Antitumor activity of recombinant antimicrobial peptide penaeidin-2 against kidney cancer cells.

Penaeidin-2 (Pen-2) is an important antimicrobial peptide derived from the Pacific white shrimp, Penaeus vannamei, and possesses both antibacterial and antifungal activities. Recent studies suggest that recombinant penaeidins show similar activities to the native Pen-2 protein. Previous researches have shown that some antimicrobial peptides (AMPs) exhibit cytotoxic activity against cancer cells. To date, there have been no studies on the antitumor effects of Pen-2. This study evaluated the potential of recombinant pen-2 (rPen-2) in the selective killing of kidney cancer cell lines ACHN and A498, and its action mechanism. MTT assays found the maximal growth inhibition of HK-2, ACHN and A498 cells treated with 100 µg/mL rPen-2 at 48 h was 13.2%, 62.4%, and 70.4%, respectively. DNA-specific fluorescent dye staining showed a high percentage of apoptosis on cancer cells. Flow cytometry revealed that the apoptosis rate of HK-2, ACHN and A498 cells was 15.2%, 55.2%, and 61.5% at 48 h respectively, suggesting that rPen-2 induced higher apoptosis rate in cancer cells than in HK-2 cells. Laser confocal scanning microscopy demonstrated that the plasma membrane was the key site where rPen-2 interacted with and destroyed tumor cells. Scanning electron microscopy showed the morphologic changes of the cell membranes of kidney cancer cells treated with rPen-2. These results suggest that rPen-2 is a novel potential therapeutic agent that may be useful in treating kidney cancers.

Production, purification, and characterization of the cecropin from Plutella xylostella, pxCECA1, using an intein-induced self-cleavable system in Escherichia coli.

Antimicrobial peptides (AMPs) are widely expressed and play an important role in innate immune defense against infectious agents such as bacteria, viruses, fungi, and parasites. Cecropins are a family of AMPs synthesized in the fat body of insects that have proven effective at killing specific pathogens. In order to fulfill their clinical potential as antimicrobial drugs, a simple, cost-effective method to express AMPs is sorely needed. In this study, we expressed and characterized the cecropin from Plutella xylostella (pxCECA1) using an intein-dependent expression system in Escherichia coli. We cloned the pxCECA1 gene from larva by RT-PCR and fused the encoding sequence of mature pxCECA1 with an intein gene and a chitin-binding domain gene (CBD) in pTWIN1 plasmid. The fusion protein CBD-intein-pxCECA1 was expressed in E. coli BL21 (DE3) and separated by flowing cell extracts through a chitin column. Subsequently, self-cleavage of the intein at its C-terminus was induced in a temperature- and pH-dependent manner, resulting in the release of mature pxCECA1. The optimal conditions for self-cleavage were determined to be pH 6.0 for 48 h at 4°C, under which 12.3 mg of recombinant pxCECA1 could be recovered from 1 l of E. coli culture. The purified pxCECA1 displayed antimicrobial activity against a broad variety of gram-positive and gram-negative bacteria. This preparation was especially effective against Staphylococcus aureus, including methicillin-resistant strains. Catalase release assays demonstrated that pxCECA1 acts as a microbicidal agent. These results show for the first time that the IMPACT-TWIN expression system is an efficient, cost-effective way to produce fully functional AMPs and that the AMP pxCECA1 is a novel microbicidal agent with promising therapeutic applications.

[Relationship between placental pathology and small-for-gestational age neonates].

OBJECTIVE: To investigate the relationship between pathological abnormalities of placenta and small-for-gestational-age neonates. METHODS: One hundred placentas of small-for-gestational-age (SGA group) and 200 appropriate-for-gestational-age (AGA group) with single living birth in third trimester were investigated by gross and microscopic examination. The AGA placentas were collected from 2 cases following every SGA placenta. All cases were collected from Shanghai Changning District Maternity and Infant Health Hospital from January 2010 to December 2011. RESULTS: The gestational week, neonatal birth weight, full-term neonatal birth weight, the preterm birth rate and vaginal spontaneous delivery rate were significantly lower in SGA group than that in AGA group (P < 0.002). Full-term placental volume, placental weight and fetal placental weight ratio were lower in SGA group than that in AGA group (P < 0.05). Unusual insertion and torsion of umbilical cord were more common in SGA group (P < 0.05). Syncytial knots increase, avascular villi and villous infarcts were significantly higher in SGA group (P < 0.005), but there were no significant difference between SGA group and AGA group in intervillous thrombi, chronic villitis and chorangiosis (P > 0.05). Gestational hypertension disease and abnormality of fetal monitoring were more common in SGA group (P < 0.05). CONCLUSIONS: Gestational hypertension disease is the main clinical cause of SGA. Some placental abnormality can affect the growth and development of intrauterine fetus.

[Invasive ductal carcinomas of breast showing partial reversed cell polarity are associated with lymphatic tumor spread].

OBJECTIVE: To investigate the relationship between partial reversed cell polarity (PRCP) and lymphatic tumor spread in invasive ductal carcinoma (IDC), not othervise specified (NOS). METHODS: Immunohistochemistry (EnVision method) was used to examine the expression of epithelial membrane antigen (EMA) and the reversed cell polarity in 199 cases of IDC. RESULTS: Of the 199 cases, including five cases with micropapillary differentiation,30 cases with PRCP and 164 cases of IDC-NOS (without micropapillary differentiation and/or PRCP), lymphovascular invasion was seen in four (4/5), 13(43.3%) and 30 cases (18.3%) respectively; nodal metastasis was seen in four (4/5), 19 (63.3%) and 56 cases (34.1%) respectively. The rates of lymphovascular invasion and nodal metastasis were significantly higher in IDC with PRCP or IMPC than IDC-NOS (P = 0.00); there was however no significant difference between IDC with PRCP and IMPC for lymphovascular invasion and nodal metastasis (P = 0.18, P = 0.64). CONCLUSIONS: IDC with PRCP, similar to IMPC, is more likely to show lymphovascular invasion and nodal metastasis. Complete or partial reversal of cell polarity may play a significant role in lymphatic tumor spread.

[Primary malignant tumor of the appendix: clinicopathological analysis of 22 cases].

OBJECTIVE: To investigate the clinicopathological and immunohistochemical features of primary malignant tumor of the appendix. METHODS: The clinical data were reviewed; and histopathological and immunohistochemical features were analyzed in 22 cases with primary malignant tumor of the appendix. RESULT: In 22 cases of primary malignant tumor of the appendix, 19 cases were carcinoid and 3 were adenocarcinoma. Immunohistochemistry showed that the carcinoid was positively reacted to the neuroendocrine markers, and the adenocarcinoma was negatively reacted to the neuroendocrine markers. CONCLUSION: Immunohistochemistry is useful in diagnosis of primary malignant tumor of the appendix, a rare type of cancer.

Use of low-dose computed tomography to assess pulmonary tuberculosis among healthcare workers in a tuberculosis hospital.

BACKGROUND: According to the World Health Organization, China is one of 22 countries with serious tuberculosis (TB) infections and one of the 27 countries with serious multidrug-resistant TB strains. Despite the decline of tuberculosis in the overall population, healthcare workers (HCWs) are still at a high risk of infection. Compared with high-income countries, the TB prevalence among HCWs is higher in low- and middle-income countries. Low-dose computed tomography (LDCT) is becoming more popular due to its superior sensitivity and lower radiation dose. However, there have been no reports about active pulmonary tuberculosis (PTB) among HCWs as assessed with LDCT. The purposes of this study were to examine PTB statuses in HCWs in hospitals specializing in TB treatment and explore the significance of the application of LDCT to these workers. METHODS: This study retrospectively analysed the physical examination data of healthcare workers in the Beijing Chest Hospital from September 2012 to December 2015. Low-dose lung CT examinations were performed in all cases. The comparisons between active and inactive PTB according to the CT findings were made using the Pearson chi-square test or the Fisher's exact test. Comparisons between the incidences of active PTB in high-risk areas and non-high-risk areas were performed using the Pearson chi-square test. Analyses of active PTB were performed according to different ages, numbers of years on the job, and the risks of the working areas. Active PTB as diagnosed by the LDCT examinations alone was compared with the final comprehensive diagnoses, and the sensitivity and positive predictive value were calculated. RESULTS: A total of 1 012 participants were included in this study. During the 4-year period of medical examinations, active PTB was found in 19 cases, and inactive PTB was found in 109 cases. The prevalence of active PTB in the participants was 1.24%, 0.67%, 0.81%, and 0.53% for years 2012 to 2015. The corresponding incidences of active PTB among the tuberculosis hospital participants were 0.86%, 0.41%, 0.54%, and 0.26%. Most HCWs with active TB (78.9%, 15/19) worked in the high-risk areas of the hospital. There was a significant difference in the incidences of active PTB between the HCWs who worked in the high-risk and non-high-risk areas (odds ratio [OR], 14.415; 95% confidence interval (CI): 4.733 - 43.896). Comparisons of the CT signs between the active and inactive groups via chi-square tests revealed that the tree-in-bud, cavity, fibrous shadow, and calcification signs exhibited significant differences (P = 0.000, 0.021, 0.001, and 0.024, respectively). Tree-in-bud and cavity opacities suggest active pulmonary tuberculosis, whereas fibrous shadow and calcification opacities are the main features of inactive pulmonary tuberculosis. Comparison with the final comprehensive diagnoses revealed that the sensitivity and positive predictive value of the diagnoses of active PTB based on LDCT alone were 100% and 86.4%, respectively. CONCLUSIONS: Healthcare workers in tuberculosis hospitals are a high-risk group for active PTB. Yearly LDCT examinations of such high-risk groups are feasible and necessary.

[Significance of galectin-3 and CD44v6 expression in differential diagnosis of thyroid nodules].

OBJECTIVE: To investigate the difference of galectin-3 and CD44v6 expression between benign and malignant thyroid nodules, and to evaluate their clinical value in distinguishing thyroid cancer from benign thyroid nodules. METHODS: The expression of galectin-3 and CD44v6 was immunohistochemically detected by the ABC method in 143 benign and malignant thyroid nodule samples. RESULTS: Expression of these two markers in benign thyroid nodules: galectin-3 was negative in 10 cases of para-cancer normal tissue and 14 cases of benign nodules found in the other benign thyroid disease. It was weakly positive in 4 of 52 nodular goiter (7.7%). Also it was weakly positive in 2 of 22 follicular adenomas (9.1%). But all three eosinophilic follicular adenomas were diffusely or focally positive for galectin-3. CD44v6 was negative in 10 cases of para-cancer normal tissue, but positive in 4 of 14 nodular lesions found in benign thyroid diseases (28.6%). It was also positive in 16 of 52 nodular goiters (30.8%), and weakly positive in 7 of 22 follicular adenomas (31.8%). The two markers in malignant lesions: galectin-3 was positive in 50 of 52 thyroid adenocarcinoma (96.2%), CD44v6 was positive in 42 of 52 thyroid adenocarcinoma (80.8%). The positive rate of galectin-3 and CD44v6 expression in thyroid cancer was significantly higher than that in benign thyroid nodule and normal tissue (P < 0.001). The sensitivity, specificity and accuracy of galectin-3 combined with CD44v6 in differentiating benign from malignant thyroid nodule were 80.8%, 93.4%, 88.8%; they were 96.2%, 90.1%, 92.3% for Galectin-3 alone. CONCLUSION: The immunohistochemical expression of galectin-3 and CD44v6 by the ABC method is significantly higher in thyroid cancers than in benign thyroid nodules, especially galectin-3 in thyrocyte being helpful in differentiating benign thyroid nodule from thyroid cancer.

Enhanced delivery of human growth hormone across cell membrane by Tat-PTD.

In this study, we utilized the HIV protein Tat protein transduction domain (Tat-PTD) to enhance the intestinal absorption of recombinant human growth hormone (rhGH) delivered by oral administration. Insulin-like growth factor 1 (IGF-1), the key factor in the GH signal transduction pathway, was differentially affected at the mRNA level by various concentrations of rhGH. At high rhGH concentrations (500 ng/ml), IGF-1 was downregulated, while low concentrations (5 ng/ml) caused IGF-1 upregulation. The addition of Tat-PTD had a significant facilitating effect on rhGH. Frozen tissue sections visualized with fluorescence microscopy, cultured cells visualized by confocal microscopy and flow cytometry all confirmed that rhGH fused to Tat-PTD demonstrated more intracellular fluorescent signal when compared to rhGH alone. ELISA showed that after 2 h of incubation, Tat-rhGH levels in the rat intestinal cavity were 1.38-fold higher than rhGH. These data indicated that Tat-PTD effectively improved the internalization of rhGH and enhance the signal transduction of rhGH, possibly laying a solid foundation for the novel oral administration of rhGH.

PcLT, a novel C-type lectin from Procambarus clarkii, is involved in the innate defense against Vibrio alginolyticus and WSSV.

Lectins play important roles in the innate immunity. In this work, a C-type lectin, PcLT, was obtained from Procambarus clarkii which contained a carbohydrate recognition domain (CRD) with the ability to bind to Vibrio alginolyticus and white spot syndrome virus (WSSV). RT-PCR and qRT-PCR analyses demonstrated PcLT was specifically expressed in the hepatopancreas and the mRNA was markedly upregulated by V. alginolyticus and WSSV challenge, although a slight difference in timing was observed. The study also revealed upregulation of the mRNA expression and activity of immunological factors, peroxinectin, phenoloxidase, and superoxide dismutase in hemolymph in response to recombinant PcLT (rPcLT). Moreover, rPcLT also enhanced the phagocytosis, facilitated the subsequent clearance of V. alginolyticus and prolonged the survival of WSSV-infected shrimp. These results suggested that PcLT not only served as a pathogen recognition receptor (PRR), but also functioned as an immune modulator, participating in host defense against invaders.

The effective evaluation on symptoms and quality of life of chronic obstructive pulmonary disease patients treated by comprehensive therapy based on traditional Chinese medicine patterns.

OBJECTIVE: To evaluate the efficacy of comprehensive interventions based on the three TCM patterns on symptoms and quality of life of COPD patients. DESIGN: An open-label, randomized, controlled trial. SETTING: Four hospitals in China. INTERVENTION: 352 patients were randomly divided into two groups. Patients in the trial group were given conventional Western medicine and Bu-Fei Jian-Pi granules, Bu-Fei Yi-Shen granules and Yi-Qi Zi-Shen granules respectively; patients in the control group were given conventional Western medicine. Data collection was performed at baseline, in the 3rd and 6th month during the treatment period, and the 12th month during the follow-up period. OUTCOMES: Symptoms, including cough, sputum, pant, chest tightness, short of breath, lassitude, cyanosis and symptom total score; quality of life, measured by the WHOQOL-BREF questionnaire and adult COPD quality of life questionnaire (COPD-QOL). RESULTS: Of the 352 patients, 306 fully completed the study. After treatment and follow-up, there were significant differences between two groups in the following: cough, sputum, pant, chest tightness, shortness of breath, lassitude score and symptom total score (P<.05); physical, psychological, social and environment domain (P<.05) of the WHOQOL-BREF; daily living ability, social activity, depression symptoms and anxiety symptoms domain (P<.05) of the COPD-QOL. There were no differences between two groups in cyanosis and adverse events. CONCLUSION: Based on the TCM patterns, Bu-Fei Jian-Pi granules, Bu-Fei Yi-Shen granules and Yi-Qi Zi-Shen granules have beneficial effects on symptoms and quality of life of COPD patients.

TAT-mediated oral subunit vaccine against white spot syndrome virus in crayfish.

White spot syndrome virus is a highly pathogenic virus that infects crayfish and other crustaceans. VP28 is one of its major envelope proteins, and plays a crucial role in viral infection. Cell-penetrating peptides are short peptides that facilitate cellular uptake of various molecular cargoes, and one well known example is TAT peptide from HIV-1 TAT protein. In this study, recombinant plasmids were constructed and transformed into Escherichia coli strain BL21 (DE3) to express TAT-VP28, VP28, TAT-VP28-EGFP and VP28-EGFP fusion proteins. Enzyme-linked immunosorbent assay (ELISA) and flow cytometry methods were used to confirm that TAT fusion proteins can translocate from the intestine to the hemolymph of the crayfish Cambarus clarkii. After immunization, activities of phenoloxidase and superoxide dismutase were analyzed, and it was found that rTAT-VP28 produced the most pronounced increase in both C. clarkii were vaccinated by oral administration of rTAT-VP28 and rVP28 for 7 and 14 days, and rTAT-VP28 resulted in the highest relative percent survival (RPS) (63.3% at 7 days, and 67.8% at 14 days), compared with rVP28 (44.4% at 7 days, and 53.6% at 14 days) following challenge with WSSV after the last day of feeding. This study reports the use of TAT-derived peptide as an oral delivery method of a subunit vaccine against WSSV in C. clarkii.

Improve bioavailability of Harpin protein on plant use PLGA based nanoparticle.

Harpins can induce systemic acquired resistance (SAR) pathway on scores of non-host plant, provide protection against a range of pathogens. In this study, we demonstrated that applied recombinant HarpinZ Pseudomonas syringae pv. tomato (rHrpZ) on tobacco with three kinds of methods: infiltrating from micro-pore into leaf; injecting into petiole, and spraying on leaf, there is great difference in assimilation of protein because of the poor osmosis of tobacco leaves, and with multi-application of rHrpZ, the stimulation effect decreased. We prepared poly d,l-lactide-co-glycolide nanoparticles containing rHrpZ (rHrpZ PLGA NPs). To study the drug effect, we analyzed the change ratio of phenylalanine ammonia lyase (PAL) activity and PR-5dB gene expression after administration of rHrpZ and rHrpZ PLGA NPs on tobacco leaves. The results show that rHrpZ could elicit a rapid and transient increase in both PAL activity and PR-5dB expression, but the effect decreased after multi-application. While sprayed rHrpZ PLGA NPs on leaves, both the change ratio of PAL activity and PR-5dB expression were comparatively smooth and durable. Our study suggested that rHrpZ NPs could help protein enter leaf epidermis and cell wall, release rHrpZ in situ continuously, and enhance the bioavailability of rHrpZ.

Oral delivery of DNA vaccine encoding VP28 against white spot syndrome virus in crayfish by attenuated Salmonella typhimurium.

Protective immune responses in shrimp induced by DNA vaccines against white spot syndrome virus (WSSV) with intramuscular injection have been reported in recent reports. In this study, we investigated the utilities of attenuated Salmonella enterica serovar Typhimurium (Salmonella typhimurium) as a bactofection vehicle for the oral delivery of a DNA vaccine plasmid to crayfish (Cambarus clarkii). The DNA vaccine plasmid pcDNA3.1-VP28, encoding viral envelope protein VP28, was transformed to an attenuated S. typhimurium strain SV4089 and the resulting recombinant bacteria named SV/pcDNA3.1-VP28 were used to orally immunize crayfish with coated feed. Successful delivery of the DNA vaccine plasmid was shown by the isolation of recombinant bacteria SV/pcDNA3.1-VP28 from the vaccinated crayfish. The distribution analysis of plasmid pcDNA3.1-VP28 in different tissues revealed the effective release of DNA vaccine plasmid into crayfish. RT-PCR and immunoflurescence results confirmed the expression of protein VP28 in the vaccinated crayfish. Challenge experiments with WSSV at 7, 15, 25 days post-vaccination demonstrated significant protection in immunized crayfish with relative survival rate 83.3%, 66.7% and 56.7%, respectively. Studies on stability and safety of SV/pcDNA3.1-VP28 showed the recombinant bacteria could exist in crayfish at least 7 days but not more than 10 days and without any observable harm to the host. Our study here demonstrates, for the first time, the ability of attenuated Salmonella as a live vector to orally deliver a DNA vaccine against WSSV into the arthropod crayfish and provides a new way to design more practical strategies for the control of WSSV and other invertebrate pathogens.

Cloning and expression of earthworm fibrinolytic enzyme PM(246) in Pichia pastoris.

We have cloned, expressed, and purified a novel earthworm fibrinolytic enzyme (EFE) of Lumbricus rubellus in Pichia pastoris. Its cDNA sequence revealed a 747bp region containing an intact ORF that encodes a protein of 246 amino acid residues, designated as EFE PM(246). While EFE PM(246) is distinct, its cDNA shows a high degree of sequence homologies with four other EFE cDNAs registered in GenBank. The recombinant EFE PM(246) was active, showing a fibrinolytic activity of 7.5 x 10(6)U/L in basal salts medium, a higher fibrinolytic activity than those produced in other expression systems. The recombinant EFE PM(246) expressed in basal salts medium was purified by a three-step purification procedure with a recovery rate of about 20%. This is the first report detailing the successful purification of a genetically engineered earthworm fibrinolytic enzyme. The main physiochemical features of the EFE PM(246), including temperature stability, pH resistance, and sensitivity to some protein inhibitors, were also characterized.