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Abstract RT-LAMP detection of SARS-CoV-2 has been shown as a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validated the optimized RT-LAMP assay for the detection of SARS-CoV-2 in raw nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings with limited economic resources.
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The recent outbreak of a novel coronavirus SARS-CoV-2 (also known as 2019-nCoV) threatens global health, given serious cause for concern. SARS-CoV-2 is a human-to-human pathogen that caused fever, severe respiratory disease and pneumonia (known as COVID-19). By press time, more than 70,000 infected people had been confirmed worldwide. SARS-CoV-2 is very similar to the severe acute respiratory syndrome (SARS) coronavirus broke out 17 years ago. However, it has increased transmissibility as compared with the SARS-CoV, e.g. very often infected individuals without any symptoms could still transfer the virus to others. It is thus urgent to develop a rapid, accurate and onsite diagnosis methods in order to effectively identify these early infects, treat them on time and control the disease spreading. Here we developed an isothermal LAMP based method-iLACO (isothermal LAMP based method for COVID-19) to amplify a fragment of the ORF1ab gene using 6 primers. We assured the species-specificity of iLACO by comparing the sequences of 11 related viruses by BLAST (including 7 similar coronaviruses, 2 influenza viruses and 2 normal coronaviruses). The sensitivity is comparable to Taqman based qPCR detection method, detecting synthesized RNA equivalent to 10 copies of 2019-nCoV virus. Reaction time varied from 15-40 minutes, depending on the loading of virus in the collected samples. The accuracy, simplicity and versatility of the new developed method suggests that iLACO assays can be conveniently applied with for 2019-nCoV threat control, even in those cases where specialized molecular biology equipment is not available.
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Surgical treatment is the most effective approach to achieving radical treatment and long-term survival of liver cancer. At present, most patients with intermediate and advanced liver cancer do not have the opportunity to receive radical surgery. The downstaging and conversion treatment strategies for intermediate and advanced liver cancer should be taken seriously in clinical practice to further improve treatment outcome. According to disease stage and conditions of patients with intermediate and advanced liver failure, multidisciplinary cooperation is needed to optimize downstaging and conversion treatment regimens, which may help inoperable patients to obtain the opportunity for surgical treatment. Palliative therapy can be changed to radical surgical resection, and patients who cannot undergo antitumor treatment can still receive palliative therapy. More patients with intermediate and advanced liver cancer are able to achieve a better clinical outcome, an improvement in prognosis, and long-term survival.
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Collective migration is the essential movement style in embryonic development.Recent stu-dies have found that collective migration is one of the main movement styles in invasion and metastasis of tumor cells.Tumor cells can invade into the stroma and microvascular in group,and the collective migration shows the unique characters when compared to the single cell movement.The potential mechanisms are closely correlated to the cell-cell connection and interaction between tumor cells and microenvironment.With the gradual improve-ment of the observation models,the mechanisms about the physical or chemical guidance,the related signaling pathways,and the epigenetic regulation in collective migration will be elucidated.
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Primary liver cancer is a common malignancy with increasing incidence and mortality. Remarkable progress has been made in the clinical research on liver cancer, but challenges to further improvement are still rigorous. This article reviews the progress and difficulties of primary liver cancer study from the following aspects: molecular classification and stem cell biology, pathological and clinical diagnosis, clinical staging, surgical treatment, loco-regional treatment, molecular targeted therapy, systemic chemotherapy, management of portal vein tumor thrombus, antiviral therapy, diagnosis and treatment of intrahepatic cholangiocarcinoma, and multimodality treatment. The future strategies to further improve primary liver cancer study are also investigated.
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BACKGROUND:Ischiogluteal bursitis has been recognized for a long time, but its treatment stil limits to local blocking injection and surgery methods that were developed 40 years ago. OBJECTIVE:To observe the efficacy of platelet-rich plasma on ischiogluteal bursitis. METHODS:Data of 15 patients with ischiogluteal bursitis were colected. Al the patients with ischiogluteal bursitis were treated with bilateral platelet-rich plasma (n=10) or local blocking injection (n=5). Patients’ outcomes were assessed by visual analogue scale, the Treatment Satisfaction Questionnaire for Medication (TSQM) Version II and recurrence rate. The folow-up time was from 6 to 14 months. RESULTS AND CONCLUSION: There was no statistical difference in visual analogue scale score between the platelet-rich plasma group and local blocking group (F=0.219,P=0.643), but the score of visual analogue scale in the platelet-rich plasma group was higher during short-term folow-up (within 1 week after treatment), but lower in the long-term folow-up. In the aspects of overal satisfaction score, clinical effectiveness and side effects, the platelet-rich plasma group was inferior to the local blocking group at short-term folow-up, especialy at 1 week after treatment; however, these scores became better in the platelet-rich plasma group than the local blocking group during the long-term folow-up period. In addition, no statistical difference in the convenience score was found between the two groups. At the last folow-up, the recurrence rate in the platelet-rich plasma group was lower than that in the local blocking group. Both the platelet-rich plasma and local blocking injection can significantly reduce the pain of patients with ischiogluteal bursitis. Local blocking injection has better short-term effectiveness. Platelet-rich plasma injection works moderately, but its effectiveness can last for longer time, and the recurrence rate is lower.
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Objective To investigate the selective oncolytic role and antitumor action of a novel recombinant adenovirus containing E1A and IL-24 on hepatocellular carcinoma cell(HCC). Methods The recombinant adenovirus expressing IL-24 (Ad. HS4. AFP. E1A/IL-24) was constructed by using modified human alpha-fetoprotein (HS4-AFP) promoter to drive adenovirus E1A gene and II-24 gene.Cell Counting Kit-8 were performed to test the selective cytotoxicity of the virus in hepatocellular carcinoma cell lines SMMC-7721, Hep3B, MHCC97-H and hepatocyte cell line L02 . The mRNA and protein expression of IL-24 gene were detected by RT-PCR and western blot. Cell growth curves and Annexin V/PI assay were used to study cell proliferation and apoptosis of MHCC97-H. The anti-metastatic effects of the recombinant adenovirus were evaluated in cell adhesion, migration, and cell motion. Matrix metalloproteinase-2 (MMP-2) expression was examined by RT-PCR and zymography.Results Selective replications of Ad. HS4. AFP. E1A/IL-24 adenovirus were observed in over expression AFP cell line MHCC97-H, a highly metastatic potential HCC cell line but not in hepatocyte cell line L02. The mRNA and protein of IL-24 were also over expressed in MHCC97-H. This recombinant adenovirus also showed the significant oncolytic action on MHCC97-H but not on L02 (P＜0. 05). Besides, the recombinant adenovirus significantly inhibited MHCC97-H metastatic potential such as cell adhesion, migration and invasion as well(P＜0.01). Conclusion The selective oncolytic adenovirus expressing E1A and II-24 has a selective antitumor effect and play an inhibitory role in metastasis of HCC.
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Objective To assess clinicopathological features and prognosis of patients with combined hepatocellular and cholangiocarcinoma (cHCC-CC). Methods Clinicopathological and follow-up data of 115 cHCC-CC patients confirmed pathologically in Liver Cancer Institute of Fudan University from 1995 to 2007 were analyzed. Kaplan-Meier method was used to calculate 1-,3- and 5-year survival rates and tumor-free survival rates. Survival curves were analyzed using the log-rank test. The factors that impacted the prognosis of cHCC-CC were estimated. Results In 115 cases, one was Allen's type A, one was Allen's type B, and the other 113 were Allen's type C. Being with male in predominance, most of the cHCC-CC patients had liver cirrhosis background. They presented with elevated AFP or CA19-9, vascular invasion, resembling hepatocellular carcinoma(HCC)as well as lymph nodes metastasis. One-, 3-, 5-year survival rates of 115 patients were 68. 1%, 38. 1% and 33.6%, respectively, with median survival time of 13.0 months. Whereas the 1-, 3-, 5-year survival rates in radical resected patients were 78.4 % ,44.4 % and 44.4 % ,respectively, with median survival time of 16.0 months. Tumor free survival time at 1-, 3- and 5-year was 57.8 %, 12.6 % and 0.0 %,respectively,with median recurrent time of 10.0 months. One-, 3-, 5-year survival rates of 10 nonsurgical patients were 10/10,10/10 and 0/10,respectively, with median survival time of 5.3 months.TNM stage was independent factor for prognosis of the patients after resection. Whereas the lymph nodes involvement was independent factor for the tumor free survival time of radical resected patients.Conclusions Although clinicopathological characteristics of cHCC-CC are more similar to those of HCC, the prognosis of cHCC-CC is more unfavorable than that of HCC. TNM stage is an independent determinant of long time outcome for patients after resection.
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Objective:The protein of missing in metastasis (MIM), a novel discovered actin-binding scaffold protein, is involved in actin cytoskeleton rearrangements, signal transduction and transcriptional activation, and has close association with tumor growth, invasion, and metastasis. Recently, much focus has been placed on the role of MIM performed in tumor progression. In recent years, more and more attention is focused on its action mechanism in various kinds of tumors, which has a wide foreground of investigation. In this paper, we make a comprehensive review of the association of MIM with cancer development, in order to provide the theoretical basis and new strategies for application of MIM proteins in cancer diagnosis and treatment.
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Objective To compare the Barcelona clinic liver cancer staging classification (BCLC), the Japan integrated staging score (JIS), the cancer of the liver Italian program score (CLIP) and Chinese staging system in terms of their ability to predict outcomes and to guide option of therapy in patients with hepatocellular carcinoma (HCC) in China.Methods Clinical data of 861 HCC patients from Zhongshan Hospital between 2001 and 2002 were retrospectively analyzed. Patients were classified acccording to different staging systems. Survival for patients in different stages and the effects of therapeutic methods on survival time were compared. Results BCLC, JIS and Chinese staging system showed the ability in predicting survival for patients in different staging. CLIP failed to show significant difference in survival rates for each subgroup. There was no significant difference in survival rate between surgery and transarterial chemoembolization (TACE)/transarterial embolization (TAE) for patients classified as BCLC stage C, CLIP scores more than 3 or Chinese stage Ⅲ a.The survival rate, however, was higher in patients received operation than those received TACE/TAE if they were classified as earlier stages. Conclusions The BCLC, JIS and Chinese staging systems show prospective ability for Chinese HCC patients in prediction outcomes, whereas the BCLC and the Chinese staging systems are better at both predicting outcomes and guiding the option of treatment.
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<p><b>OBJECTIVE</b>To compare gene expression profile of human hepatocellular carcinoma (HCC) cell lines with different metastatic potentials, so as to screen for metastasis-related genes.</p><p><b>METHODS</b>Gene expression profile of MHCC97-L and HCCLM3, two HCC cell lines with similar genetic background but different in spontaneous metastatic potentials, were studied by cDNA microarray.</p><p><b>RESULTS</b>From 1,626 screened genes, 25 differentially expressed genes were found, 18 showed decreased expression including the decreased expression of cell cycle control genes Rb2, mismatch repair gene hMSH2, and signal transduction gene protein kinase C beta 2 and 7 increased expression including signal transduction gene MAP kinase kinase 6, cell proliferation gene E25, immunity related gene SP40, 40, etc in HCCLM3.</p><p><b>CONCLUSION</b>The genes, being closely associated with cancer metastasis, could be considered as potential markers to predict metastasis and targets for anti-metastasis intervention.</p>
Subject(s)
Humans , Blotting, Northern , Carcinoma, Hepatocellular , Genetics , Pathology , Gene Expression , Gene Expression Profiling , Liver Neoplasms , Genetics , Pathology , Neoplasm Metastasis , Genetics , Oligonucleotide Array Sequence Analysis , RNA, Neoplasm , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To study the anti-tumor effects of combined IL-12 and granalocyte-macrophage-colong scimulating factor (GM-CSF) gene therapy on murine hepatocellular carcinoma.</p><p><b>METHODS</b>Twenty-four mice received subcutaneous inoculation of 1 x 10(6) BNL hepatoma cells were randomly divided into the following four groups with different cytokine encoding plasmids (6 mice for each group): (1)pXX-GM-CSF 12.5 microg and pXX-IL-12 12.5 microg; (2)pXX-IL-12 25 microg; (3)pXX-GM-CSF 25 microg; (4)pXX-Neo 25 microg. The plasmids were given through tail vein using a versatile hydrodynamics-based DNA delivery method on day 3 and day 6 after tumor challenge. The growth of tumor and cellular immune response were observed intensively. The changes in serum concentration of IL-12, GM-CSF, and IFN-gamma after plasmids injection were also observed.</p><p><b>RESULTS</b>Co-delivery of IL-12 and GM-CSF could mount stronger anti-tumor effects, longer term enhanced IL-12 expression and lower level of IFN-gamma than did IL-12 alone.</p><p><b>CONCLUSIONS</b>Combined IL-12 and GM-CSF can render a strong anti-tumor effect as well as a potential to lower the side effects.</p>
Subject(s)
Animals , Male , Mice , CD4-Positive T-Lymphocytes , Cell Biology , CD8-Positive T-Lymphocytes , Cell Biology , Cell Division , Genetics , Physiology , Genetic Therapy , Methods , Granulocyte-Macrophage Colony-Stimulating Factor , Blood , Genetics , Physiology , Interferon-gamma , Blood , Interleukin-2 , Blood , Genetics , Physiology , Liver Neoplasms, Experimental , Genetics , Therapeutics , Lymphocyte Count , Mice, Inbred BALB C , Neoplasm Transplantation , Plasmids , Genetics , Time Factors , Tumor Cells, CulturedABSTRACT
<p><b>OBJECTIVE</b>To study the immunoprotective effect of IL-2 and B7-1 gene co-transfected liver cancer vaccine on hepatocarcinogenesis in mice.</p><p><b>METHODS</b>The murine liver cancer cell strain Hepal-6 was transfected with IL-2 and/or B7-1 gene via recombinant adenovirus vectors and the liver cancer vaccines were prepared. C57BL/6 mice were immunized with the vaccines and challenged with the parental Hepal-6 cells afterwards. The immunoprotection was investigated and the reactive T cell line was assayed.</p><p><b>RESULTS</b>The effect of the Hep6-IL2/B7 vaccine on the onset of tumor formation was the strongest. The media survival time of the mice was the longest (68 days, P<0.05) and the implanted tumor was the smallest (P<0.05). The effect of single IL-2 or B7-1 gene-transfected vaccine was next to the co-transfected gene group with the mean survival time being 59 and 54 days, respectively. The mean survival time of wild or BGFP gene modified vaccine immunized group was 51 and 48 days, respectively. The control group all died within 38 days and the implanted tumor was the largest (P<0.05). The cellular immunofunction test and cytotoxicity study showed that the mice immunized with the Hep6-IL2/B7 vaccine gained significantly increased NK, LAK and CTL activity (29.0% +/- 2.5%, 65.0% +/- 2.9%, 83.1% +/- 1.5% respectively, compared with other groups P<0.05).</p><p><b>CONCLUSIONS</b>The IL-2 and B7-1 gene co-transfected liver cancer vaccines can induce the mice to produce activated and specific CTL against the parental tumor cells, and demonstrate stronger effect on the hepatocarcinogenesis than single gene modified or the regular tumor vaccine. Therefore, the vaccines may become a novel potential therapy for recurrence and metastases of HCC.</p>
Subject(s)
Animals , Female , Humans , Mice , Adenoviridae , Genetics , B7-1 Antigen , Genetics , Allergy and Immunology , Cancer Vaccines , Genetics , Allergy and Immunology , Cell Division , Allergy and Immunology , Genetic Vectors , Genetics , Allergy and Immunology , Interleukin-2 , Genetics , Allergy and Immunology , Killer Cells, Lymphokine-Activated , Allergy and Immunology , Killer Cells, Natural , Allergy and Immunology , Liver Neoplasms, Experimental , Allergy and Immunology , Pathology , Mice, Inbred C57BL , Neoplasm Transplantation , Survival Analysis , T-Lymphocytes, Cytotoxic , Allergy and Immunology , Time Factors , Transfection , Tumor Cells, CulturedABSTRACT
Objective To explore the expression and significance of p21WAF1 and p53 in HCC. Methods Immunohistochemical method (IHC) was used to localize and semi-quantitate the proteins of p21WAF1 and p53 and to observe the relationship between the expression of p21WAF1 and the different histopathologic characters in 38 patients of HCC and their peri-cancer tissue as well as 5 normal liver tissue. Results Of all 38 cases, both p21WAF1 and p53 expression were significantly higher in tumor than that in corresponding non-tumors liver tissue; 14 (36.8 %) of 38 cases showed p21WAF1 positive staining, 28 cases (73.7 %) were p53 positive, p21WAF1＋/p53＋ or p21WAF1-/p53－ were observed in 18, while 20 cases showed p53＋/p21WAF1－ or p53－/p21WAF1＋. p21WAF1＋ was seen in 1 of 38 (2.6 %) corresponding non-cancerous tissue and 2 of 5 normal liver tissue. p53 protein was not detected neither in the non-tumorous tissue nor in normal liver. No significant association was found between the expressions of p21WAF1 and p53(P＞0.05) in HCC. Their was no significant correlation between p21WAF1 or p53 expression and the different histopathologic characters of tumor (differentiating grades, intrahepatic metastasis and/or cancerous thrombi within portal veins). Conclusion Both p21WAF1 and p53 proteins are over expressed in HCC than that in corresponding non-tumorous liver tissue, but there is no relationship between them. Both p53-independent and p53-dependent mechanism may play a role in regulating p21WAF1 expression in HCC. p21WAF1 immunostaining cannot be used to assess the status of p53 in any given cell or tissue.
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Objective To assess the role of helical CT in the long-term follow-up of patients with malignant hepatic tumors treated with RFA. Methods CT findings of patients with liver malignant tumors (29 hepatocellular carcinomas and 7 metastases) were reviewed retrospectively, who underwent percutaneous ultrasound-guided radiofrequency ablation (RFA). CT images consisted of nonenhanced and dual-phase contrast-enhanced helical CT scan, the effect of ablation therapy and tumor recurrence were detected. Results The major patterns of tumor residual or local recurrence were the lesions with thick rim or nodular peripheral enhancement in arterial phase or the lesions with gross enlargement on follow-up CT images. The complete necrosis lesions in 28 cases (77.8%) were seen on the initial postablation CT scans after a single session RFA. On the subsequent follow-up CT examination, the local intra-hepatic tumor recurrences were seen in 2 cases (5.6%) as well as the remote intra-hepatic tumor recurrences were seen in 4 cases (11.1%).Conclusion For certain patients with hepatic malignant tumors, RFA is an effective therapeutic method, and dual-phase contrast enhanced helical CT scans play a important role in long-term assessment and follow-up of patients.
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Objective To study severe or rare complications following radiofrequency ablation (RFA) for liver cancer. Methods Clinical records of severe or rare complications following RFA in 272 cases of liver cancer from January 2002 to December 2004 were retrospectively studied. Results A total of 301 RFA procedures were performed in the 272 cases. The incidence of severe or rare complications was 3.32% (10/301), and the death rate was 0.66% (2/301). Complications included 1 case of intraperitoneal hemorrhage, 2 cases of infection (1 case of peritonitis with sepsis and 1 case of liver Abscess superimposed upon bilioma), 3 cases of upper gastrointestinal bleeding (including 1 case of hemobilia), 1 case of hepatic arteriovenous fistula, 1 case of hemo-pneumothorax, 1 case of esophagopleural fistula and 1 case of needle-tract implantation of tumor. Conclusions In order of frequency,severe complications following RFA are upper gastrointestinal bleeding,infection and intraperitoneal hemorrhage.
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Objective: To investigate the safety of transgenic human lung adenocarcinoma cell line SPC-A-1/IL-2 as tumor vaccine. Methods: IL-2 gene was introduced into human lung adenocarcinoma cell line SPC-A-1 and was expressed stably on the basis of the construction of retroviral packing cell line PA317/pLIL-2SN.The mutagenesis of both the DNA and supernatant of SPC-A-1/IL-2 in the and in vitro was tested by means of genetic toxicological techniques.Results:The result indicated that mutagenesis of both the DNA and the supernatant of transgenic cell SPC-A-1/IL-2 was not observed. Conclusion: The initial experiment suggested that the application of transgenic cell SPC-A-l/IL-2 as tumor vaccine was bisically safe and reliable.
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To clone the full length cDNA of the tumor rejection gene MAGE-1 from hepatocellular carcinoma(HCC) tissues. This MAGE-1 gene and the tumor rejection antigen encoded by it may be useful in subsequent studies aiming at exploring new strategies for the immunotherapy for HCC. Methods: The full length MAGE-1 cDNA was amplified by RT-PCR method using a pair of primers designed according to the encoding sequence of MAGE-1 gene. The PCR products were then digested by restriction endonucleases and inserted into the plasmid PUC19. After primary selection of the recombinants by endonuclease digestion, the sequences of the inserted gene fragments were confirmed by DNA sequence analysis. Results; Using the same pair of primers, we obtained three clones of different MAGE genes, which were a full length MAGE-1 gene, a 750 bp fragment of MAGE-3 gene and a gene highly homologous to MAGE-6 and MAGE-12 but not identical to any reported MAGE genes. Conclusion: These data suggested that some MAGE genes are expressed in heptocellular carcinoma probably including some unknown genes, which might introduce potential new targets for immune attacks.
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In this study, the RT-PCR method was employed to detect the expression of AFP in mRNA level in tissue samples form 52 patients suffered from hepatocellular carcinoma (HCC) . The results revealed that the positive rate of AFPmRNA was 76.9% in the HCC tumor tissues and 69.4% in the paratumortissues from the HCC patients with severe cirrhosis . Meanwhile, in HCC patients without cirrhosis, the positive rate reached 50% in tumor tissues, but no AFPmRNA expression was found in related paratumor tissues. The study suggested that the AFP protein was specially expressed by hepatoma cells and mutating hepatocytes. The relationships between AFPmRNA and tumoor size, capsule status and tumor metastasis were also demonstrated.
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The 5'-flanking region of the human ?-fetoprotein(AFP) gene contains transcriptional regulatory sequences(TRSs) which up-regulate AFP gene expression with cell-specific enhancer activity in hepatoma cells. A couple of primers(Primerl: 5'-TGCAAGCTTATGATTCCCAAATATC-3'; Primer 2: 5'-GTCGAATTCGTGGCCTGGA TAAAGCTGAGT-3') were designed for synthesis and purification according to the known sequences of 5'-flanking region. AFPTRSs of 416 base pairs were amplified from human chromosome DNA by PCR, The identification of the AFPTRSs was confirmed to be consistent with reported sequences. The AFPTRSs can be applied to regulating the specific expression of cytokines in hepatoma cells.
