Safe and efficient gene transfer systems are the basis of gene therapy applications. Non-integrating lentiviral (NIL) vectors are among the most promising candidates for gene transfer tools, because they exhibit high transfer efficiency in both dividing and non-dividing cells and do not present a risk of insertional mutagenesis. However, non-integrating lentiviral vectors cannot introduce stable exogenous gene expression to dividing cells, thereby limiting their application. Here, we report the design of a non-integrating lentiviral vector that contains the minimal scaffold/matrix attachment region (S/MAR) sequence (SNIL), and this SNIL vector is able to retain episomal transgene expression in dividing cells. Using SNIL vectors, we detected the expression of the eGFP gene for 61 days in SNIL-transduced stable CHO cells, either with selection or not. In the NIL group without the S/MAR sequence, however, the transduced cells died under selection for the transient expression of NIL vectors. Furthermore, Southern blot assays demonstrated that the SNIL vectors were retained extrachromosomally in the CHO cells. In conclusion, the minimal S/MAR sequence retained the non-integrating lentiviral vectors in dividing cells, which indicates that SNIL vectors have the potential for use as a gene transfer tool.
Penicillium decumbens T. is an important filamentous fungus for the production of cellulases to effectively degrade lignocellulose for second generation biofuel production. In order to enhance the capability of Penicillium decumbens to produce cellulases, we constructed a creB (a deubiquitinating enzyme encoding gene) deletion cassette, and generated a creB knockout strain with homologous double crossover recombination. This mutation resulted in a detectable decrease of carbon catabolite repression (CCR) effect. The filter paper activity, endoglucanase activity, xylanase activity and exoglucanase activity of the deltacreB strain increased by 1.8, 1.71, 2.06 and 2.04 fold, respectively, when comparing with the parent strain Ku-39. A 2.68 fold increase of extracellular protein concentration was also observed. These results suggest that the deletion of creB results in CCR derepression. These data also suggest that CREB influences cellulase production of Penicillium decumbens. In generation, this study provides information that can be helpful for constructing cellulase hyper-producing strain.
Objectives: Conducted a prospective cohort study to understand characteristics of reinfection of SARS-COV-2, to explore risk factors of developing reinfection and to provide prospectives for protecting susceptible population. Methods: Constructed a prospective cohort involving 2007 current employees from 24 units of Centers for Disease Control and Prevention (CDCs) in Chengdu city and its jurisdictional districts. Weekly follow-ups began in February 2023 and individuals would receive retrospective surveys if reinfection confirmed. Charlson comorbidity index (CCI) was used to measure individuals’ health status. Chi-square test and logistic regression were used to compare incidence and identify risk factors. Results: Reinfection incidence among people in worse health status was higher than those in better health status (27.06% versus 18.15%), the difference was statistically significant (χ2= 4.276, P=0.039). Individuals with higher CCI, indicating worse health status, was identified as a risk factor for reinfection (odds ratio [OR]=1.878, 95% confidence interval [CI]=1.137-3.100). Conclusions: Reinfection was not unusual with the progress of fading herd immunity, especially those in worse health status were more likely to develop reinfection. It suggested that the vulnerable required additional attention and protection.
Neutral cellulase has been broadly used in a lot of industries like food,textile,forage and pharmacy. We screened a positive mutant strain H14 from Humicola insolens H31-3 by ion implantation. And then UV irradiation on the protoplasts from Humicola insolens H14 was performed. After regeneration and screening we obtained another mutant strain,Humicola insolens H14-2,in which CMC enzyme activity and filter paper enzyme activity are raised 78.57% and 106.81% compared with the parent strain of Humicola insolens H31-3.
Two TaqI endonuclease (hereafter referred to as TaqI) isoschizomer genes, tsp32IR from Thermus species SM32 of Azores and tfiTok6A1I from T. filiformis Tok6A1 of New Zealand, were cloned in Escherichia coli. The overexpressed enzymes were partly purified and their thermostability was determined. In the medium-salt buffer, Tsp32IR, TfiTok6A1I and one previously cloned TaqI isoschizomer (TthHB8I) were more thermostable than TaqI. Tsp32IR remained partly active up to 90 °C in the low-salt buffer. Six amino acid residues that are identical in the three high thermostability isoschizomers (Tsp32IR, TfiTok6A1I and TthHB8I) but differ in TaqI might provide added rigidity for thermostabilization. These include four proline residues located in or near loop regions, and one alanine and one arginine located at helix regions in the predicted TaqI endonuclease secondary structure. The possible role of these residues in thermostabilization was evaluated by mutagenizing the TaqI enzyme. Mutants generated at these six positions were less thermostable than wild-type TaqI. The results suggest that the surrounding sequence or structural context might be as important as the mutation itself. The nucleotide sequence data reported in this paper for TfiTok6A1I and Tsp32IR appear in the GenBank Database under the accession numbers U86869 and U86870 respectively.
In this study, we examined the complete genome of coxsackievirus A6 (CVA6) from hand, foot, and mouth disease in Guangdong Province from 2013,and explored the genetic similarities and differences in epidemic and non-epidemic stains of CVA6.Eighteen strains of CVA6 were included in complete genome sequencing, and the sequences were subject to phylogenetic analysis,sequence alignment analysis and genetic recombination analysis using the software DNASTAR6.0,MEGA5.2and SimPlot3.5.1.The results showed that the complete genome of 18 Guangdong CVA6strains ranged from 7390bp to 7392bp.No insertions or deletions were detected in the coding region. There were several insertions and deletions in 5′UTR and 3′UTR.Phylogenetic analysis indicated that the nucleotide and amino acid sequence identity between the 18 complete genomes were 90.5%-99.6% and 97.5%-99.9%,respectively.The strains isolated in2013 could be further divided into two clusters, III and IV, while the strains isolated in 2011 were only present in the IV cluster. Genetic recombination analysis revealed that the Guangdong representative strain of CVA6,GD870/2013,had gene recombination in the P2 and P3regions,while the GD839/2013 strain did not show obvious genetic recombination. Genome-wide analysis of CVA6 revealed that there are two possible transmitted chains, III and IV, in epidemic strains from Guangdong Province in 2013.The transmitted chain Ⅲ originated from the strain with genetic recombination in the P2 and P3regions,whichwas completely different from the chain IV. Transmission of chain IV of CVA6 was only observed in the nonepidemic 2011 strain.
To the Editor: In the People’s Republic of China, >3,000 persons die of rabies each year; most were infected by dog bites (1). Since 2000, the dog population in Beijing has increased dramatically, and the exact vaccination coverage and immunization status of dogs are not known.
During 2006–2009, to assist with governmental rabies control, Fengtai District was selected as a geographically representative area in Beijing in which to conduct a survey of rabies antibody titers in domestic dogs. Blood samples were randomly collected from 4,775 dogs in Fengtai District, which account for 3% of all registered dogs in the district. Rabies virus neutralization antibody (VNA) titers were detected by fluorescent antibody virus neutralization (2). In brief, VNA titers >0.5 IU indicated positive immunization, implying that the dog had an adequate level of antibody, and VNA <0.5 IU indicated negative immunization (3). The data were analyzed by 2-tailed χ2 test; p<0.05 was considered significant. Vaccination coverage and antibody levels were categorized either by dog’s function (guard or pet) or residence (urban or suburban) (Figure).
Figure
Rabies immunization status of dogs, Fengtai, Beijing, China. Immunization status and vaccination coverage were categorized according to dog vaccination background and rabies antibody level in each dog. A) Vaccinated, B) never vaccinated, C) unclear vaccination ...
Most dogs with a history of vaccination were positively immunized (68.1%) (Figure, bar A), compared with 16.4% in the unvaccinated group (Figure, bar B), demonstrating that compulsory immunization is crucial to rabies control (4). Of 944 dogs with unclear vaccination history, 221 (23.4%) (Figure, bar C) had adequate antibody levels, possibly from undocumented vaccination or contact with rabies hosts. However, for 2006, 2007, 2008, and 2009, immunization coverage in the district was 55.0%, 53.8%, 67.4%, and 54.4%, respectively, all below the >70% criterion recommended by the World Health Organization (5). The results imply that much work still needs to be done by the Beijing government, not only to meet the World Health Organization immunization baseline but also to keep risk for a rabies epidemic in Beijing low.
Immunization coverage ratios differed significantly (p<0.05) between guard (39.3%) and pet dogs (69.5%) (Figure, bars D, E) and between urban (81.7%) and suburban areas (27.6%) (Figure, bars F, G). Consequently, the number of negatively immunized guard dogs was 1.68× lower than that for pet dogs (Figure, bars D, E) (p<0.05), and the number of positively immunized dogs in urban areas was 2.5× higher than that in suburban areas (Figure, bars F, G) (p<0.05).
In Beijing, guard dogs are usually raised by villagers to protect the house, whereas pet dogs are usually raised by city dwellers who treat dogs as friends. As a result, in urban areas dogs are registered and vaccinated in a timely manner by authorized pet hospitals (6). In suburban areas, however, dog management is deficient. For example, guard dogs in suburban areas are sometimes not vaccinated because the owner or veterinarian cannot safely restrain the dog for vaccination.
According to our study, >10% of unregistered dogs with no clear history of vaccination are not vaccinated during yearly vaccination programs. In Beijing during 2007–2009, of 9 cases of rabies in humans, 6 were associated with stray dogs (7), and most stray dogs were found in suburban areas. Hence, strategies to either reduce stray dogs in the city or to get such dogs under official management (e.g., include stray dogs in compulsory annual vaccination programs) are urgently needed.
In our opinion, policies related to dog registration, vaccination recording, and vaccination strategies need improvement in Beijing, especially in suburban areas. Although our report only focused on the Fengtai District, the findings could be helpful for the Beijing government for establishing strategies to control the rabies epidemic in the entire city.
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