format is constantly being improved. For instance, standard polyclonal antisera have been replaced by monoclonal antibodies (MAbs) for catching and detecting antibodies, and inactive viruses have been replaced by virus-like particles (VLPs). To the best of current knowledge, however, no researchers have evaluated the performances of different MAbs as tracers. In previous studies, we successfully identified site 1 and site 2 MAbs Q10E and P11A. In this study, following the established screening platform, the VLPs of putative escape mutants from sites 1 to 5 were expressed and used to demonstrate that S11B is a site 3 MAb. Additionally, the vulnerability of VLPs prompted us to assess another diagnostic antigen: unprocessed polyprotein P1. Therefore, we established and evaluated the performance of blocking ELISA (bELISA) systems based on VLPs and P1, pairing them with Q10E, P11A, S11B, and the non-neutralizing TSG MAb as tracers. The results indicated that the VLP paired with S11B demonstrated the highest correlation with the SN titers (R2 = 0.8071, n = 63). Excluding weakly positive serum samples (SN = 16-32, n = 14), the sensitivity and specificity were 95.65% and 96.15% (kappa = 0.92), respectively. Additionally, the P1 pairing with Q10E also demonstrated a high correlation (R2 = 0.768). We also discovered that these four antibodies had steric effects on one another to varying degrees, despite recognizing distinct antigenic sites. This finding indicated that MAbs as tracers could not accurately detect specific antibodies, possibly because MAbs are bulky compared to a protomeric unit. However, our results still provide convincing support for the application of two pairs of bELISA systems: VLP:S11B-HRP and P1:Q10E-HRP.

Full-genome characterization of a novel Felis catus papillomavirus 4 subtype identified in a cutaneous squamous cell carcinoma of a domestic cat.

The present study describes two full-genome sequences of Felis catus papillomavirus type 4 (FcaPV4) identified in squamous cell carcinoma (SCC) of two domestic cats. Two full-genome sequences of FcaPV4 were detected and characterized by PCR and sequencing. The L1 nucleotide sequence homology of one case showed 95.70% sequence identity to the reference FcaPV4, suggesting that this isolate should be classified as a subtype. Reverse-transcriptase PCR (RT-PCR) of two oncogenes, E6 and E7 was performed to confirm mRNA expression. Expression of E6 and E7 mRNA was detected in both cases, suggesting that FcaPV4 contributes to the development of SCC. This is the first report of FcaPV4 subtype. The present study will update the genomic features of FcaPV4 and contribute to deepening our knowledge about the etiological roles of FcaPV4 in feline cutaneous SCC.

Isotypes of autoantibodies against novel differential 4-hydroxy-2-nonenal-modified peptide adducts in serum is associated with rheumatoid arthritis in Taiwanese women.

BACKGROUND: Rheumatoid arthritis (RA) is an autoimmune disorder with systemic inflammation and may be induced by oxidative stress that affects an inflamed joint. Our objectives were to examine isotypes of autoantibodies against 4-hydroxy-2-nonenal (HNE) modifications in RA and associate them with increased levels of autoantibodies in RA patients. METHODS: Serum samples from 155 female patients [60 with RA, 35 with osteoarthritis (OA), and 60 healthy controls (HCs)] were obtained. Four novel differential HNE-modified peptide adducts, complement factor H (CFAH)1211-1230, haptoglobin (HPT)78-108, immunoglobulin (Ig) kappa chain C region (IGKC)2-19, and prothrombin (THRB)328-345, were re-analyzed using tandem mass spectrometric (MS/MS) spectra (ProteomeXchange: PXD004546) from RA patients vs. HCs. Further, we determined serum protein levels of CFAH, HPT, IGKC and THRB, HNE-protein adducts, and autoantibodies against unmodified and HNE-modified peptides. Significant correlations and odds ratios (ORs) were calculated. RESULTS: Levels of HPT in RA patients were greatly higher than the levels in HCs. Levels of HNE-protein adducts and autoantibodies in RA patients were significantly greater than those of HCs. IgM anti-HPT78-108 HNE, IgM anti-IGKC2-19, and IgM anti-IGKC2-19 HNE may be considered as diagnostic biomarkers for RA. Importantly, elevated levels of IgM anti-HPT78-108 HNE, IgM anti-IGKC2-19, and IgG anti-THRB328-345 were positively correlated with the disease activity score in 28 joints for C-reactive protein (DAS28-CRP). Further, the ORs of RA development through IgM anti-HPT78-108 HNE (OR 5.235, p < 0.001), IgM anti-IGKC2-19 (OR 12.655, p < 0.001), and IgG anti-THRB328-345 (OR 5.761, p < 0.001) showed an increased risk. Lastly, we incorporated three machine learning models to differentiate RA from HC and OA, and performed feature selection to determine discriminative features. Experimental results showed that our proposed method achieved an area under the receiver operating characteristic curve of 0.92, which demonstrated that our selected autoantibodies combined with machine learning can efficiently detect RA. CONCLUSIONS: This study discovered that some IgG- and IgM-NAAs and anti-HNE M-NAAs may be correlated with inflammation and disease activity in RA. Moreover, our findings suggested that IgM anti-HPT78-108 HNE, IgM anti-IGKC2-19, and IgG anti-THRB328-345 may play heavy roles in RA development.

Antibacterial and Antibiofilm Activities of Novel Antimicrobial Peptides against Multidrug-Resistant Enterotoxigenic Escherichia Coli.

Post-weaning diarrhea due to enterotoxigenic Escherichia coli (ETEC) is a common disease of piglets and causes great economic loss for the swine industry. Over the past few decades, decreasing effectiveness of conventional antibiotics has caused serious problems because of the growing emergence of multidrug-resistant (MDR) pathogens. Various studies have indicated that antimicrobial peptides (AMPs) have potential to serve as an alternative to antibiotics owing to rapid killing action and highly selective toxicity. Our previous studies have shown that AMP GW-Q4 and its derivatives possess effective antibacterial activities against the Gram-negative bacteria. Hence, in the current study, we evaluated the antibacterial efficacy of GW-Q4 and its derivatives against MDR ETEC and their minimal inhibition concentration (MIC) values were determined to be around 2~32 µg/mL. Among them, AMP Q4-15a-1 with the second lowest MIC (4 µg/mL) and the highest minimal hemolysis concentration (MHC, 256 µg/mL), thus showing the greatest selectivity (MHC/MIC = 64) was selected for further investigations. Moreover, Q4-15a-1 showed dose-dependent bactericidal activity against MDR ETEC in time-kill curve assays. According to the cellular localization and membrane integrity analyses using confocal microscopy, Q4-15a-1 can rapidly interact with the bacterial surface, disrupt the membrane and enter cytosol in less than 30 min. Minimum biofilm eradication concentration (MBEC) of Q4-15a-1 is 4× MIC (16 µg/mL), indicating that Q4-15a-1 is effective against MDR ETEC biofilm. Besides, we established an MDR ETEC infection model with intestinal porcine epithelial cell-1 (IPEC-1). In this infection model, 32 µg/mL Q4-15a-1 can completely inhibit ETEC adhesion onto IPEC-1. Overall, these results suggested that Q4-15a-1 may be a promising antibacterial candidate for treatment of weaned piglets infected by MDR ETEC.

Type-3 Hyaluronan Synthase Attenuates Tumor Cells Invasion in Human Mammary Parenchymal Tissues.

The microenvironment for tumor growth and developing metastasis should be essential. This study demonstrated that the hyaluronic acid synthase 3 (HAS3) protein and its enzymatic product hyaluronic acid (HA) encompassed in the subcutaneous extracellular matrix can attenuate the invasion of human breast tumor cells. Decreased HA levels in subcutaneous Has3-KO mouse tissues promoted orthotopic breast cancer (E0771) cell-derived allograft tumor growth. MDA-MB-231 cells premixed with higher concentration HA attenuate tumor growth in xenografted nude mice. Human patient-derived xenotransplantation (PDX) experiments found that HA selected the highly migratory breast cancer cells with CD44 expression accumulated in the tumor/stroma junction. In conclusion, HAS3 and HA were detected in the stroma breast tissues at a high level attenuates effects for induced breast cancer cell death, and inhibit the cancer cells invasion at the initial stage. However, the highly migratory cancer cells were resistant to the HA-mediated effects with unknown mechanisms.

Genomic characterization of a novel bovine papillomavirus type 28.

Infection of bovine papillomavirus (BPV) has been associated with mucosal and/or cutaneous tumor development in bovids. To date, up to 27 genotypes of BPVs have been identified and classified based on the nucleotide sequence identity of L1 open reading frame. In the present study, the complete sequence of a novel BPV concurrently identified with BPV1 and BPV2 in the facial cutaneous papilloma lesion of a domestic cattle was characterized. The whole genome of the unclassified BPV was 7263 base pairs in full length with GC ratio of 42.9%. In comparison with published BPV sequences, L1 nucleotide sequence of the novel BPV shared 75% identity with BPV15, and was suggested to be classified in the genus, Xipapillomavirus. According to the criteria established by the International Committee on the Taxonomy of Viruses, the novel BPV was designated as BPV type 28.

Expression of HIF-1α and VEGF in feline mammary gland carcinomas: association with pathological characteristics and clinical outcomes.

BACKGROUND: The microenvironment within solid malignant tumors, including feline mammary gland carcinomas (FMGCs), is commonly hypoxic, possibly due to the lack of functional blood vessels in rapidly proliferating neoplastic tissue. Malignant cells can undergo genetic and adaptive changes that prevent them from dying due to oxygen deprivation through expressions of hypoxia-inducible factor 1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF). Therefore, HIF-1α and VEGF are ideal biomarkers for cancer therapy and prognostic evaluation. The aims of this study were to evaluate the expression of HIF-1α and VEGF in feline mammary carcinomas and analyze their correlations with clinical and pathological factors, such as clinical stage, histologic grading, regional metastasis, and overall survival rate. RESULTS: Paraffin-embedded tissue samples collected from 72 cats with FMGCs were retrospectively studied. Histologic pattern and histologic grading (Elston and Ellis grading system) of these FMGCs were determined. Our data indicated that grade II tubulopapillary carcinomas (43/72, 59.7%) prevailed in this study, and most FMCGs showed apparent necrosis, squamous metaplasia, and intratumoral stromal response. According to the results of immunohistochemical (IHC) stainings performed in tissue microarrays (TMAs), HIF-1α and VEGF overexpressions were respectively noted in 69.4% (50/72) and 77.8% (56/72) of FMGC cases. Chi-square test showed no correlation of HIF-1α overexpression with clinical and pathological factors. VEGF overexpression was significantly correlated with histologic pattern (p = 0.021), stromal response (p = 0.048), squamous metaplasia (p = 0.001), and lymphovascular invasion (p = 0.007). However, neither HIF-1α nor VEGF overexpression was correlated with histologic grading and metastasis. Of 38 cats with 1-year follow-up, IHC stainings of HIF-1α and VEGF were performed on whole tissue sections. The results showed that overexpression of HIF-1α was significantly correlated with the overall survival rate (p < 0.05) (log-rank test), whereas there was no significant correlation between VEGF overexpression and overall survival rate. CONCLUSIONS: This study suggests that the overexpression of HIF-1α may indicate poor prognosis/overall survival rate in cats with FMGCs. Developing compounds that inhibit HIF-1α may be a potential approach to FMGC treatment.

DNA primase polypeptide 1 (PRIM1) involves in estrogen-induced breast cancer formation through activation of the G2/M cell cycle checkpoint.

The DNA primase polypeptide 1 (PRIM1) is responsible for synthesizing small RNA primers for Okazaki fragments generated during discontinuous DNA replication. PRIM1 mRNA expression levels in breast tumor samples were detected by real-time PCR analysis. Xenografted tumor model was established to study the carcinogenic role of PRIM1 and its potential therapeutic applications. The average PRIM1 mRNA (copy number × 103 /µg) expression was 4.7-fold higher in tumors than in normal tissue (*p = 0.005, n = 254). PRIM1 was detected preferentially at a higher level (>40-fold) in poorly differentiated tumor tissues (n = 46) compared with more highly differentiated tumors tissues (n = 10) (*p = 0.005). Poor overall survival rate was correlated to the estrogen receptor positive (ER+, n = 20) patients with higher PRIM1 expression when compare to the ER- (n = 10) patients (Chi Square test, p = 0.03). Stable expression of PRIM1-siRNA in the ER+ BT-474 cells-xenograft tumors significantly reduced tumor volume in SCID mice (*p = 0.005). The anti-tumoral effects of inotilone isolated from Phellinus linteus was tested and had significant effects on the inhibition of PRIM1 protein expression in ER+ breast cancer cells. In vivo study was performed by administering inotilone (10 mg/kg, twice a week for 6 weeks), which resulted in significantly reduced BT-474-xenografted tumor growth volume compared with control (n =5 per group, *p < 0.05). This study provides evidences for the prognostic effects of PRIM1 with poor overall survival rate in the ER+ patients and will be valuable to test for therapeutic purpose.

Therapeutic efficacy of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys.

Human immunodeficiency virus type 1 (HIV-1)-specific monoclonal antibodies with extraordinary potency and breadth have recently been described. In humanized mice, combinations of monoclonal antibodies have been shown to suppress viraemia, but the therapeutic potential of these monoclonal antibodies has not yet been evaluated in primates with an intact immune system. Here we show that administration of a cocktail of HIV-1-specific monoclonal antibodies, as well as the single glycan-dependent monoclonal antibody PGT121, resulted in a rapid and precipitous decline of plasma viraemia to undetectable levels in rhesus monkeys chronically infected with the pathogenic simian-human immunodeficiency virus SHIV-SF162P3. A single monoclonal antibody infusion afforded up to a 3.1 log decline of plasma viral RNA in 7 days and also reduced proviral DNA in peripheral blood, gastrointestinal mucosa and lymph nodes without the development of viral resistance. Moreover, after monoclonal antibody administration, host Gag-specific T-lymphocyte responses showed improved functionality. Virus rebounded in most animals after a median of 56 days when serum monoclonal antibody titres had declined to undetectable levels, although, notably, a subset of animals maintained long-term virological control in the absence of further monoclonal antibody infusions. These data demonstrate a profound therapeutic effect of potent neutralizing HIV-1-specific monoclonal antibodies in SHIV-infected rhesus monkeys as well as an impact on host immune responses. Our findings strongly encourage the investigation of monoclonal antibody therapy for HIV-1 in humans.

Role of nuclear factor-kappa B in feline injection site sarcoma.

BACKGROUND: Chronic inflammation has been implicated in sarcomagenesis. Among various factors, activation of nuclear factor-kappa B (NF-κB) signaling pathway has been documented being able to target genes associated with tumor progression and up-regulate the expression of tumor-promoting cytokines and survival genes in several human solid tumors. Feline injection sites sarcomas (FISS) are malignant entities derived from the mesenchymal origin. The disease has been considered to be associated with vaccine adjuvant, aluminum, which serves as a stimulus continuously inducing overzealous inflammatory and immunologic reactions. To understand the contribution of NF-κB in FISS, detection of activated NF-κB in paraffin-embedded specimens, in vitro establishment of primary cells derived from FISS, and evaluation of the effects of the NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), on primary tumor cells were conducted. RESULTS: In this study, nuclear expression of NF-κB p65 was detected in 83.3% of FISS cases and not correlated with tumor grading, sex, and age. Primary cells derived from FISS in three cats exhibiting same immunohistochemical characteristics as their original tumor were successfully established. The NF-κB inhibitor, DHMEQ, was able to prevent nuclear translocation of NF-κB p65, inhibit cell proliferation, migration, and colonization in dosage-dependent manners, and induce cell apoptosis in these primary FISS cells. CONCLUSIONS: High expression rate of nuclear NF-κB p65 in FISS cases and dose-dependent inhibitory effects on the growth of FISS primary cells treated with NF-κB inhibitor suggested that NF-κB might be a potential molecular therapeutic target for FISS.

Development and comparison of enzyme-linked immunosorbent assays based on recombinant trimeric full-length and truncated spike proteins for detecting antibodies against porcine epidemic diarrhea virus.

BACKGROUND: Since 2010, outbreaks of genotype 2 (G2) porcine epidemic diarrhea virus (PEDV) have caused high mortality in neonatal piglets and have had devastating impacts on the swine industry in many countries. A reliable serological assay for evaluating the PEDV-specific humoral and mucosal immune response is important for disease survey, monitoring the efficacy of immunization, and designing strategies for the prevention and control of PED. Two PEDV spike (S) glycoprotein-based indirect enzyme-linked immunosorbent assays (ELISAs) were developed using G2b PEDV-Pintung 52 (PEDV-PT) trimeric full-length S and truncated S1-501 proteins derived from the human embryonic kidney (HEK)-293 cell expression system. The truncated S1-501 protein was selected from a superior expressed stable cell line. The sensitivity and specificity of these two ELISAs were compared to immunostaining of G2b PEDV-PT infected cells and to a commercial nucleocapsid (N)-based indirect ELISA kit using a panel of PEDV negative and hyperimmune sera. RESULTS: The commercial N-based ELISA exhibited a sensitivity of 37%, a specificity of 100%, and a fair agreement (kappa = 0.37) with the immunostaining result. In comparison, the full-length S-based ELISA showed a sensitivity of 97.8%, a specificity of 94%, and an almost perfect agreement (kappa = 0.90) with the immunostaining result. Interestingly, the S1-501-based ELISA had even higher sensitivity of 98.9% and specificity of 99.1%, and an almost perfect agreement (kappa = 0.97) with the immunostaining result. A fair agreement (kappa< 0.4) was seen between the commercial N-based ELISA and either of our S-based ELISAs. However, the results of the full-length S-based ELISA shared an almost perfect agreement (kappa = 0.92) with that of S1-501-based ELISA. CONCLUSIONS: Both full-length S-based and S1-501-based ELISAs exhibit high sensitivity and high specificity for detecting antibodies against PEDVs. Considering the high protein yield and cost-effectiveness, the S1-501-based ELISA could be used as a reliable, sensitive, specific, and economic serological test for PEDV.

Study of the antitumor mechanisms of apiole derivatives (AP-02) from Petroselinum crispum through induction of G0/G1 phase cell cycle arrest in human COLO 205 cancer cells.

BACKGROUND: Apiole was isolated from the leaves of various plants and vegetables and has been demonstrated to inhibit human colon cancer cell (COLO 205 cells) growth through induction of G0/G1 cell cycle arrest and apoptotic cell death. This study further explored the antitumor effects of apiole derivatives AP-02, 04, and 05 in COLO 205 cancer cells. METHODS: Human breast (MDA-MB-231, ZR75), lung (A549, PE089), colon (COLO 205, HT 29), and hepatocellular (Hep G2, Hep 3B) cancer cells were treated with apiole and its derivatives in a dose-dependent manner. Flow cytometry analysis was subsequently performed to determine the mechanism of AP-02-induced G0/G1 cell cycle arrest. The in vivo antitumor effect of AP-02 (1 and 5 mg/kg, administered twice per week) was examined by treating athymic nude mice bearing COLO 205 tumor xenografts. The molecular mechanisms of AP-02-induced antitumor effects were determined using western blot analysis. RESULTS: AP-02 was the most effective compound, especially for inhibition of COLO 205 colon cancer cell growth. The cytotoxicity of AP-02 in normal colon epithelial (FHC) cells was significantly lower than that in other normal cells derived from the breast, lung or liver. Flow cytometry analysis indicated that AP-02-induced G0/G1 cell cycle arrest in COLO 205 cells but not in HT 29 cells (< 5 µM for 24 h, **p < 0.01). Tumor growth volume was also significantly inhibited in AP-02 (> 1 mg/kg)-treated athymic nude mice bearing COLO 205 tumor xenografts compared to control mice (*p < 0.05). Furthermore, G0/G1 phase regulatory proteins (p53 and p21/Cip1) and an invasion suppressor protein (E-cadherin) were significantly upregulated, while cyclin D1 was significantly downregulated, in AP-02-treated tumor tissues compared to the control group (> 1 mg/kg, *p < 0.05). CONCLUSIONS: Our results provide in vitro and in vivo molecular evidence of AP-02-induced anti-proliferative effects on colon cancer, indicating that this compound might have potential clinical applications.

Long-term exposure to extremely low-dose of nicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induce non-malignant breast epithelial cell transformation through activation of the a9-nicotinic acetylcholine receptor-mediated signaling pathway.

Breast cancer (BC) is the most common cancer affecting women worldwide and has been associated with active tobacco smoking. Low levels of nicotine (Nic) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), have been detected in cases of second-hand smoke (SHS). However, the correlation between SHS and BC risk remains controversial. In this study, we investigated whether the physiological SHS achievable dose of Nic and tobacco specific nitrosamine, NNK act together to induce breast carcinogenesis using an in vitro breast cell carcinogenesis model. Immortalized non-tumorigenic breast epithelial cell line, HBL-100 used for a time-course assay, was exposed to very low levels of either Nic or NNK, or both. The time-course assay consisted of 23 cycles of nitrosamines treatment. In each cycle, HBL-100 cells were exposed to 1pM of Nic and/or 100 femtM of NNK for 48 hours. Cells were passaged every 3 days and harvested after 10, 15, and 23 cycles. Our results demonstrated that the tumorigenicity of HBL-100, defined by soft agar colony forming, proliferation, migration and invasion abilities, was enhanced by co-exposure to physiologically SHS achievable doses of Nic and NNK. In addition, α9-nAChR signaling activation, which plays an important role in cellular proliferation and cell survival, was also observed. Importantly, an increase in stemness properties including the prevalence of CD44+/CD24- cells, increase Nanog expression and mammosphere-forming ability were also observed. Our results indicate that chronic and long term exposure to environmental tobacco smoke, may induce breast cell carcinogenesis even at extremely low doses.