Fusion with CTP increases the stability of recombinant neuritin.

Neuritin is a vital neurotrophin that plays an essential role in recovery from nerve injury and neurodegenerative diseases and may become a new target for treating these conditions. However, improving neuritin protein stability is an urgent problem. In this study, to obtain active and stable neuritin proteins, we added a carboxyl-terminal peptide (CTP) sequence containing four O-linked glycosylation sites to the C-terminus of neuritin and cloned it into the Chinese hamster ovary (CHO) expression system. The neuritin-CTP protein was purified using a His-Tag purification strategy after G418 screening of stable high-expression cell lines. Ultimately, we obtained neuritin-CTP protein with a purity >90%. Functional analyses showed that the purified neuritin-CTP protein promoted the neurite outgrowth of PC12 cells, and stability experiments showed that neuritin stability was increased by adding CTP. These results indicate that neuritin protein-CTP fusion effectively increases stability without affecting secretion and activity. This study offers a sound strategy for improving the stability of neuritin protein and provides material conditions for further study of the function of neuritin.

A tick bite patient with fever and meningitis co-infected with Rickettsia raoultii and Tacheng tick virus 1: a case report.

BACKGROUND: Increasing numbers of tick-borne pathogens are being discovered, including those that infect humans. However, reports on co-infections caused by two or more tick-borne pathogens are scarce. CASE PRESENTATION: A 38-year-old male farmer was bitten by a hard tick, presented with fever (37.7 °C), severe headache and ejection vomiting. Lumbar puncture was performed in the lateral decubitus. The cerebrospinal fluid (CSF) was clear, and analysis showed severe increased pressure (320 mm H2O), mild leukocytosis (126.0 × 106/L, mononuclear cells accounting for 73%) and elevated total protein concentration (0.92 g/L). Bacterial cultures of CSF and blood were negative. The diagnosis of Rickettsia raoultii and Tacheng tick virus 1 (TcTV-1) co-infection was confirmed by amplifying four rickettsial genetic markers and the partial small (S) RNA segment of TcTV-1 from the patient's blood. The patient gradually recovered after treatment with levofloxacin and ribavirin. CONCLUSIONS: This is the first reported co-infection case with fever and meningitis caused by R. raoultii and TcTV-1. It is vital to screen for multiple pathogens in tick-bitten patients, especially in those with severe complex symptoms.

Rickettsiae in red fox (Vulpes vulpes), marbled polecat (Vormela peregusna) and their ticks in northwestern China.

BACKGROUND: Previously, twelve Rickettsia species were identified in ticks, fleas, sheep keds (Melophagus ovinus), bats (Pipistrellus pipistrellus) and a tick-bitten patient in the Xinjiang Uygur Autonomous Region (XUAR) in northwestern China. Here we aimed to molecularly detect rickettsial agents in red fox (Vulpes vulpes), marbled polecat (Vormela peregusna) and their ticks. METHODS: During 2018-2019, 12 red foxes, one marbled polecat and their ticks were sampled in two counties and a city of the XUAR. The heart, liver, spleen, lung and kidney of these 13 carnivores were dissected, followed by DNA extraction. Hard ticks were identified both morphologically and molecularly. All samples were examined for the presence of rickettsiae by amplifying four genetic markers (17-kDa, gltA, ompA, sca1). RESULTS: A total of 26 adult ticks and 28 nymphs (38 Ixodes canisuga, nine Ixodes kaiseri, six Haemaphysalis erinacei and one Dermacentor marginatus) were collected from red foxes, and four Ha. erinacei ticks were removed from the marbled polecat. Analysis of cytochrome c oxidase subunit I (COI) gene sequences indicated that 2-32 nucleotides differed between I. canisuga, I. kaiseri and Ha. erinacei from northwestern China and Europe. Rickettsia raoultii was detected in three red foxes, Candidatus Rickettsia barbariae in a red fox, Rickettsia sibirica in a red fox and a marbled polecat, and R. raoultii in two tick species (I. canisuga and D. marginatus). CONCLUSIONS: To the best of our knowledge, I. canisuga and I. kaiseri have not been previously reported from red foxes in China. The DNA of R. sibirica and R. raoultii was detected for the first time in the organs of red foxes, and R. sibirica in the organs of a marbled polecat. This is also the first molecular evidence for the presence of R. raoultii in I. canisuga. Our findings expand the range of tick-borne pathogens in wildlife species and associated ticks in China.

SOX5 Regulates Cell Proliferation, Apoptosis, Migration and Invasion in KSHV-Infected Cells.

Kaposi's sarcoma (KS) originates from vascular endothelial cells, with KS-associated herpesvirus (KSHV) as the etiological agent. SRY-box transcription factor 5 (SOX5) plays different roles in various types of cancer, although its role in KS remains poorly understood. In this study, we identified the role of SOX5 in KS tissues and KSHV-infected cells and elucidated the molecular mechanism. Thirty-two KS patients were enrolled in this study. Measurement of SOX5 mRNA and protein levels in human KS tissues and adjacent control tissues revealed lower levels in KS tissues, with KS patients having higher SOX5 level in the early stages of the disease compared to the later stages. And SOX5 mRNA and protein was also lower in KSHV-infected cells (iSLK-219 and iSLK-BAC) than normal cells (iSLK-Puro). Additionally, SOX5 overexpression inhibited cell proliferation and promoted apoptosis and decreased KSHV-infected cell migration and invasion. Moreover, we found that SOX5 overexpression suppressed the epithelial-to-mesenchymal transition of KSHV-infected cells. These results suggest SOX5 is a suppressor factor during KS development and a potential target for KS treatment.

A case with neurological abnormalities caused by Rickettsia raoultii in northwestern China.

BACKGROUND: The number of new rickettsial species are rapidly increasing, and increasing numbers of Rickettsia raoultii (R. raoultii) infection cases have been detected in humans. However, neurological abnormalities caused by R. raoultii are rarely reported, especially in northwestern China. CASE PRESENTATION: A 36-year-old Kazakh shepherd with an attached tick on part temporalis, presented with right eyelid droop, lethargy, fever, headache, fever (38.0-41.0 °C) and erythematous rash. The examination of cerebrospinal fluid (CSF) showed cerebrospinal pressure of 200 mm H2O, leukocyte count of 300.0 × 106/L, adenosine deaminase of 2.15 U/L, and total protein concentration of 0.93 g/L. The diagnosis of R. raoultii infection was confirmed by six genetic markers, and semi-quantified by enzyme-linked immunosorbent assay for rickettsial antigen. The patient gradually recovered after treatment with doxycycline and ceftriaxone. R. raoultii DNA was found both in a tick detached from this patient and in 0.18% (2/1107) of blood samples collected from local shepherds. CONCLUSIONS: This is the first reported case with neurological abnormalities caused by R. raoultii in northwestern China. It is vital to detect rickettsial agents both in blood and CSF for tick bite patients with neurological abnormalities. Public health workers and physicians should pay attention to neurological abnormalities caused by Rickettsia.

Production of polyclonal antibody against human Neuritin and its application of immunodetection.

OBJECTIVE: To date, a commercial antibody to human Neuritin for immunoprecipitation is still limited. In this study, we aimed to develop a specific antibody for further research on the potential function of Neuritin. METHODS AND RESULTS: By epitope prediction of recombinant human Neuritin, the active fragment of human Neuritin that could be used as an excellent immunogen. Soluble His-tagged Neuritin was expressed and purified from Pichia pastoris. Polyclonal antibody against Neuritin was obtained by immunizing Sprague-Dawley rats with purified recombinant human Neuritin. Affinity-purified polyclonal antibody against Neuritin was characterized with indirect enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, and immunofluorescence. The results demonstrated that the polyclonal antibody against Neuritin had been prepared successfully. The prepared antibody bound to both exogenous and endogenous Neuritin. Importantly, the anti-Neuritin polyclonal antibody could be used in immunoprecipitation assays. CONCLUSIONS: The prepared polyclonal antibody could be used in immunoprecipitation and provide researchers with a useful tool for further investigating the function and mechanism of Neuritin.

Rickettsia raoultii and Rickettsia sibirica in ticks from the long-tailed ground squirrel near the China-Kazakhstan border.

Spotted fever group (SFG) rickettsiae cause infection in humans, domestic animals and wildlife. To date, no rickettsial agents have been reported in hard ticks from the long-tailed ground squirrel (Spermophilus undulatus). A total of 50 adult ticks and 48 nymphs were collected from S. undulatus in the border region of northwestern China. Tick species (identified according to morphological and molecular characteristics) included Dermacentor nuttalli, Dermacentor silvarum and Ixodes kaiseri. Based on the cytochrome c oxidase subunit I (COI) haplotype analysis, I. kaiseri from S. undulatus belongs to an ancestral. In addition, all tick samples were analyzed for the presence of rickettsiae by PCR amplification and sequencing of six genetic markers. Rickettsia raoultii and Rickettsia sibirica subsp. sibirica were shown to occur in adults and nymphs of D. nuttalli and D. silvarum. Rickettsia sibirica subsp. sibirica was also detected in an I. kaiseri adult. Dermacentor silvarum and I. kaiseri were found for the first time on S. undulatus. Rickettsia raoultii and R. sibirica subsp. sibirica were detected in two Dermacentor and one Ixodes species, respectively, suggesting that these rickettsiae circulate in the region of the China-Kazakhstan border by hard ticks infesting S. undulatus.

The generation and biological activity of a long-lasting recombinant human interferon-λ1.

The previously generated recombinant human (rh) interferon (IFN)-λ1 protein has a short half-life, and this feature makes it challenging to conduct studies on potential clinical applications for rhIFN-λ1. In an attempt to overcome this difficulty, we constructed a 'long-life' version of rhIFN-λ1. This modified rhIFN-λ1, named rhIFN-λ1-CTPON, has a human chorionic gonadotropin ß subunit carboxyl-terminal peptide (CTP) and an N-glycosylation sequence linked to its C-terminus. We confirmed the sequence of rhIFN-λ1-CTPON by mass spectrometry and then measured its biological activities. The results show that rhIFN-λ1-CTPON had antiviral activity and anti-proliferation activity in vitro that were similar to those of rhIFN-λ1 and that it similarly promoted natural killer cell cytotoxicity. Notably, the in vivo half-life of rhIFN-λ1-CTPON was determined to be 3-fold higher than that of rhIFN-λ1. We also assessed the anti-hepatitis B virus activity of rhIFN-λ1-CTPON; it was able to inhibit the production of the antigens HBs-Ag and HBe-Ag and induce antiviral gene expression. In conclusion, rhIFN-λ1-CTPON has a longer half-life than rhIFN-λ1 and has similar biological activities, so rhIFN-λ1-CTPON is an appropriate substitute for rhIFN-λ1 in the further study of potential clinical applications for rhIFN- λ1.

Genetic Diversity of Echinococcus granulosus Genotype G1 in Xinjiang, Northwest of China.

Cystic echinococcosis (CE) caused by E. granulosus is a serious helminthic zoonosis in humans, livestock and wildlife. Xinjiang is one of high endemic province for CE in China. A total of 55 sheep and cattle livers containing echinococcal cysts were collected from slaughterhouses in Changji and Yining City, northern region of Xinjiang. PCR was employed for cloning 2 gene fragments, 12S rRNA and CO1 for analysis of phylogenetic diversity of E. granulosus. The results showed that all the samples collected were identified as G1 genotype of E. granulosus. Interestingly, YL5 and CJ75 strains were the older branches compared to those strains from France, Argentina, Australia. CO1 gene fragment showed 20 new genotype haploids and 5 new genotype haplogroups (H1-H5) by the analysis of Network 5.0 software, and the YLY17 strain was identified as the most ancestral haplotype. The major haplotypes, such as CJ75 and YL5 strains, showed identical to the isolates from Middle East. The international and domestic trade of livestock might contribute to the dispersal of different haplotypes for E. granulosus evolution.

Molecular evidence of Rickettsia raoultii, "Candidatus Rickettsia barbariae" and a novel Babesia genotype in marbled polecats (Vormela peregusna) at the China-Kazakhstan border.

In this study, two road-killed marbled polecats (Vormela peregusna) were molecularly analysed for tick-borne pathogens. Rickettsia raoultii, "Candidatus Rickettsia barbariae" and a novel Babesia genotype have been identified, for the first time in marbled polecat. DNA of this Babesia sp. genotype was also present in four out of 15 Haemaphysalis erinacei ticks collected from the Babesia PCR-positive marbled polecat. Results of this study suggest that marbled polecats may serve as reservoirs for these bacteria and protozoans.

Clinical value of Naa10p and CEA levels in saliva and serum for diagnosis of oral squamous cell carcinoma.

BACKGROUND: N-α-acetyltransferase 10 protein (Naa10p) is a potential prognostic biomarker that modulates the phenotypes of several cancer types. Carcinoembryonic antigen (CEA) is currently the most well-known biomarker for the detection of epithelial malignancies. Our objective was to evaluate the clinical value of Naa10p, CEA, and their combined detection for diagnosis of oral squamous cell carcinoma (OSCC). METHODS: This study included 202 individuals: 112 patients with OSCC, 30 patients with oral premalignant lesions (OPMLs), and 60 cancer-free and without OPML patients as control. Naa10p and CEA were determined in serum and saliva samples utilizing enzyme-linked immunosorbent assays. RESULTS: Salivary and serum levels of Naa10p and CEA in OSCC patients were significantly higher than those detected in OPML and the control groups, although patients with OPMLs also showed increased salivary and serum Naa10p and CEA levels as compared to the control group. Salivary Naa10p level in OSCC patients is correlated with the degree of differentiation and lymph node metastasis, and serum Naa10p level is specifically correlated with patient age. Additionally, salivary CEA level is correlated with the clinical stage and lymph node metastasis, whereas serum CEA level is correlated with lymph node metastasis. The sensitivity, specificity, positive predictive value, and negative predictive value of combined detection were greater than any single detection. CONCLUSIONS: Combined use of salivary Naa10p and CEA as tumor markers for OSCC was more sensitive than serum Naa10p and CEA. These results indicated that combined detection of salivary Naa10p and CEA improved diagnostic performance and early detection rate for OSCC.

IFN-λ1 in CHO cells: its expression and biological activity.

BACKGROUND: Many studies have investigated the characteristics and biological activities of type III interferon (IFN), finding that it has similar features to type I IFN but also unique actions because it is recognized by a different receptor. RESULTS: A full-length recombinant human IFN-λ1 (rhIFN-λ1) cDNA was cloned into the pDF expression vector and stably expressed in Flp-In-CHO cells. After four purification steps (ammonium sulfate precipitation, SP Sepharose chromatography, Blue Sepharose 6 fast flow affinity chromatography and molecular sieve chromatography), the rhIFN-λ1 had a purity of about 90% and was found to have the predicted biological activities. The anti-viral activity of rhIFN-λ1 was determined as 106 IU/mg using the vesicular stomatitis virus (WISH-VSV) assay system. The anti-proliferation activity of rhIFN-λ1 was measured using the MTS method and the growth inhibition ratio was 57% higher than that for recombinant human IFN-α2b (rhIFN-α2b) when the rhIFN-λ1 concentration was 1000 IU/ml. rhIFN-λ1 had lower natural killer cell cytotoxicity than rhIFN-α2b. CONCLUSION: The Flp-In-CHO system is suitable for stably expressing rhIFN-λ1 that possesses the predicted anti-viral, anti-proliferation and natural killer cell cytotoxicity-promoting activities.

Recombinant neuritin affects the senescence, apoptosis, proliferation, and migration of rat bone marrow-derived mesenchymal stem cells.

OBJECTIVE: To study the effects of recombinant neuritin expressed by Pichia pastoris GS115 on the senescence, apoptosis, proliferation, and migration associated with rat bone marrow-derived mesenchymal stem cells (BMSCs). RESULTS: Recombinant neuritin was purified by Ni-affinity chromatography and identified by western blot and MALDI-TOF spectrometry. The effects of recombinant neuritin on senescence, apoptosis, proliferation, and migration of rat BMSCs WERE investigated. ß-Galactosidase staining indicated that recombinant neuritin administration significantly inhibited BMSCs senescence at 1 µg neuritin/ml. Additionally, recombinant neuritin reduced the number of apoptotic cells at the early stage according to Annexin V/propidium iodide staining and inhibited cell proliferation according to MTT assay results. Moreover wound healing assay results showed that recombinant neuritin promoted BMSCs migration in the neuritin-treatment group. CONCLUSION: Recombinant neuritin affects the senescence, apoptosis, proliferation, migration of rat BMSCs. Our findings offer insight into neuritin function outside of the nervous system.

Corrigendum: Recombinant hNeuritin Promotes Structural and Functional Recovery of Sciatic Nerve Injury in Rats.

[This corrects the article on p. 589 in vol. 10, PMID: 28066172.].

Recombinant hNeuritin Promotes Structural and Functional Recovery of Sciatic Nerve Injury in Rats.

Neuritin is a new neurotropic factor implicated in nervous system development and plasticity. Studies have shown that Neuritin is upregulated in injured nerves, suggesting that it is involved in nerve repair. To test this hypothesis, we investigated whether recombinant human Neuritin could restore nerve structure and function in a rat model of sciatic nerve injury. Neuritin treatment had a dose-dependent effect on functional recovery 4 weeks after injury, as determined by the walking-track test. Similar trends were observed for gastrocnemius muscular strength and nerve conduction velocity. Additionally, sciatic nerve fiber density and organization as well as degree of remyelination were increased, while growth-associated protein 43 and neurofilament 200 expression was upregulated upon treatment with Neuritin. These findings demonstrate that Neuritin stimulates nerve regeneration and functional recovery and thus promotes the repair of injured sciatic nerves.

Human polyomavirus type six in respiratory samples from hospitalized children with respiratory tract infections in Beijing, China.

BACKGROUND: HPyV6 is a novel human polyomavirus (HPyV), and neither its natural history nor its prevalence in human disease is well known. Therefore, the epidemiology and phylogenetic status of HPyV6 must be systematically characterized. METHODS: The VP1 gene of HPyV6 was detected with an established TaqMan real-time PCR from nasopharyngeal aspirate specimens collected from hospitalized children with respiratory tract infections. The HPyV6-positive specimens were screened for other common respiratory viruses with real-time PCR assays. RESULTS: The prevalence of HPyV6 was 1.7 % (15/887), and children ≤ 5 years of age accounted for 80 % (12/15) of cases. All 15 HPyV6-positive patients were coinfected with other respiratory viruses, of which influenza virus A (IFVA) (8/15, 53.3 %) and respiratory syncytial virus (7/15, 46.7 %) were most common. All 15 HPyV6-positive patients were diagnosed with lower respiratory tract infections, and their viral loads ranged from 1.38 to 182.42 copies/µl nasopharyngeal aspirate specimen. The most common symptoms were cough (100 %) and fever (86.7 %). The complete 4926-bp genome (BJ376 strain, GenBank accession number KM387421) was amplified and showed 100 % identity to HPyV6 strain 607a. CONCLUSIONS: The prevalence of HPyV6 was 1.7 % in nasopharyngeal aspirate specimens from hospitalized children with respiratory tract infections, as analyzed by real-time PCR. Because the coinfection rate was high and the viral load low, it was not possible to establish a correlation between HPyV6 and respiratory diseases.

The cloning, expression and purification of recombinant human neuritin from Escherichia coli and the partial analysis of its neurobiological activity.

Neuritin (Nrn1) is a neurotrophic factor that plays various roles in neural development and synaptic plasticity. In this study, the NRN1 gene was cloned and expressed in Escherichia coli and then recombinant neuritin protein was purified so that its neurobiological activity could be evaluated. The protein, which was obtained at a concentration of 0.45 mg/ml and > 90% purity, had the predicted molecular weight of 30 kDa, as determined via sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Western blot analysis confirmed that an anti-neuritin antibody could recognize the fusion protein. Subsequent functional analyses revealed that recombinant neuritin promoted neurite outgrowth in embryonic chicken dorsal root ganglia and PC12 cells. These results suggest that recombinant neuritin protein could be a valuable tool for inducing neurite regeneration, for instance in cases of spinal cord injury or neurological diseases.

[The expression of interferon-lambda1 in CHO cell].

OBJECTIVE: To construct the eukaryotic expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which linked the enhancer SP163 with interferon lambda1. Then express the interferon lambda1 in CHO (dhfr-) cells. METHODS: Using PCR method to introduce the restriction enzyme sites and through the fusion PCR binding the enhancer with the interferon Lambda1. After sequenced, lambda1 and SP163-lambda1 was inserted into PCI-dhfr forming the expression vector PCI-dhfr-lambda1 and PCI-dhfr-SP163-lambda1 which was constructed successfully confirming by sequencing. Then the expressing vectors were transfected into CHO (dhfr-) cells using liposome transfection method and interferon lambda1 protein was assayed with indirect immunofluorescence and Western Blot. Using cytopathic effect inhibition evaluated the antiviral activity of interferon lambda1. RESULTS: Successfully constructing the eukaryotic expression vectors of interferon lambda and the vectors could express interferon lambda1. The result of immunofluorescence showed the enhancer developed the expression of interferon lambda1. Detecting the interferon lambda1 in CHO (dhfr-) cells after transfecting 48 hour using Western Blot. The cytopathic effect inhibition showed the expressed interferon lambda1 has the antiviral activity. CONCLUSION: Successfully expressed the interferon lambda1 in CHO (dhfr-) cells and the protein possesses antiviral activity, which may supply a valuable basis for building the stable cell line of interferon lambda1.

[Preparation of the monoclonal antibody against human Bocavirus VP2 protein].

OBJECTIVE: To express and purify HBoV VP2 protein, and the monoclonal antibody against HBoV VP2 protein was prepared with hybridoma technique. METHODS: The HBoV VP2 cloned into vector pET-30a was expressed in E. coil. After purified by immobilized metal affinity chromatography, the BALB/c mouse was immunized with purified protein as antigen. The positive hybridoma cells were screened with hybridoma technique and ELISA assay. Isotype and titer of the monoclonal antibody were detected. RESULTS: The recombinant HBoV VP2 protein was expressed and purified, and then the monoclonal antibody was obtained with hybridoma technique. The titer of the IgG monoclonal antibody was up to 1:4 x 10(5). CONCLUSION: Monoclonal antibody against recombinant HBoV VP2 protein was prepared and the antibody titer was high. This work may provide a new method in rapid diagnosis and study of HBoV.

Drug-induced apoptosis of Echinococcus granulosus protoscoleces.

Parasitic infection by Echinococcus granulosus in humans induces hydatidosis (echinococcosis), which is a zoonotic disease that seriously endangers public health. This study was to determine the status of cell apoptosis in the protoscoleces of E. granulosus, which were isolated from hydatid cysts in livers or lungs of sheep. Those protoscoleces were incubated with drugs (at the concentration of 1 mmol L(-1) H(2)O(2) and 5 mmol L(-1)dexamethasone) for 8 h, the apoptosis were examined by transmission electron microscopy and TUNEL assay, the expression of caspase-1 and caspase-3 were detected by immunohistochemistry and caspase-3 activity was detected by colorimetric assay. Our results have clearly demonstrated the presence of cell apoptosis in protoscoleces in the absence or presence of drug (H(2)O(2), dexamethasone) treatment, but drug-induced apoptosis rate, caspase-1 and caspase-3 expression levels were higher than no-drug induce and caspase-3 activity were significantly increasing. We found H(2)O(2) and dexamethasone can induce the cell apoptosis of protoscoleces. Our results implied the existence of a CED-3 like apoptosis gene in protoscolces and provide a rationale for further exploring the induction of apoptosis as non-surgical treatment method in treating this parasitic disease.