Improvements in advanced hepatocellular carcinoma to repeat implementation of primary protocol after cancer progression occurs following sequential systemic therapy and a clinical trial: A case report.

INTRODUCTION: Systemic therapy is recommended for patients with advanced hepatocellular carcinoma (aHCC). However, drug resistance occurs over time when patients receive systemic therapy, resulting in cancer progression. Due to the lack of relevant clinical trials, optimizing subsequent treatments after cancer progression remains elusive. PATIENT CONCERNS: A 52-year-old male patient presented with epigastric discomfort and fatigue for almost 1 month with a past history of chronic hepatitis B virus infection for 30 years. DIAGNOSIS: Based on the patient's performance status, tumor status assessed by computed tomography, liver function, he was diagnosed with HCC at BCLC stage C. INTERVENTIONS AND OUTCOMES: He first received transarterial chemoembolization (TACE) combined with sintilimab and lenvatinib as first-line treatment and experienced 10-month progression-free survival. After cancer progression, the patient participated in a clinical trial of ABSK-011, a novel fibroblast growth factor receptor 4 inhibitor, with a frustrating result. Then, the patient underwent TACE and received sintilimab plus lenvatinib again. Surprisingly, the tumor had a partial response, and the patient's serum alpha-fetoprotein returned to normal. LESSONS: The combined treatment of TACE plus systemic therapy might be an appropriate subsequent treatment.

MicroRNA-188-5p inhibits hepatocellular carcinoma proliferation and migration by targeting forkhead box N2.

BACKGROUND: This study aimed to identify the biological functions, expression modes, and possible mechanisms underlying the relationship between metastatic human hepatocellular carcinoma (HCC) and MicroRNA-188-5p (miR-188) dysregulation using cell lines. METHODS: A decrease in miR-188 was detected in low and high metastatic HCC cells compared to that in normal hepatic cells and non-invasive cell lines. Gain- and loss-of-function experiments were performed in vitro to investigate the role of miR-188 in cancer cell (Hep3B, HepG2, HLF, and LM3) proliferation and migration. RESULTS: miR-188 mimic transfection inhibited the proliferation of metastatic HLF and LM3 cells but not non-invasive HepG2 and Hep3B cells; nonetheless, miR-188 suppression promoted the growth of HLF and LM3 cells. miR-188 upregulation inhibited the migratory rate and invasive capacity of HLF and LM3, rather than HepG2 and Hep3B cells, whereas transfection of a miR-188 inhibitor in HLF and LM3 cells had the opposite effects. Dual-luciferase reporter assays and bioinformatics prediction confirmed that miR-188 could directly target forkhead box N2 (FOXN2) in HLF and LM3 cells. Transfection of miR-188 mimics reduced FOXN2 levels, whereas miR-188 inhibition resulted in the opposite result, in HLF and LM3 cells. Overexpression of FOXN2 in HLF and LM3 cells abrogated miR-188 mimic-induced downregulation of proliferation, migration, and invasion. In addition, we found that miR-188 upregulation impaired tumor growth in vivo. CONCLUSIONS: In summary, this study showed thatmiR-188 inhibits the proliferation and migration of metastatic HCC cells by targeting FOXN2.

Alkaline phosphatase combined with γ-glutamyl transferase is an independent predictor of prognosis of hepatocellular carcinoma patients receiving programmed death-1 inhibitors.

Background: Immunotherapy plays an increasingly critical role in the systemic treatment of HCC. This current study aimed to establish a novel prognostic predictor of Programmed death 1 (PD-1) inhibitor therapy in hepatocellular carcinoma (HCC) independent of Child-Pugh grade. Methods: Our study screened patients with HCC who received PD-1 inhibitors at Tongji Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology from January 2018 to December 2020. ALG grade was determined by the patient's serum ALP and GGT levels before the initiation of PD-1 inhibitors. The endpoints of our study were overall survival (OS) and progression free survival (PFS). Follow-up ended at May 31, 2022. Results: Eighty- five patients (77 with Child-Pugh grade A, 8 with Child-Pugh grade B at baseline) were enrolled according to the inclusion criteria. Patients with Child-Pugh grade A achieved longer PFS and OS than those with Child-Pugh grade B. Patients with ALG grade 3 at baseline showed worse tumor response and poorer survival, and ALG grade could stratify patients with Child-Pugh grade A into subgroups with significantly different prognosis. Conclusions: ALG grade, combining ALP and GGT, is a novel and readily available prognostic marker and the predictive effect of ALG grade on patient prognosis is independent of Child-Pugh grade.

Postoperative outcomes and recurrence patterns of intermediate-stage hepatocellular carcinoma dictated by the sum of tumor size and number.

BACKGROUND: The selection criteria for Barcelona Clinic Liver Cancer (BCLC) intermediate-stage hepatocellular carcinoma (HCC) patients who would truly benefit from liver resection (LR) remain undefined. AIM: To identify BCLC-B HCC patients more suitable for LR. METHODS: We included patients undergoing curative LR for BCLC stage A or B multi-nodular HCC (MNHCC) and stratified BCLC-B patients by the sum of tumor size and number (N + S). Overall survival (OS), recurrence-free survival (RFS), recur-rence-to-death survival (RTDS), recurrence patterns, and treatments after recurrence in BCLC-B patients in each subgroup were compared with those in BCLC-A patients. RESULTS: In total, 143 patients who underwent curative LR for MNHCC with BCLC-A (n = 25) or BCLC-B (n = 118) were retrospectively analyzed. According to the N + S, patients with BCLC-B HCC were divided into two subgroups: BCLC-B1 (N + S ≤ 10, n = 83) and BCLC-B2 (N + S > 10, n = 35). Compared with BCLC-B2 patients, those with BCLC-B1 had a better OS (5-year OS rate: 67.4% vs 33.6%; P < 0.001), which was comparable to that in BCLC-A patients (5-year OS rate: 67.4% vs 74.1%; P = 0.250), and a better RFS (median RFS: 19 mo vs 7 mo; P < 0.001), which was worse than that in BCLC-A patients (median RFS: 19 mo vs 48 mo; P = 0.022). Further analysis of patients who developed recurrence showed that both BCLC-B1 and BCLC-A patients had better RTDS (median RTDS: Not reached vs 49 mo; P = 0.599), while the RTDS in BCLC-B2 patients was worse (median RTDS: 16 mo vs not reached, P < 0.001; 16 mo vs 49 mo, P = 0.042). The recurrence patterns were similar between BCLC-B1 and BCLC-A patients, but BCLC-B2 patients had a shorter recurrence time and a higher proportion of patients had recurrence with macrovascular invasion and/or extrahepatic metastasis, both of which were independent risk factors for RTDS. CONCLUSION: BCLC-B HCC patients undergoing hepatectomy with N + S ≤ 10 had mild recurrence patterns and excellent OS similar to those in BCLC-A MNHCC patients, and LR should be considered in these patients.

Exploring synthetic lethal network for the precision treatment of clear cell renal cell carcinoma.

The emerging targeted therapies have revolutionized the treatment of advanced clear cell renal cell carcinoma (ccRCC) over the past 15 years. Nevertheless, lack of personalized treatment limits the development of effective clinical guidelines and improvement of patient prognosis. In this study, large-scale genomic profiles from ccRCC cohorts were explored for integrative analysis. A credible method was developed to identify synthetic lethality (SL) pairs and a list of 72 candidate pairs was determined, which might be utilized to selectively eliminate tumors with genetic aberrations using SL partners of specific mutations. Further analysis identified BRD4 and PRKDC as novel medical targets for patients with BAP1 mutations. After mapping these target genes to the comprehensive drug datasets, two agents (BI-2536 and PI-103) were found to have considerable therapeutic potentials in the BAP1 mutant tumors. Overall, our findings provided insight into the overview of ccRCC mutation patterns and offered novel opportunities for improving individualized cancer treatment.

Generation of Premature Termination Codon (PTC)-Harboring Pseudorabies Virus (PRV) via Genetic Code Expansion Technology.

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.

Hypothyroidism is a predictive factor of superior antitumour efficacy of programmed death 1 inhibitors in hepatocellular carcinoma.

Programmed death 1 (PD-1) inhibitors are widely used for treatment of hepatocellular carcinoma (HCC). Hypothyroidism is commonly associated with this therapy, although the mechanism underlying this complication and effects on patient prognosis remain unclear. We retrospectively analysed the data of patients with HCC who received anti-PD-1 therapy at Tongji Hospital, Tongji Medical College of Huazhong University of Science and Technology between January 2018 and May 2020. Based on thyroid function evaluation, patients were categorised into hypothyroidism group and nonhypothyroidism group. Follow-up was completed on February 28, 2021. The primary endpoint of our study was progression free survival (PFS). The study included 74 patients, and the disease control rate was higher in hypothyroidism group (62.7%, 27/43) than in nonhypothyroidism group (36.4%, 11/31) (P = .020). The PFS was longer in hypothyroidism group (7.44 months) than in nonhypothyroidism group (5.68 months) (P = .006). Additionally, the PFS of patients with hypothyroidism before immunotherapy (6.27 months) was also longer than that in nonhypothyroidism group (5.68 months), although the difference was statistically nonsignificant (P = .527). Cox regression analysis showed that the hazard ratios of hypothyroidism, Child-Pugh grade B at initial admission and serum gamma-glutamyl transferase levels >71 U/L before immunotherapy were 0.404 (95% confidence interval [CI]: 0.207-0.791, P = .008), 2.753 (95%CI: 1.127-6.455, P = .026) and 2.469 (95%CI: 1.155-5.277, P = .020), respectively. Hypothyroidism was associated with prognosis in patients with HCC treated with PD-1 inhibitors, and prognosis was more favourable in patients with hypothyroidism than in those without hypothyroidism. Hypothyroidism and the Child-Pugh grade at initial admission were independently associated with patient prognosis.

A novel approach for monitoring TGF-ß signaling in vivo in colon cancer.

The TGF-ß receptor kinase inhibitors (TRKI) have been reported to inhibit tumorigenicity in colon cancer. However, there is no direct evidence showing that these inhibitors function through inhibiting the TGF-ß- mediated tumor-promoting effects in vivo. We established a TGF-ß inducible reporter system by inserting a luciferase reporter gene to the vector downstream of TGF-ß-inducible promoter elements, and transfected it into colon cancer cell lines. TRKIs SB431542 and LY2109761 were used to treat TGF-ß inducible cells in vitro and in vivo. The luciferase activity was induced 5.24-fold by TGF-ß in CT26 inducible cells, while it was marginally changed in MC38 inducible cells lacking Smad4 expression. Temporary treatment of mice with SB431542 inhibited the TGF-ß pathway and TGF-ß induced bioluminescence activity in vivo. Long-term treatment with LY2109761 inhibited tumorigenicity and liver metastasis in vivo in concomitant with reduced luciferase activity in the tumor. In this study, we established a model to monitor the TGF-ß pathway in vivo and to compare the antitumor effects of TRKIs. Based on this novel experimental tool, we provided direct evidences that LY2109761 inhibits tumorigenicity and liver metastasis by blocking the pro-oncogenic functions of TGF-ß in vivo.

Preoperative circulating tumor cells to predict microvascular invasion and dynamical detection indicate the prognosis of hepatocellular carcinoma.

BACKGROUND: This study explored the diagnostic power of preoperative circulating tumor cells (CTCs) for the presence of microvascular invasion (MVI) and the relationship between dynamic changes in postoperative CTCs and prognosis. METHODS: A total of 137 patients were recruited for the study. Preoperative blood samples were collected from all patients to detect CTCs. The time points for blood collection were before the operation, during the operation, and at 1 week, 1 month, 2 months, 3 months, 6 months, and 1 year after surgery. The predictive power of CTC count for the presence of MVI was analyzed by receiver operating characteristic (ROC) curve analysis. According to recurrence status, 137 patients were divided into three groups: no recurrence, early recurrence, and non-early recurrence groups. RESULTS: A threshold CTC count of 5 showed the most significant power for predicting the existence of MVI. In multivariate analysis, the parameters of preoperative CTC count, alpha-fetoprotein (AFP) and tumor diameter were independent predictors of MVI (P <  0.05). A CTC count greater than or equal to 5 had better predictive value than AFP > 400 µg/L and tumor diameter > 5 cm. The number of intraoperative CTCs in the three groups did not increase compared to that before surgery (P > 0.05). The number of CTCs in the nonrecurrence group and the non-early recurrence group decreased significantly 1 week after surgery compared with the intraoperative values (P <  0.001), although there was no significant difference in the early recurrence group (P = 0.95). Patients with mean CTC count ≥5 had significantly worse long-term outcomes than those with mean CTC count < 5 (P <  0.001). CONCLUSION: The preoperative CTC counts in the peripheral blood of patients with HCC are closely correlated with MVI. The intraoperative manipulation of the lesion by the surgeon does not increase the number of CTCs in peripheral blood. Surgical removal of the tumor decreases the number of CTCs. The persistence of CTCs at a high level (≥ 5) after surgery suggests a risk of early recurrence. CLINICAL TRIAL REGISTRATION: Registration number is ChiCTR-OOC-16010183 , date of registration is 2016-12-18.

Two novel recombinant porcine reproductive and respiratory syndrome viruses belong to sublineage 3.5 originating from sublineage 3.2.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an agent of porcine reproductive and respiratory syndrome (PRRS), which causes substantial economic losses to the swine industry. PRRSV displays rapid variation, and five lineages coexist in mainland China. Lineage 3 PRRSVs emerged in mainland China in 2005 and prevailed in southern China after 2010. In the present study, two lineage 3 PRRSV strains, which are named SD110-1608 and SDWH27-1710, were isolated from northern China in 2017. To explore the characteristics and origins of the two strains, we divided lineage 3 into five sublineages (3.1-3.5) based on 146 open reading frame (ORF) 5 sequences. Both strains and the strains isolated from mainland China were classified into sublineage 3.5. Lineage 3 PRRSVs isolated from Taiwan and Hong Kong were classified into sublineages 3.1-3.3 and sublineage 3.4, respectively. Recombination analysis revealed that SD110-1608 and SDWH27-1710 were derived from recombination of QYYZ (major parent strain) and JXA1 (minor parent strain). Sequence alignment showed that SD110-1608 and SDWH27-1710 shared a 36-aa insertion in Nsp2 with QYYZ isolated from Guangdong Province in 2010. Based on the evolutionary relationship among GP2a, GP3, GP4, GP5 and N proteins between sublineages 3.2 (FJ-1) and 3.5 (FJFS), we speculated that sublineage 3.5 (mainland China) originated from sublineage 3.2 (Taiwan, China). This study provides important information regarding the classification and transmission of lineage 3 PRRSVs.

Sustainable Green Pavement Using Bio-Based Polyurethane Binder in Tunnel.

As a closed space, the functional requirements of the tunnel pavement are very different from ordinary pavements. In recent years, with the increase of requirements for tunnel pavement safety, comfort and environmental friendliness, asphalt pavement has become more and more widely used in long tunnels, due to its low noise, low dust, easy maintenance, and good comfort. However, conventional tunnel asphalt pavements cause significant safety and environmental concerns. The innovative polyurethane thin overlay (PTO) has been developed for the maintenance of existing roads and constructing new roads. Based on the previous study, the concept of PTO may be a feasible and effective way to enrich the innovative functions of tunnel pavement. In this paper, the research aims to evaluate the functional properties of PTO, such as noise reduction, solar reflection and especially combustion properties. Conventional asphalt (Open-graded Friction Course (OGFC) and Stone Mastic Asphalt (SMA)) and concrete pavement materials were used as control materials. Compared with conventional tunnel pavement materials, significant improvements were observed in functional properties and environmental performance. Therefore, this innovative wearing layer can potentially provide pavements with new eco-friendly functions. This study provides a comprehensive analysis of these environmentally friendly materials, paving the way for the possible application in tunnels, as well as some other fields, such as race tracks in stadiums.

Small hepatocellular carcinoma suppressed by chemotherapy for synchronous gastric carcinoma after laparoscopy-assisted radical distal gastrectomy: A case report and literature review.

RATIONALE: Synchronous gastric carcinoma and hepatocellular carcinoma (HCC) is rare. It is hard to distinguish synchronous HCC from metastatic liver cancer in this condition. The treatment and prognosis is quite different for synchronous HCC of gastric carcinoma and liver metastasis of gastric carcinoma. PATIENT CONCERNS: A 68-year-old man with a chief complaint of epigastric pain for 1 year, accompanied by reflux and belching. The patient was diagnosed with gastric carcinoma (cT4NxM0) and laparoscopy-assisted radical distal gastrectomy was performed. This was followed by chemotherapy of FOLFOX regimen. However, a liver nodule growth was observed after postoperative systemic treatment. DIAGNOSIS: The initial diagnosis was liver metastasis of gastric carcinoma. However after hepatectomy of segment VI and VII as well as thrombectomy of right hepatic vein, histology revealed intermediate to poor differentiated HCC. Hence this case was diagnosed as synchronous gastric carcinoma and HCC. INTERVENTIONS: A preventive transcatheter arterial chemoembolization (TACE) was conducted at 4 weeks after hepatectomy. Another FOLFOX regimen was suggested, but was refused by the patient. OUTCOMES: The patient survived without tumor recurrence for 9 months after the second surgery. LESSONS: Synchronous HCC should be routinely distinguished from gastric carcinoma liver metastasis, especially for patients with hepatitis B virus (HBV) infection. The FOLFOX4 regimen for treating gastric carcinoma liver metastasis may have inhibited the progression of primary HCC in this case. This patient with HCC benefited from liver resection, inspite of hepatic vein tumor thrombosis.

Emergence of novel porcine reproductive and respiratory syndrome viruses (ORF5 RFLP 1-7-4 viruses) in China.

Porcine reproductive and respiratory syndrome viruses (PRRSVs) pose a serious threat to the porcine industry of China, and the importation of novel strain(s) makes it challenging to control these viruses. Several NADC30-like PRRSV outbreaks have occurred in mainland China since 2013. In the current study, we report two novel PRRSVs, designated LNWK96 and LNWK130, which belong to lineage 1 and are closely related to US strains with ORF5 restriction fragment length polymorphism (RFLP) 1-7-4. The two viruses had a 100-aa deletion in the nsp2 gene corresponding to positions 328-427 in the VR-2332 strain, which was consistent with most of the ORF5 RFLP 1-7-4 viruses. Recombination analyses indicated that both viruses derived from the recombination of 1-7-4 isolates and ISU30 or NADC30, which were isolated in the United States. Taken together, these results demonstrate the emergence of ORF5 RFLP 1-7-4-like (NADC34-like) PRRSVs in China for the first time.

Adaptions of field PRRSVs in Marc-145 cells were determined by variations in the minor envelope proteins GP2a-GP3.

The recent rapid evolution of PRRSVs has resulted in certain biological characteristic changes, such as the fact that an increasing number of field PRRSVs can be isolated from PAMs but not from Marc-145 cells. In this study, we first isolated Marc-145-unadaptive field PRRSV strains from PAMs; sequence analysis showed that these PRRSVs belong to the HP-PRRSV (lineage 8) branch or NADC30-Like (lineage 1) branch. We further found major variations in ORF2-4 regions. To explore the viral adaptation mechanisms in detail, we constructed a full-length cDNA clone of MY-376, a Marc-145-unadaptive PRRSV. Construction of serially chimeric viruses of HuN4-F112 (a Marc-145-adaptive strain) and MY-376 demonstrated that variation in the minor envelope protein (GP2a and GP3) complex is a main determinant of PRRSV tropism for Marc-145 cells.

n-3 fatty acid-based parenteral nutrition improves postoperative recovery for cirrhotic patients with liver cancer: A randomized controlled clinical trial.

BACKGROUND & AIMS: A new lipid emulsion enriched in n-3 fatty acid has been reported to prevent hepatic inflammation in patients following major surgery. However, the role of n-3 fatty acid-based parenteral nutrition for postoperative patients with cirrhosis-related liver cancer is unclear. We investigated the safety and efficacy of n-3 fatty acid-based parenteral nutrition for cirrhotic patients with liver cancer followed hepatectomy. METHODS: A prospective randomized controlled clinical trial (Registered under ClinicalTrials.gov Identifier no. NCT02321202) was conducted for cirrhotic patients with liver cancer that underwent hepatectomy between March 2010 and September 2013 in our institution. We compared isonitrogenous total parenteral nutrition with 20% Structolipid and 10% n-3 fatty acid (Omegaven, Fresenius-Kabi, Germany) (treatment group) to Structolipid alone (control group) for five days postoperatively, in the absence of enteral nutrition. RESULTS: We enrolled 320 patients, and 312 (97.5%) were included in analysis (155 in the control group and 157 in the treatment group). There was a significant reduction of morbidity and mortality in the treatment group, when compared with the control group (total complications 78 [50.32%] vs. 46 [29.30%]; P < 0.001, total infective complications, 30 [19.35%] vs. 15 [9.55%]; P = 0.014), overall mortality (5 [3.23%] vs. 1 [0.64%]; P = 0.210), and hospital stay (12.56 ± 3.21 d vs. 10.17 ± 3.15 d; P = 0.018). CONCLUSIONS: We found that addition of n-3 fatty acid-based parenteral nutrition significantly improved postoperative recovery for cirrhotic patients with liver cancer following hepatectomy, with a significant reduction in overall mortality and length of hospital stay.

An integrin beta4-EGFR unit promotes hepatocellular carcinoma lung metastases by enhancing anchorage independence through activation of FAK-AKT pathway.

Anoikis, a form of programmed cell death, occurs when the cells are detached from the appropriate extracellular matrix. Anoikis resistance or anchorage independence is necessary for distant metastases of cancer. The mechanisms by which hepatocellular carcinoma (HCC) cells become resistant to anoikis are not fully understood. Integrin beta4 (ITGB4, also known as CD104) is associated with progression of many human cancers. In this study, we demonstrate that ITGB4 is over-expressed in HCC tissues and aggressive HCC cell lines. To explore the role of ITGB4 in HCC, we inhibited its expression using small interfering RNA in two HCC cell lines: HCCLM3 and HLF. We show that knockdown of ITGB4 significantly enhanced susceptibility to anoikis through inhibition of AKT/PKB signaling. Moreover, ITGB4 interacts with epidermal growth factor receptor (EGFR) in a ligand independent manner. Inactivation of EGFR inhibits the anchorage independence and AKT pathway promoted by ITGB4. Further investigation proved that the ITGB4-EGFR unit triggers the focal adhesion kinase (FAK) to activate the AKT signaling pathway. Finally, we demonstrate that over-expression of ITGB4 is positively associated with tumor growth and lung metastases of HCC in vivo. Collectively, we demonstrate for the first time that ITGB4 is overexpressed in HCC tissues and promotes metastases of HCC by conferring anchorage independence through EGFR-dependent FAK-AKT activation.

Smad4 sensitizes colorectal cancer to 5-fluorouracil through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade.

5-Fluorouracil (5-FU), a cell cycle-specific antimetabolite, is one of the most commonly used chemotherapeutic agents for colorectal cancer (CRC). Yet, resistance to 5-FU-based chemotherapy is still an obstacle to the treatment of this malignancy. Mutation or loss of Smad4 in CRC is pivotal for chemoresistance. However, the mechanism by which Smad4 regulates the chemosensitivity of CRC remains unclear. In the present study, we investigated the role of Smad4 in the chemosensitivity of CRC to 5-FU, and whether Smad4-regulated cell cycle arrest is involved in 5-FU chemoresistance. We used Smad4-expressing CT26 and Smad4-null SW620 cell lines as experimental models, by knockdown or transgenic overexpression. Cells or tumors were treated with 5-FU to determine chemosensitivity by cell growth, tumorigenicity assay and a mouse model. Cell cycle distribution was examined with flow cytometric analysis, and cell cycle-related proteins were examined by western blotting. Smad4 deficiency in CT26 and SW620 cells induced chemoresistance to 5-FU both in vitro and in vivo. Smad4 deficiency attenuated G1 or G2 cell cycle arrest by activating the PI3K/Akt/CDC2/survivin pathway. The PI3K inhibitor, LY294002, reversed the activation of the Akt/CDC2/survivin cascade in the Smad4-deficient cells, while it had little effect on cells with high Smad4 expression. In conclusion, we discovered a novel mechanism mediated by Smad4 to trigger 5-FU chemosensitivity through cell cycle arrest by inhibiting the PI3K/Akt/CDC2/survivin cascade. The present study also implies that LY294002 has potential therapeutic value to reverse the chemosensitivity of CRC with low Smad4 expression.

Characterization of two newly emerged isolates of porcine reproductive and respiratory syndrome virus from Northeast China in 2013.

A newly emerged porcine reproductive and respiratory syndrome virus (PRRSV) that has caused severe reproductive losses in sows appeared in some regions of China in 2013. To explore the biology of this new PRRSV and understand more fully genetic diversity in PRRSV isolates from China, the complete genome of the two 2013 Chinese isolates, designated HLJA1 and HLJB1, were analyzed. Genomic sequence analysis showed that HLJA1 and HLJB1 shared 88.6-98.3% nucleotide identity with genotype 2 (North American type, NA-type) isolates, but only 61.1% with the genotype 1 (European type, EU-type) isolate of Lelystad virus, indicating that both these isolates belong to the NA-type PRRSV genotype. Phylogenetic analysis showed that the NA-type PRRSV isolates formed three subgroups (1, 2 and 3); representatives of these subgroups are VR-2332, CH-1a and HUN4, respectively. HLJA1 and HLJB1 belong to subgroup 2. Analysis of NSP2 revealed that HLJA1 has a 48-amino acid deletion at positions 473-480 and 482-521, unlike other HP-PRRSV isolates, while HLJB1 has only a 1-amino acid deletion at position 481 compared with CH-1a. Interestingly, HLJA1 replicated in PAM cells but not in MARC-145 cells, whereas HLJB1 replicated in both cell types. The neutralizing antibody titer of pig hyperimmune sera against HUN4 was significantly higher than that of HLJA1 or HLJB1. Additionally, genetic variability in GP5 and GP3 proteins and in the novel ORF5a protein was evident. In addition to elucidating the genetic relationships between PRRSV isolates, our results suggest that Chinese PRRSV will remain a pandemic virus.

Pseudorabies virus variant in Bartha-K61-vaccinated pigs, China, 2012.

The widely used pseudorabies virus (PRV) Bartha-K61 vaccine has played a key role in the eradication of PRV. Since late 2011, however, a disease characterized by neurologic symptoms and a high number of deaths among newborn piglets has occurred among Bartha-K61-vaccinated pigs on many farms in China. Clinical samples from pigs on 15 farms in 6 provinces were examined. The PRV gE gene was detectable by PCR in all samples, and sequence analysis of the gE gene showed that all isolates belonged to a relatively independent cluster and contained 2 amino acid insertions. A PRV (named HeN1) was isolated and caused transitional fever in pigs. In protection assays, Bartha-K61 vaccine provided 100% protection against lethal challenge with SC (a classical PRV) but only 50% protection against 4 challenges with strain HeN1. The findings suggest that Bartha-K61 vaccine does not provide effective protection against PRV HeN1 infection.

Expression of short peptide by an improved isocaudamer tandem repeat strategy.

An improved isocaudamer tandem repeat strategy for the production of short functional peptide was demonstrated in the study. The coding sequence of short peptide was codon optimized, and two isocaudamers were induced into the end of coding sequence. By re-cutting with isocaudamers and re-ligating, the coding sequence of short peptide in the expression vector was increased in a multiple manner (21, 22, 23, 24, 25 …….). In the present study, an 8 amino-acidresidue peptide of porcine reproductive and respiratory syndrome virus (PRRSV) was effectively expressed in 8 copies and 16 copies by this approach, then the proteins in 8 copies and 16 copies were used to generate antibody against this epitope in rabbits. The results showed that PRRSVs were well recognized by the antibody in indirect immunofluorescence assay. The technology using isocaudamer to insert multiple tandem repeats in the vector provides an important approach for the studies of small molecule peptides.