Rapid screening of monoclonal antibodies against porcine circovirus type 2 using colloidal gold-based paper test.

A proof of concept for using paper test as a suitable method in the production of monoclonal antibodies (MAbs) is reported. The paper test which detects antibodies against porcine circovirus type 2 (PCV2) using colloidal gold-labelled capsid protein as the antigen probe was applied exclusively in the screening of anti-PCV2 MAbs. It allowed the detection of 118 single cell clones within 30 min using naked eyes. MAbs with specific binding to authentic epitopes on the virus were selected using a blocking strategy in which the antibody was pre-incubated with PCV2 viral sample before applying to the test paper. Five hybridomas secreting MAbs against the capsid protein were obtained, with only three of them capable of binding to PCV2. The results were validated and confirmed using enzyme-linked immunosorbent assay and immunofluorescence assay. The paper test is simple, rapid, and independent on professional technicians and proves to be an excellent approach for the screening of MAbs against specific targets.

Hemalin from Haemaphysalis flava ticks: cloning, expression and antithrombogenicity.

Hemalin, initially described in Haemaphysalis longicornis, is a protein with anticoagulant activity. We retrieved a gene fragment functionally annotated as hemalin from H. flava salivary gland transcriptomic library, but its full-length complementary DNA (cDNA) and antithrombogenicity have not been investigated in the species. Here we cloned the full length of hemalin (Hf-hemalin) by 3'-end rapid-amplification of cDNA ends, and the open reading frame (ORF) of Hf-hemalin was expressed in Escherichia coli. The recombinant protein (rHf-Hemalin) was tested for antithrombogenicity. The full-length of Hf-hemalin was 607 bp with an ORF of423 bp. Protein encoded by Hf-hemalin was predicted to contain 2 Kunitz domains and a signal peptide. The expression of Hf-hemalin in salivary glands, midguts and ovaries was higher in the semi-engorged than the fully engorged. Prokaryotic expression yielded a product of 40 kDa containing a glutathione S-transferase (GST) tag. Incubation of rHf-Hemalin with rat plasma significantly extended prothrombin time and activated partial thromboplastin time compared with normal saline and GST controls. Our data demonstrated that Hemalin from H. flava shared a similar primary structure with that from H. longicornis, and was also anticoagulant. Further investigations are needed to test its feasibility to be an antigen candidate for the development of vaccines against ticks.

Cloning of four HSPA multigene family members in Haemaphysalis flava ticks.

The heat shock protein 70 (HSPA) family and their genes have been studied in ticks and are considered as possible antigen candidates for the development of anti-tick vaccines. However, knowledge about their members, structure and function in ticks is incomplete. Based on our transcriptomic data, the full length of four HSPA genes in Haemaphysalis flava (Acari: Ixodidae) was cloned via rapid amplification of cDNA ends. The open reading frame of HSPA2A, HSPA2B, HSPA5 and HSPA9 was 1920, 1911, 1983 and 2088 bp in length, respectively. Three family signatures and one localization motif were in the encoding proteins. HSPA2A and HSPA2B were predicted to be located at cytoplasm/nucleus, whereas HSPA5 and HSPA9 were at endoplasmic reticulum and mitochondria, respectively. In silico simulation demonstrated that those proteins had distinct numbers of α-helixes, extended strands and coils, and different antigenic epitopes. Expression of HSPA5 and HSPA9 in the salivary gland was significantly higher in partially-engorged female adult ticks than the fully-engorged (P < 0.01) as shown by a quantitative polymerase chain reaction. Our data indicated that H. flava ticks had at least four HSPA genes encoding proteins with different cellular locations, structures and expression profiles, suggesting their diverse roles in tick biology.

[Study on the sensitivity of multi-slice spiral CT in diagnosis of lymph node metastasis in different lymph node stations of gastric cancer].

Objective: To study the sensitivity of multi-slice spiral CT in the diagnosis of lymph node metastasis in different lymph node stations of gastric cancer. Methods: A retrospective series of case study was employed in the research. Inclusion criteria: (1) patients undergoing preoperative abdominal CT scan plus enhanced examination, and data in the image archiving and communication system of Sun Yat-sen University Cancer Center; (2) patients undergoing total or subtotal gastrectomy plus D2 or D1+ lymphadenectomy, with more than 15 harvested lymph nodes and more than 1 metastatic lymph node confirmed by postoperative pathology; (3) WHO pathological classification defined as gastric adenocarcinoma; (4) no history of lymph node tuberculosis, giant lymph node hyperplasia, lymphoma or other diseases resulting in enlarged lymph nodes; (5) no history of gastrectomy; (6) no preoperative neoadjuvant therapy. Clinicopathologic data of gastric cancer patients at the Department of Gastric and Pancreatic Surgery, Sun Yat-sen University Cancer Center from January 2009 to December 2012 were retrospectively analyzed. Using the pathologically positive lymph nodes as a reference, the sensitivity of CT-positive lymph nodes was calculated (total number of positive image lymph nodes/total number of positive pathological lymph nodes) and complete coincidence rate (number of case defined as complete coincidence/number of case with positive pathologic lymph nodes; complete coincidence indicated that the number of positive image lymph nodes was consistent with the number of positive pathologic lymph nodes in each lymph node station). The χ(2) test was used to compare the sensitivity of CT in the diagnosis of lymph node metastasis in each lymph node station. Results: A total of 228 patients with pathology-proven gastric cancer were enrolled in the study, including 147 male and 81 female. The overall sensitivity of CT in diagnosis of metastatic lymph nodes in gastric cancer was 68.7% (1769/2576). The sensitivity of CT in diagnosis of lymph node metastasis of groups 1 to 8 from high to low was group 3 [81.1% (506/624)], group 7 [73.9% (246/333)], group 2 [70.3% (111/158)], group 6 [68.7% (248/361)], group 4 [68.1% (262/385)], group 8 [60.4% (116/192)], group 1 [53.8% (155/288)], group 5 [47.1% (82/174)]. The CT diagnostic sensitivity of group 3 was significantly higher than the overall level (χ(2)=37.689, P<0.001). The CT diagnostic sensitivity of group 5 was significantly lower than the overall level (χ(2)=34.387, P<0.001). The CT diagnostic sensitivity of group 1 was also significantly lower than the overall level (χ(2)=25.918, P<0.001). Significant differences were not found in the CT diagnostic sensitivity of group 2, 4, 6, 7, 8 compared with the overall level (all P>0.05). The complete coincidence rate was 56.9% (536/942) between pathological positive lymph nodes and CT positive lymph nodes. The highest complete coincidence rate was observed in group 3 (68.0%, 123/181) and the lowest was in group 1 (41.4%, 46/111), whose difference was statistically significant (χ(2)=9.673, P=0.002). Conclusion: The sensitivity of CT in diagnosis of lymph nodes in different lymph node stations of gastric cancer is different.

[Value of DCE-MRI and DWI in the differential diagnosis of inflammatory pseudotumor and lymphoma in the lacrimal gland].

Objective: To evaluate the value of dynamic contrast-enhanced MR (DCE-MRI) and diffusion weighted MR (DWI) in differential diagnosis of inflammatory pseudotumor and lymphoma in the lacrimal gland. Methods: In this retrospective study, a total of 24 cases of inflammatory pseudotumor and 22 cases of lymphoma in the lacrimal gland at Beijing Tongren Hospital confirmed by histological results were enrolled from January 2010 to January 2015.DCE-MRI and DWI were performed in these cases, and the type of time-signal intensity curve (time-intensity curve, TIC), the peak contrast index (CIpeak), maximum enhancement ratio (ERmax), washout ratio (WR) and ADC value were analyzed.Differences of these parameters between inflammatory pseudotumor and lymphoma in the lacrimal gland was evaluated by independent samples t test or Mann-Whitney U test.The receiver operating characteristic analysis was used to evaluate the utility of these parameters in discriminating the two diseases. Results: Type of TIC, CIpeak, WR, ERmax and ADC values were statistically different (all P<0.05) between the inflammatory pseudotumor and lymphoma in the lacrimal gland.ERmax, CIpeak and ADC values of inflammatory pseudotumor were greater than those of lymphoma, but the WR values of inflammatory pseudotumor was less than those of lymphoma.The area under the ROC of the CIpeak, WR, ERmax and ADC values was 0.68±0.08, 0.70±0.08, 0.70±0.08, 0.84±0.81 respectively.Using an ADC(b=0, 1000) value of 1.005×10(-3) mm(2)/s as the diagnostic threshold, the sensitivity, specificity and accuracy to differentiate benign lesions from malignant lesions were 84.2%, 65.0% and 75.0% respectively.The diagnostic ability of DWI combined with DCE-MRI was superior to that of DCE-MRI(P=0.000) or DWI alone(P=0.008) with overall sensitivity, specificity and accuracy of 86.3%, 87.5% and 86.9%. Conclusion: DCE-MRI and DWI may improve the diagnostic accuracy in differentiation of the inflammatory pseudotumors and lymphomas in the lacrimal gland, and play an important role in differentiation among them.

[Cloning, sequencing and in vitro expression of human RANTES gene].

An expected 276 bp fragment of the gene precursor encoding the signal peptide and mature protein of human beta-chemokine RANTES was amplified by reverse transcription-polymerase chain reaction (RT-PCR) from RNA of PHA-activated human peripheral blood lymphocytes. This putative interested gene was inserted directly into a T-vector and the ligation was confirmed by restriction enzyme digestion. The sequence data of the cloned fragment showed that it was almost identical with published sequences of RANTES gene, except for only one nucleotide substitution within the signal peptide region. The in vitro expressed recombinant RANTES protein was detected by the chemiluminescence enzyme-linked immune Dot blotting assay after combining the recombinant plasmid with the in vitro SP6/T7 transcription and translation system. The successful cloning and expression of RANTES gene should shed light on future's gene therapy of AIDS.

Determination of the Hubble constant.

Establishing accurate extragalactic distances has provided an immense challenge to astronomers since the 1920s. The situation has improved dramatically as better detectors have become available, and as several new, promising techniques have been developed. For the first time in the history of this difficult field, relative distances to galaxies are being compared on a case-by-case basis, and their quantitative agreement is being established. New instrumentation, the development of new techniques for measuring distances, and recent measurements with the Hubble Space telescope all have resulted in new distances to galaxies with precision at the +/-5-20% level. The current statistical uncertainty in some methods for measuring H(0) is now only a few percent; with systematic errors, the total uncertainty is approaching +/-10%. Hence, the historical factor-of-two uncertainty in the value of the H(0) is now behind us.

SRPK1 and Clk/Sty protein kinases show distinct substrate specificities for serine/arginine-rich splicing factors.

Serine/arginine-rich (SR) proteins are essential for pre-mRNA splicing, and modify the choice of splice site during alternative splicing in a process apparently regulated by protein phosphorylation. Two protein kinases have been cloned that can phosphorylate SR proteins in vitro: SRPK1 and Clk/Sty. Here, we show that these two kinases phosphorylate the same SR proteins in vitro, but that SRPK1 has the higher specific activity toward ASF/SF2. SRPK1, like Clk/Sty, phosphorylates ASF/SF2 in vitro on sites that are also phosphorylated in vivo. Tryptic peptide mapping of ASF/SF2 revealed that three of the phosphopeptides from full-length ASF/SF2 phosphorylated in vitro contain consecutive phosphoserine-arginine residues or phosphoserine-proline residues. In vitro, the Clk/Sty kinase phosphorylated Ser-Arg, Ser-Lys, or Ser-Pro sites, whereas SRPK1 had a strong preference for Ser-Arg sites. These results suggest that SRPK1 and Clk/Sty may play different roles in regulating SR splicing factors, and suggest that Clk/Sty has a broader substrate specificity than SRPK1.