MUC13 promotes the development of esophageal cancer by upregulating the expression of O-glycan process-related molecules.

BACKGROUND: Esophageal cancer is one of the most common malignant tumors in the world, which is characterized by poor prognosis, aggressiveness, and poor survival. Mucin 13 (MUC13) is a member of the membrane-bound mucin and located on chromosome 3q21.2 and consists of α and ß subunits. It has been found that MUC13 is overexpressed in a variety of tumor cells and acts a vital role in the invasiveness and malignant progression of several types of tumors. However, the role and regulatory mechanism of MUC13 in the progression of esophageal cancer remain unclear. METHODS: The expression level of MUC13 was detected in 15 esophageal cancer tissues and 15 pairs of adjacent nontumor tissues by immunohistochemistry (IHC). In addition, the expression of MUC13 mRNA level in human esophageal cancer cell lines (EC9706 and ECA109 and TE-1) was measured by qRT-PCR. In vitro, after silencing MUC13 with lentiviral interference technology, CCK8 assay, clone formation assay, and flow cytometry were applied to investigate the proliferation activity, clone formation ability and anti-apoptosis ability of EC9706 and ECA109 cells. The tumor xenograft growth assay was used to confirm the influence of MUC13 knockdown on the growth of esophageal tumors in vivo. The qRT-PCR assay and western blot experiments were taken to study the mechanism of MUC13 regulating the proproliferation and antiapoptotic of esophageal cancer. RESULTS: The results showed that MUC13 was overexpressed in esophageal cancer tissues and cell lines (EC9706 and ECA109 and TE-1), especially in EC9706 and ECA109 cells, but low expressed in human esophageal epithelial cell line (HEEC). Next, silencing MUC13 inhibits proliferation, blocks cell cycle progression, and promotes cell apoptosis in vitro, and restrains the growth of esophageal cancer tissues in vivo. Finally, MUC13 affects the proproliferation and antiapoptotic by regulating the expression of GLANT14, MUC3A, MUC1, MUC12, and MUC4 that closely related to O-glycan process. CONCLUSIONS: This study proved that MUC13 is an important molecule that regulates the O-glycan process and then affects the progress of esophageal cancer. MUC13 may be a novel therapeutic target for patients with esophageal cancer.

The Spectrum of Tigecycline-Induced Pancreatitis in Clinical Characteristics, Diagnosis, and Management.

Introduction: Tigecycline-induced acute pancreatitis (AP) has been frequently increasingly reported in solid organ transplant patients. This review aimed to summarize the characteristics, possible mechanisms, and management of tigecycline-induced AP. Methods: Case reports of tigecycline-induced AP published in Chinese or English were collected until February 2023 for retrospective analysis. Results: Thirty-four patients from 29 articles were included. Fifteen patients (46.9%) had solid organ transplantation, and 4 patients (12.5%) had malignant tumors. Twenty-five patients (89.3%) received a recommended maintenance dose of tigecycline (50 mg q12 h). The median age was 50 years (range 9-87). Compared to the nontransplant patients, the median age of the transplant patients was significantly younger, 44 years (range 12.5-61) versus 57.5 years (range 9-87) (P=0.03). The median time of symptom onset was 7 days (range 2-29), and 91.2% (31/34) were less than 14 days. Typical initial symptoms included abdominal pain (90.6%), nausea (46.9%), vomiting (43.8%), and abdominal distention (21.9%). Most cases were accompanied by elevated levels of pancreatic enzymes. The main radiological features included edematous infiltrate and acute pancreatitis on computed tomography (CT) scan and abdominal ultrasound. Except for one patient who continued tigecycline treatment, all patients discontinued treatment and received symptomatic support such as fasting, acid suppression, and enzyme suppression. The median time to recover pancreatic enzymes to the normal range was 5 days (range 1-43), and the median time to relieve symptoms was 4 days (range 1-12). Four patients died, of whom two died of severe pancreatitis complications and two of cardiogenic shock and septicemia. Conclusion: Tigecycline-induced AP was a rare and serious complication that occurred mainly within two weeks of the medication. This serious side effect should be kept in mind while treating severe infections especially in transplant recipients.

Evaluation of humoral and cellular immune responses induced by a cocktail of recombinant African swine fever virus antigens fused with OprI in domestic pigs.

BACKGROUND: African swine fever (ASF) is a highly fatal disease in domestic pigs caused by ASF virus (ASFV), for which there is currently no commercial vaccine available. The genome of ASFV encodes more than 150 proteins, some of which have been included in subunit vaccines but only induce limited protection against ASFV challenge. METHODS: To enhance immune responses induced by ASFV proteins, we expressed and purified three fusion proteins with each consisting of bacterial lipoprotein OprI, 2 different ASFV proteins/epitopes and a universal CD4+ T cell epitope, namely OprI-p30-modified p54-TT, OprI-p72 epitopes-truncated pE248R-TT, and OprI-truncated CD2v-truncated pEP153R-TT. The immunostimulatory activity of these recombinant proteins was first assessed on dendritic cells. Then, humoral and cellular immunity induced by these three OprI-fused proteins cocktail formulated with ISA206 adjuvant (O-Ags-T formulation) were assessed in pigs. RESULTS: The OprI-fused proteins activated dendritic cells with elevated secretion of proinflammatory cytokines. Furthermore, the O-Ags-T formulation elicited a high level of antigen-specific IgG responses and interferon-γ-secreting CD4+ and CD8+ T cells after stimulation in vitro. Importantly, the sera and peripheral blood mononuclear cells from pigs vaccinated with the O-Ags-T formulation respectively reduced ASFV infection in vitro by 82.8% and 92.6%. CONCLUSIONS: Our results suggest that the OprI-fused proteins cocktail formulated with ISA206 adjuvant induces robust ASFV-specific humoral and cellular immune responses in pigs. Our study provides valuable information for the further development of subunit vaccines against ASF.

Identification of p72 epitopes of African swine fever virus and preliminary application.

African swine fever virus (ASFV) causes a highly lethal hemorrhagic viral disease (ASF) of pigs that results in serious losses in China and elsewhere. The development of a vaccine and diagnosis technology for ASFV is essential to prevent and control the spread of ASF. The p72 protein of ASFV is highly immunogenic and reactive, and is a dominant antigen in ASF vaccine and diagnostic research. In this study, 17 p72 monoclonal antibodies (mAbs) were generated. Epitope mapping by a series of overlapping peptides expressed in Escherichia coli showed that these mAbs recognized a total of seven (1-7) linear B cell epitopes. These mAbs did not show significant neutralizing activity. Epitopes 1 (249HKPHQSKPIL258), 2 (69PVGFEYENKV77), 5 (195VNGNSLDEYSS205), and 7 (223GYKHLVGQEV233) are novel. Sequence alignment analysis revealed that the identified epitopes were highly conserved among 27 ASFV strains from nine genotypes. Preliminary screening using known positive and negative sera indicated the diagnostic potential of mAb-2B8D7. The results provide new insights into the antigenic regions of ASFV p72 and will inform the diagnosis of ASFV.

The role of anxiety and depressive symptoms in mediating the relationship between subjective sleep quality and cognitive function among older adults in China.

BACKGROUND: Deterioration of cognitive function has a significant impact on the unavoidable burden on individuals, families, and society. This study aimed to examine the serial multiple mediating effects of anxiety and depressive symptoms on the relationship between subjective sleep quality and cognitive function among older adults in China. METHODS: We selected 6442 Chinese older adults aged 65 years and older from the 2018 Chinese Longitudinal Healthy Longevity Survey. The SPSS PROCESS macro was employed to perform simple and serial multiple mediation analyses. RESULTS: Subjective sleep quality, depressive symptoms, anxiety symptoms, and cognitive function were significantly related (P < 0.01). Poor sleep quality can have a direct negative influence on cognitive function among older adults (effect = -0.110; 95 % CI = [-0.166, -0.053]), but it can also have an indirect negative impact via three pathways: the independent mediation of anxiety symptoms (effect = -0.028; 95 % CI = [-0.048, -0.011]), the independent mediation of depressive symptoms (effect = -0.014; 95 % CI = [-0.026, -0.002]), and the serial mediation of anxiety and depressive symptoms (effect = -0.009; 95 % CI = [-0.017, -0.001]). LIMITATIONS: This study used a cross-sectional design, which restricts the ability to infer causal relationships. CONCLUSIONS: The effect of subjective sleep quality on cognitive function was serially mediated by anxiety and depressive symptoms among older adults. Diverse therapies targeted at improving sleep quality in older adults may improve mood and cognitive functioning.

A vesicular stomatitis virus-based African swine fever vaccine prototype effectively induced robust immune responses in mice following a single-dose immunization.

Introduction: African swine fever (ASF) is a highly contagious hemorrhagic fever disease in pigs caused by African swine fever virus (ASFV). It is very difficult to control and prevent ASF outbreaks due to the absence of safe and effective vaccines. Methods: In order to develop a safe and effective ASF vaccine for the control and prevention of ASF, two ASFV recombinant vesicular stomatitis virus (VSV) live vector vaccine prototypes, containing the gene of p72, and a chimera of p30 and p54, were developed based on the replication-competent VSV, and named VSV-p72 and VSV-p35. The immune potency of VSV-p72 or VSV-p35 alone and in combination was evaluated in BALB/c mice via intramuscular and intranasal vaccination. Results: The results indicated that whether administered alone or in combination, the two vaccine prototypes showed acceptable safety in mice and, more importantly, induced high-level specific antibodies against p72, p30, and p54 of ASFV and a strong cellular immune response 28 days after vaccination. The sera from mice vaccinated with the vaccine prototypes significantly inhibited ASFV from infecting porcine alveolar macrophages (PAMs) in vitro. Most notably, the immunized sera from a mixture of VSV-p35 and VSV-p72 inhibited ASFV from infecting PAMs, with an inhibition rate of up to 78.58%. Conclusion: Overall, our findings suggest that ASFV recombinant VSV live vector vaccine prototypes may become a promising candidate vaccine for the control and prevention of ASF.

Formulation enhanced the stability of Foot-and-mouth virus and prolonged vaccine storage.

Foot-and-mouth disease (FMD) is a highly contagious viral disease that affects cloven-hoofed animals. Vaccination is the most effective measure to control FMD. However, FMDV particles are prone to dissociation, leading to insufficient potency of vaccine. Based on this characteristic, a combination of twenty percentage trehalose, 500 mM NaCl and 3 mM CuSO4·5H2O was developed to increase viral stability. Heating-resistance test showed that FMDV infectivity was maintained when formulated with formulation. Additionally, the half-life of FMDV inactivation was prolonged remarkably. Sequencing analysis demonstrated that viral genome could not be altered in serial passages. Vaccine stability was monitored for up to 1 year at 4 °C, with a higher level of 146S content remained. This study suggested that the formulation could protect FMDV against massive structural breakdown and extend the shelf life of vaccine. Our findings could provide strategy to develop more solutions for the stabilization of viral vaccine.

Health-related quality of life and its association with socioeconomic status and diet diversity in Chinese older adults.

Objectives: The study aimed at examining the combined association of socioeconomic status (SES) and diet diversity (DD) with health-related quality of life (HRQoL) and exploring whether DD played a mediating role in the relationship between varied SES and HRQoL among Chinese older persons. Method: A multi-stage random sampling method was conducted in Shanxi Province of China, with 3,250 older adults participating in this cross-sectional survey. SES was divided into groups by quartiles and DD by means, and these variable groups were combined in pairs to generate a total of eight combinations. The PROCESS macro developed by Hayes was employed for the simple mediation analysis. Results: Compared with the reference group (those with both high SES and high DD), older adults who were classified to have lower SES or DD had elevated odds of having worse HRQoL: low SES/ low DD (OR = 1.65, 95% CI 1.41-2.92); low SES/ high DD (OR = 1.45, 95% CI 1.17-1.80); middle low SES/ low DD (OR = 1.43, 95% CI 1.24-1.65); middle low SES/ high DD (OR = 1.23, 95% CI 1.03-1.47); upper high SES/ low DD (OR = 1.41, 95% CI 1.21-1.65); and high SES/ low DD (OR = 1.30, 95%CI 1.10-1.53). The mediation analysis revealed that DD mediated the relationship between SES and HRQoL (B=0.011, 95% CI 0.008-0.013), with its indirect effects accounting for 39.29% of the total effects. Conclusions: These findings highlighted the role of DD as a mediator of the relationship between SES and HRQoL. As DD could be protective, modifiable, and easy for older adults to understand and implement, village clinics and community health stations should work collaboratively to design proper DD intervention measures for better HRQoL.

A retrospective study of the antibiotic-resistant phenotypes and genotypes of Helicobacter pylori strains in China.

Helicobacter pylori antibiotic resistance is a serious concern in China, where it severely influences treatment for H. pylori infection. To overcome this, it is essential to apply personalized therapies based on local or individual data on antibiotic-resistant phenotypes or genotypes. We conducted a large-scale multi-center study with a retrospective cross-sectional observational design to investigate the antibiotic-resistant phenotypes and genotypes of H. pylori in China. Strains were isolated from the gastric biopsy samples of H. pylori-infected patients from five different regions in China. The strains were tested for antibiotic-resistant phenotypes and genotypes, and the agreement between the two was assessed. In total, 4242 H. pylori strains were isolated and cultured, with an 84.43% success rate. The primary and secondary antibiotic resistance rates of H. pylori were 37.00% and 76.93% for clarithromycin, 34.21% and 61.58% for levofloxacin, 2.20% and 6.12% for amoxicillin, 1.61% and 3.11% for furazolidone, 1.18% and 3.31% for tetracycline, and 87.87% and 93.48% for metronidazole, respectively. The dual-resistance patterns for metronidazole/clarithromycin, metronidazole/levofloxacin, and clarithromycin/levofloxacin were 43.6%, 38.4%, and 26.1%, respectively. Clarithromycin- and levofloxacin-resistant H. pylori phenotypes and genotypes showed satisfactory agreement. Based on these findings, clarithromycin- and levofloxacin-resistant genotype testing could partially replace traditional antibiotic susceptibility testing in China. Continuous monitoring and personalized treatments based on individual and local H. pylori antibiotic-resistance data remain necessary.

68Ga-Labeled GX1 Dimer: A Novel Probe for PET/Cerenkov Imaging Targeting Gastric Cancer.

PURPOSE: To synthesize the dimer of GX1 and identify whether its affinity and targeting are better than those of GX1. To prepare 68Ga-DOTA-KEK-(GX1)2 and to apply it to PET and Cerenkov imaging of gastric cancer. METHODS: 68Ga-DOTA-KEK-(GX1)2 was prepared, and the labeling yield and stability were determined. Its specificity and affinity were verified using an in vitro cell binding assay and competitive inhibition test, cell immunofluorescence, and cell uptake and efflux study. Its tumor-targeting ability was determined by nano PET/CT and Cerenkov imaging, standardized uptake value (SUV), signal-to-background ratio (SBR) quantification, and a biodistribution study in tumor-bearing nude mice. RESULTS: 68Ga-DOTA-KEK-(GX1)2 was successfully prepared, and the labeling yield was more than 97%. It existed stably for 90 min in serum. The binding of 68Ga-DOTA-KEK-(GX1)2 to cocultured HUVECs (Co-HUVECs) was higher than that to human umbilical vein endothelial cells (HUVECs), BGC823 cells, and GES cells. It was also higher than that of 68Ga-DOTA-GX1, indicating that the dimer did improve the specificity and affinity of GX1. The binding of KEK-(GX1)2 to Co-HUVECs was significantly higher than that of GX1. Additionally, the uptake of 68Ga-DOTA-KEK-(GX1)2 by Co-HUVECs was higher than that of 68Ga-DOTA-GX1 and reached a maximum at 60 min. Nano PET/CT and Cerenkov imaging showed that the tumor imaging of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 was clear, and the SUV and SBR value of the tumor sites were significantly higher than those of the nude mice injected with 68Ga-DOTA-GX1, indicating that the probe had better targeting in vivo. Finally, the biodistribution showed quantitatively that when organs such as the kidney and liver metabolized rapidly, the radioactivity of the tumor site of the nude mice injected with 68Ga-DOTA-KEK-(GX1)2 decreased relatively slowly. At the same time, the percentage of injected dose per gram (%ID/g) of the tumor site was higher than that of other normal organs except the liver and kidney at 60 min, which indicated that the tumor had good absorption of the probe. CONCLUSION: GX1 was modified successfully, and the in vivo and in vitro properties of the GX1 dimer were significantly better than those of GX1. The imaging probe, 68Ga-DOTA-KEK-(GX1)2, was successfully prepared, which provides a candidate probe for PET and Cerenkov diagnosis of gastric cancer.

Antigenicity and immunogenicity of recombinant proteins comprising African swine fever virus proteins p30 and p54 fused to a cell-penetrating peptide.

African swine fever (ASF) is a highly fatal swine disease threatening the global pig industry. Currently, vaccine is not commercially available for ASF. Hence, it is desirable to develop effective subunit vaccines against ASF. Here, we expressed and purified two recombinant fusion proteins comprising ASFV proteins p30 and p54 fused to a novel cell-penetrating peptide Z12, which were labeled as ZPM (Z12-p30-modified p54) and ZPMT (Z12-p30-modified p54-T cell epitope). Purified recombinant p30 and modified p54 expressed alone or fused served as controls. The transduction capacity of these recombinant proteins was assessed in RAW264.7 cells. Both ZPM and ZPMT exhibited higher transduction efficiency than the other proteins. Subsequently, humoral and cellular immune responses elicited by these proteins were evaluated in mice. ZPMT elicited the highest levels of antigen-specific IgG responses, cytokines (interleukin-2, interferon-γ, and tumor necrosis factor-α) and lymphocyte proliferation. Importantly, sera from mice immunized with ZPM or ZPMT neutralized greater than 85% of ASFV in vitro. Our results indicate that ZPMT induces potent neutralizing antibody responses and cellular immunity in mice. Therefore, ZPMT may be a suitable candidate to elicit immune responses in swine, providing valuable information for the development of subunit vaccines against ASF.

188Re-labeled GX1 dimer as a novel dual-functional probe targeting TGM2 for imaging and antiangiogenic therapy of gastric cancer.

PURPOSE: The GX1 peptide (CGNSNPKSC) can specifically bind to TGM2 and possesses the ability to target the blood vessels of gastric cancer. This study intends to develop an integrated dual-functional probe with higher affinity, specificity and targeting and to characterize it in vivo and in vitro. METHODS: The dimer and tetramer of GX1 were prepared using cross-linked PEG and labeled with 99mTc. The best targeting probe [PEG-(GX1)2] was selected by gamma camera imaging in nude mouse models of gastric cancer. 188Re-PEG-(GX1)2 was prepared and characterized through cell binding analysis and competitive inhibition experiments, gamma camera imaging, MTT analysis and flow cytometry, BLI, immunohistochemistry, HE staining and biochemical analysis. RESULTS: PEG-(GX1)2 bound specifically to Co-HUVEC with higher affinity than GX1. 188Re-PEG-(GX1)2 had better ability to target gastric cancer in tumor-bearing nude mice and higher T/H ratios than 188Re-GX1. 188Re-PEG-(GX1)2 inhibited the growth of Co-HUVEC and induced apoptosis, and its effects were more robust than those of 188Re-GX1. BLI showed that 188Re-PEG-(GX1)2 inhibited tumor proliferation in vivo with a stronger effect than 188Re-GX1. Compared with 188Re-GX1, 188Re-PEG-(GX1)2 suppressed tumor angiogenesis and tumor cell proliferation and induced tumor cell apoptosis in vivo. The 188Re-PEG-(GX1)2 group did not cause visible changes in liver and kidney morphology and function in vivo. CONCLUSION: The dimer of GX1 was synthesized by using cross-linked PEG, and then 188Re-PEG-(GX1)2 was prepared. This radiopharmaceutical played both diagnostic and therapeutic functions, and gamma camera imaging could be utilized to detect the distribution of drugs in vivo during treatment. Through a series of experiments in vitro and in vivo, the feasibility of the drug was confirmed, and these results laid the foundation for the subsequent development and application of GX1.

Thermus thermophilus DNA Ligase Connects Two Fragments Having Exceptionally Short Complementary Termini at High Temperatures.

Thermus thermophilus DNA ligase (Tth DNA ligase) is widely employed for cloning, enzymatic synthesis, and molecular diagnostics at high temperatures (e.g., 65 °C). It has been long believed that the complementary ends must be very long (e.g., >30 bp) to place two DNA fragments nearby for the ligation. In the current study, the length of the complementary portion was systematically varied, and the ligation efficiency was evaluated using the high resolution melting (HRM) method. Unexpectedly, very short oligonucleotides (7-10 nt) were successfully ligated on the complementary overhang attached to a dsDNA at 70 °C. Furthermore, sticky ends with the overhang of only 4 nt long, available after scission with many restriction enzymes, were also efficiently ligated at 45-70 °C. The ligation yield for the 6-nt-long sticky ends was as high as 80%. It was concluded that Tth DNA ligase can be used as a unique tool for DNA manipulation that cannot be otherwise easily accomplished.

Diversity evolution of functional bacteria and resistance genes (CzcA) in aerobic activated sludge under Cd(II) stress.

An activated sludge sequencing batch reactor (SBR) was used to treat divalent cadmium (Cd(II)) wastewater for 60 d to investigate the overall treatment performance, evolution of the bacterial community, and abundance of the Cd(II) resistance gene CzcA and shifts in its potential host bacteria. During stable operation with a Cd(II) concentration of 20 mg/L, the average removal efficiencies of Cd(II) and chemical oxygen demand (COD) were more than 85% and that of total phosphorus was greater than 70%, while the total nitrogen (TN) was only about 45%. The protein (PN) content in the extracellular polymeric substances (EPS) increased significantly after Cd(II) addition, while polysaccharides displayed a decreasing trend (p < 0.05), indicating that EPS prefer to release PN to adsorb Cd(II) and protect bacteria from damage. Three-dimensional fluorescence spectral analysis showed that fulvic acid-like substances were the most abundant chemical components of EPS. The addition of Cd(II) adversely affected most denitrifying bacteria (p < 0.05), which is consistent with the low TN removal. In addition, quantitative polymerase chain reaction analysis revealed that CzcA gene abundance decreased as the Cd(II) concentration increased, possibly because expression of the CzcA gene was inhibited by Cd(II) stress. The majority of CzcA gene sequences were carried by Pseudomonas, making it the dominant genus among Cd(II)-resistant bacteria.

Evolution and resistance of a microbial community exposed to Pb(II) wastewater.

This study investigated the treatment performance of activated sludge on Pb(II)-containing wastewater, including contaminant removal efficiency, extracellular polymeric substances, pbrT gene content and the microbial community. The average removal efficiencies of ammonia nitrogen, chemical oxygen demand, total phosphorus, total nitrogen and Pb(II) were 40% ±â€¯4%, 91% ±â€¯3%, 95% ±â€¯3%, 51% ±â€¯5% and 92% ±â€¯9% during the stable operation stage, respectively. Moreover, the extracellular polymeric substance -protein contents increased significantly from day 0 to day 60 (p < 0.05). The most abundant fluorescent component in extracellular polymeric substances was a humic acid-like substance, and its fluorescence intensity increased significantly from day 0 to day 60 (p < 0.05). Adsorption of negatively charged organic functional groups in extracellular polymeric substances was identified as a major component of the removal of Pb(II). Most of the denitrifying bacteria associated with nitrogen removal showed an increasing trend during the acclimation stage, which may have resulted in high total nitrogen removal efficiency. In addition, pbrT uptake protein was found to be responsible for the uptake of Pb(II) into cells. The abundance of the pbrT gene showed a downward trend (p < 0.05) after adding Pb(II), probably because expression of the pbrT gene was inhibited under Pb(II) stress. Sphingopyxis containing the pbrT gene was the dominant resistance genus, and its relative abundance increased significantly (p < 0.05) from day 0 to day 60. This study provided a theoretical basis for the treatment of Pb(II)-containing wastewater.

Chemical composition of extracellular polymeric substances and evolution of microbial community in activated sludge exposed to ibuprofen.

Ibuprofen (IBU) containing wastewater with a concentration of 1-5 mg/L was treated in an activated sludge sequencing batch reactor (SBR), for 60 days, in order to investigate the overall performance of the SBR, the parameter variations during a typical cycle, the chemical composition and content of extracellular polymeric substances (EPS) and the evolution of microbial community. The average removal efficiencies of COD, NH4+-N and TN were >85%, while >40% of the IBU was removed and the removal efficiencies of TP fluctuated around ~ 75%. The EPS content increased significantly with IBU addition (p < 0.01). Fulvic acid-like substances in the chemical composition of EPS increased during the stable operation phase. Proteobacteria associated with nitrogen removal was the dominant phylum, which can also resist IBU stress. For the denitrifying bacteria, the OTUs of both Rhodobacter and Pseudomonas increased from day 1-30 and reduced on day 60 (p < 0.01), which was opposite to the results observed for Rhodocyclaceae (phosphorus-accumulating bacteria). The OTUs of Acidovorax showed an increasing trend (p < 0.01), whereas the OTUs for Nitrospira (nitrite oxidizers) and Nitrosomonas (ammonia oxidizers) decreased significantly (p < 0.05).

Cyclization of secondarily structured oligonucleotides to single-stranded rings by using Taq DNA ligase at high temperatures.

Single-stranded DNA rings play important roles in nanoarchitectures, molecular machines, DNA detection, etc. Although T4 DNA ligase has been widely employed to cyclize single-stranded oligonucleotides into rings, the cyclization efficiency is very low when the oligonucleotides (l-DNAs) take complicated secondary structures at ambient temperatures. In the present study, this problem has been solved by using Thermus aquaticus DNA ligase (Taq DNA ligase) at higher temperatures (65 and 70 °C) where the secondary structures are less stable or completely destroyed. This method is based on our new finding that this ligase successfully functions even when the splint strand is short and forms no stable duplex with l-DNA (at least in the absence of the enzyme). In order to increase the efficiency of cyclization, various operation factors (lengths and sequences of splint, as well as the size of the DNA ring) have been investigated. Based on these results, DNA rings have been successfully synthesized from secondarily structured oligonucleotides in high yields and high selectivity. The present methodology is applicable to the preparation of versatile DNA rings involving complicated secondary structures, which should show novel properties and greatly widen the scope of DNA-based nanotechnology.

Chemiluminescence Immunoassay for the Detection of Antibodies against the 2C and 3ABC Nonstructural Proteins Induced by Infecting Pigs with Foot-and-Mouth Disease Virus.

The potential diagnostic value of chemiluminescence immunoassays (CLIAs) has been accepted in recent years, although their use for foot-and-mouth disease (FMD) diagnostics has not been reported. Full-length 3ABC and 2C proteins were expressed in bacteria and purified by affinity chromatography to develop a rapid and accurate approach to distinguish pigs infected with foot-and-mouth disease virus (FMDV) from vaccinated pigs. The recombinant proteins were then used as antigens to develop two CLIAs for the detection of antibodies against nonstructural viral proteins. The diagnostic performance of the two assays was compared by analyzing serum from pigs (naive pigs, n = 63; vaccinated, uninfected pigs, n = 532; naive, infected pigs, n = 117) with a known infection status. The 3ABC-2C CLIA had a higher accuracy rate, with a diagnostic sensitivity of 100% and a diagnostic specificity of 96.5%, than the 3ABC CLIA, which had a diagnostic sensitivity of 95.7% and a diagnostic specificity of 96.0%. The results of the 3ABC-2C CLIA also had a high rate of concordance with those of two commercial FMDV enzyme-linked immunosorbent assay (ELISA) kits used to assess serum collected from 962 pigs in the field (96.2% and 97.8%, respectively). The 3ABC-2C CLIA detected infection in serum samples from infected pigs earlier than the commercial ELISA kits. In addition, the 3ABC-2C CLIA produced results within 15 min. On the basis of these findings, the 3ABC-2C CLIA could serve as the foundation for the development of penside FMD diagnostics and offers an alternative method to detect FMDV infections.

Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves.

The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R2 = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 µg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.