Global, regional and national trends in tuberculosis incidence and main risk factors: a study using data from 2000 to 2021.

BACKGROUND: Despite the significant progress over the years, Tuberculosis remains a major public health concern and a danger to global health. This study aimed to analyze the spatial and temporal characteristics of the incidence of tuberculosis and its risk factors and to predict future trends in the incidence of Tuberculosis. METHODS: This study used secondary data on tuberculosis incidence and tuberculosis risk factor data from 209 countries and regions worldwide between 2000 and 2021 for analysis. Specifically, this study analyses the spatial autocorrelation of Tuberculosis incidence from 2000 to 2021 by calculating Moran's I and identified risk factors for Tuberculosis incidence by multiple stepwise linear regression analysis. We also used the Autoregressive Integrated Moving Average model to predict the trend of Tuberculosis incidence to 2030. This study used ArcGIS Pro, Geoda and R studio 4.2.2 for analysis. RESULTS: The study found the global incidence of Tuberculosis and its spatial autocorrelation trends from 2000 to 2021 showed a general downward trend, but its spatial autocorrelation trends remained significant (Moran's I = 0.465, P < 0.001). The risk factors for Tuberculosis incidence are also geographically specific. Low literacy rate was identified as the most pervasive and profound risk factor for Tuberculosis. CONCLUSIONS: This study shows the global spatial and temporal status of Tuberculosis incidence and risk factors. Although the incidence of Tuberculosis and Moran's Index of Tuberculosis are both declining, there are still differences in Tuberculosis risk factors across countries and regions. Even though literacy rate is the leading risk factor affecting the largest number of countries and regions, there are still many countries and regions where gender (male) is the leading risk factor. In addition, at the current rate of decline in Tuberculosis incidence, the World Health Organization's goal of ending the Tuberculosis pandemic by 2030 will be difficult to achieve. Targeted preventive interventions, such as health education and regular screening of Tuberculosis-prone populations are needed if we are to achieve the goal. The results of this study will help policymakers to identify high-risk groups based on differences in TB risk factors in different areas, rationalize the allocation of healthcare resources, and provide timely health education, so as to formulate more effective Tuberculosis prevention and control policies.

Variational Bayesian Inference for Robust Identification of PWARX Systems With Time-Varying Time-Delays.

This article presents a robust variational Bayesian (VB) algorithm for identifying piecewise autoregressive exogenous (PWARX) systems with time-varying time-delays. To alleviate the adverse effects caused by outliers, the probability distribution of noise is taken to follow a t -distribution. Meanwhile, a solution strategy for more accurately classifying undecidable data points is proposed, and the hyperplanes used to split data are determined by a support vector machine (SVM). In addition, maximum-likelihood estimation (MLE) is adopted to re-estimate the unknown parameters through the classification results. The time-delay is regarded as a hidden variable and identified through the VB algorithm. The effectiveness of the proposed algorithm is illustrated by two simulation examples.

[Corrigendum] NLRP3 inflammasome is responsible for Hantavirus inducing interleukin­1ß in THP­1 cells.

Subsequently to the publication of the above article, the authors have realized that an error was introduced in the preparation of Fig. 2B for publication. In Fig. 2B, the lanes shown for the western blot were misannotated. Additionally, in the legend of Fig. 2C, '24 h post­infection' should have been written as '12 h post­infection'. Furthermore, the description of the data shown in Fig. 2B in the Results section (sentence commencing on p. 1635, the subsection 'HTNV activates caspase­1 and pro­IL­1ß in THP­1 cells', line 10), was incomplete. The sentence here should have read as follows (the added text is highlighted in bold): 'In order to investigate whether caspase­1 was activated during HTNV infection, the culture supernatant of HTNV­infected THP­1 was ultra­ï¬ltered and an increased concentration of secreted caspase­1 was detected post­infection compared with the Mock group; similar results were also observed in the LPS­ and ATP­stimulated groups (Fig. 2B)'. The correct version of Fig. 2, with the lanes of the western blot in Fig. 2B labelled correctly and the appropriate changes having been made to the Figure legend, is shown opposite. The errors associated with this Figure did not have an impact on the overall meaning of the paper, or on the reported conclusions of this study. The authors regret that this Figure was not corrected prior to the publication of this article, and apologize to the readership for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 35: 1633­1640, 2015; DOI: 10.3892/ijmm.2015.2162].

NLRP3 inflammasome is responsible for Hantavirus inducing interleukin-1ß in THP-1 cells.

Persistent high fever is one typical clinical symptom of hemorrhagic fever with renal syndrome (HFRS) and circulating interleukin-1ß (IL-1ß) is elevated throughout HFRS. The mechanisms responsible for viral induction of IL-1ß secretion are unknown. In the present study, Hantaan virus (HTNV) induced the secretion of IL-1ß in the human monocytic cell line THP-1. Induction of IL-1ß by HTNV relies on the activation of caspase-1. Small hairpin RNA knockdown in HTNV-infected THP-1 cells indicated that nucleotide-binding domain, leucine-rich repeat containing protein 3 (NLRP3) recruits the adaptor apoptosis-associated speck-like protein and caspase-1 to form an NLRP3 inflammasome complex, crucial for the induction of IL-1ß. In HTNV-infected THP-1 cells, reactive oxygen species release, but not extracellular adenosine triphosphate, was crucial for IL-1ß production. In conclusion, Hantavirus induces the formation of the NLRP3 inflammasome in THP-1 cells and this may be responsible for the elevated IL-1ß levels in HFRS patients.

A recombinant pseudotyped lentivirus expressing the envelope glycoprotein of hantaan virus induced protective immunity in mice.

BACKGROUND: Hantaviruses cause acute hemorrhagic fever with renal syndrome (HFRS). Currently, several types of inactivated HFRS vaccines are widely used, however the limited ability of these immunogen to elicit neutralizing antibodies restricts vaccine efficacy. Development of an effective vaccine to overcome this weakness is must. METHODS: In the present study, a recombinant pseudotyped lentivirus bearing the hantaan virus (HTNV) envelope glycoproteins (GP), rLV-M, was constructed. C57BL/6 mice were immunized with the rLV-M and a series of immunological assays were conducted to determine the immunogenicity of the recombinant pseudotyped lentivirus. The humoral and cell-mediated immune responses induced by rLV-M were compared with those of the inactivated HFRS vaccine. RESULTS: Indirect immunofluorescence assay (IFA) showed the rLV-M expressed target proteins in HEK-293 cells. In mice, the rLV-M efficiently induced GP-specific humoral responses and protection against HTNV infection. Furthermore, the rLV-M induced higher neutralizing antibody titers than the inactivated HFRS vaccine control. CONCLUSIONS: The results indicated the potential of using a pseudotyped lentivirus as a delivery vector for a hantavirus vaccine immunogen.

Induction of Hantaan virus-specific immune responses in C57BL/6 mice by immunization with a modified recombinant adenovirus containing the chimeric gene, GcS0.7.

Hantavirus glycoprotein Gc is one of the main components that contribute to the generation of humoral immune responses, while the nucleocapsid protein (NP) is involved in cellular immune responses through the induction of antibody-dependent cytotoxic T cells. In this study, a chimeric gene, GcS0.7, which encodes a fusion protein containing Gc and truncated NP, was constructed as a candidate for Hantaan virus (HTNV) vaccine development. The chimeric gene was cloned into an adenoviral vector in conjunction with the powerful hybrid cytomegalovirus (CMV) enhancer/chicken ß-actin (CAG) promoter or the woodchuck hepatitis virus (WHV) post-transcriptional regulatory element (WPRE), or both. Both elements increased the expression level of the fusion protein. The rAd-GcS0.7-pCAG group demonstrated the highest fusion protein expression level, with a 2.3-fold increase compared with the unmodified adenoviral vector. To further evaluate the humoral and cellular immunity induced by the recombinant adenovirus, the antibody titers, interferon (IFN)-γ secretion level and cytotoxic T cell ratio were detected in immunized mice. The strongest HTNV­specific humoral and cellular immune responses were detected in the rAd-GcS0.7­pCAG group. The immunogenicity of these recombinant adenoviruses was compared with that of the inactivated vaccine through a series of immunological assays. In terms of the cellular immune responses, the rAd-GcS0.7-pCAG group even exceeded those induced by the vaccine control. The CAG hybrid promoter improved not only the expression level, but also the immunogenicity of the fusion protein, and may thus provide a promising strategy for HTNV vaccine research.

Modification of the adenoviral transfer vector enhances expression of the Hantavirus fusion protein GnS0.7 and induces a strong immune response in C57BL/6 mice.

Hantavirus glycoproteins (Gn and Gc) are significant components of vaccines for haemorrhagic fever with renal syndrome (HFRS); however, they are not effective due to weak immunogenicity and low levels of production in expression systems. To circumvent this problem, a 0.7-kb fragment of the S segment was fused to Gn, and a hybrid CAG promoter/enhancer in conjunction with (or without) the WPRE (Woodchuck hepatitis virus post-transcriptional regulatory element) was used to improve the expression of fusion protein GnS0.7 in the adenoviral expression system. The expression level of the fusion protein as well as the response of mice immunized with recombinant adenoviruses containing GnS0.7 was investigated. In addition, a series of immunological assays were conducted to determine the immunogenicity of the recombinant adenoviruses. The results showed that the recombinant adenovirus with the CAG promoter/enhancer (rAd-GnS0.7-pCAG) expressed approximately 2.1-fold more GnS0.7 than the unmodified recombinant adenovirus containing GnS0.7 (rAd-GnS0.7-pShuttle). This enhanced expression level was also higher than for other modified recombinant adenoviruses studied. Animal experiments showed that rAd-GnS0.7-pCAG induced a stronger Hantaan virus (HTNV)-specific humoral and cellular immune response in mice, with the cellular immune response to the GnS0.7 being stronger than the HFRS vaccine control. These results demonstrate that the CAG promoter/enhancer improved significantly the expression of the chimeric gene GnS0.7 in the adenovirus expression system. These findings may have significant implications for the development of genetically engineered vaccines for HFRS.

Downregulation of NDRG1 promotes invasion of human gastric cancer AGS cells through MMP-2.

The N-myc downstream-regulated gene-1 (NDRG1) has recently been proposed as a metastasis suppressor, but its precise role remains unclear. To investigate whether NDRG1 can indeed influence the metastasis progress, expression of endogenous NDRG1 was knocked down in human AGS gastric adenocarcinoma cells using RNA interference. Stable NDRG1 "silenced" transfectants showed similar growth rates as their control counterparts. By contrast, invasive ability in Matrigel invasion activity and Gelatinolytic activity by matrix metalloproteinase-2 (MMP-2) were markedly increased in NDRG1 "silenced" cells. Moreover, re-expression of NDRG1 by recombinant adenovirus Ad-NDRG1 in NDRG1 "silenced" cells inhibited the increased invasive ability. Further study, we found the induction of MMP-2 by downregulation of NDRG1 was mediated by MT1-MMP. Altogether, our results imply that NDRG-1 could play a key role in the regulation of cellular invasion and metastasis, which may involve the upregulation of matrix metalloproteinases.

[Construction and immunogenic study of recombinant adenovirus containing chimeric gene G2S0.7 and CTL epitopes of Hantaan virus].

AIM: To express G2 fragment of M segment and 0.7 kb fragment of S segment and several CTL epitopes of S segment in adenovirus expression system and investigate the immunological properties of hantaan virus chimeric gene. METHODS: The recombinant adenovirus was constructed and the recombinant adenovirus was obtained after transfecting HEK293 cells. The titer of it was determined and the expressed product was detected by IFA and ELISA. Further, BALB/c mice were vaccinated by the recombinant adenovirus and the immune response was tested by ELISA, microcell-culture neutralizing experiment, T lymphocyte proliferation test (MTT assay) and cell-mediated cytotoxicity assay. RESULTS: The recombinant adenovirus AG2S0.7CTL1, AG2S0.7CTL2 were constructed successfully and the titer of it was about 10¹°-10¹¹ pfu/mL. The expressed protein could be recognized by the hantaan virus NP-specific mAb and glycoprotein G2-specific mAb. The recombinant adenovirus containing CTL epitopes could elicit effectively the cellular immune response aimed to the NP and GP of hantaan virus in BALB/c mice. CONCLUSION: The recombinant adenovirus containing CTL epitopes could induce the higher cellular immune response than the group that not containing CTL epitopes.

[Construction and identification of a new type of recombinant adenovirus containing S0.7 gene of Hantavirus].

AIM: To construct a adenovirus vector containing the 0.7 kb fragment of S gene of Hantavirus, CAG promoter, and WPRE (mRNA-stabilizing post-transcriptional regulatory element from the woodchuck hepatitis virus). METHODS: The fragments of CAG and WPRE were synthesized according to GenBank, and inserted into the plasmid pShuttle-S0.7 to create a transfer vector pShuttle-S0.7-CAG-WPRE. The S0.7-CAG-WPRE fragment was then cloned into Adeno-X; Viral DNA by PI-Sce I and I-Ceu I digestion. The recombinant adenovirus DNA was linearized by Pac I, transfected into HEK 293 cells via Lipofectamine; 2000, and the titer of the recombinant adenovirus was determined by Adeno-X; Rapid Titer Kit. The expressed product of S0.7-CAG-WPRE fragment was detected by immunofluorescence assay. RESULTS: The sequence of S0.7-CAG-WPRE fragment was confirmed by sequencing, and the recombinant adenovirus containing S0.7-CAG-WPRE was titered at 10(13); pfu/L. HEK293 cells transfected with recombinant adenoviruses were detected positive by immunofluorescence assay using a specific mAb 1A8 against Hantavirus nucleoprotein. CONCLUSION: The recombinant adenovirus containing the 0.7 kb fragment of S segment of Hantavirus, CAG promoter, WPRE was constructed.

Determination of microRNAs in mouse preimplantation embryos by microarray.

Expression profile of microRNA (miRNA) in mouse oocytes and preimplantation embryos has been revealed by a novel high throughput microarray assay. A total of 97 (43 "new" and 54 known) including mouse, human, and predicted miRNAs have been discovered in the preimplantation mouse embryos which can be classified into developmental stage-dependent groups and non-stage-dependent group according to the statistical analysis of the expression patterns. Potential gene targets of each group of miRNAs are estimated by TargetsScan system and significantly changed signaling pathways and biological processes underlying these gene targets are searched by PANTHER classification system between the stage-dependent miRNAs and the non-stage-dependent miRNAs. Expression of some miRNAs is confirmed by reverse transcriptase-polymerase chain reaction. It is shown that dynamic synthesis and degradation of miRNAs coexists in the preimplantation development of mouse embryos. However, the overall quantity of miRNAs and percentage of the stage-dependent miRNAs increase as the preimplantation embryos develop.

Expression and purification of Mycobacterium tuberculosis ESAT-6 and MPT64 fusion protein and its immunoprophylactic potential in mouse model.

The completion of Mycobacterium tuberculosis genome sequence has opened a new way for the identification and characterization of bacterial antigens, such as ESAT-6, CFP10, MPT64, and Ag85 complex, which are helpful for tuberculosis control. In this work, genes of ESAT-6 and MPT64 were fused and expressed in Escherichia coli in form of inclusion bodies with a histidine tag. The expressed fusion protein was purified by nitrilotriacetic acid (Ni-NTA) affinity chromatography under denaturing conditions, and the yield was 18mg/L of culture. In mice, the purified ESAT-6-MPT64 fusion protein elicited stronger humoral response, greater splenic lymphocyte stimulated index, and higher levels of IFN-gamma and IL-12 production than that of the single MPT64 inoculation group, and rendered modest protection on the experimental tuberculosis mouse models. In short, the ESAT-6-MPT64 fusion protein might be a potential candidate vaccine for tuberculosis.

[Transformation of full-length gene of mouse/human chimeric antibody against Hantaan virus into Arabidopsis thaliana].

AIM: To construct the transgenic Arabidopsis thaliana containing full-length gene of mouse/human chimeric antibody(3G1MH) against Hantaan virus. METHODS: The recombinant plasmid 3G1MH-pCAMBIA2301 was transformed into Agrobacterium tumefaciens GV3101 by TSS freeze-thaw method, and then the recombinant was transferred into wild Arabidopsis thaliana by vacuum-transgenic method. The regenerated transgenic plants were selected with kanamycin, and confirmed by PCR and Northern blot. RESULTS: PCR result showed stable integration of the 3G1MH gene IN Arabidopsis thaliana genome in 7 stains of the transformed plants. Northern blot analyses confirmed the transcription of heavy and light chains in the transgenic plants. CONCLUSION: The successful establishment of 3G1MH transgenic Arabidopsis thaliana plants pave the way for further research on expressing therapeutic antibody in transgenic plants.

Short hairpin RNA targeting survivin inhibits growth and angiogenesis of glioma U251 cells.

Survivin is a novel tumor-associated gene, its overexpression mostly associates with carcinogenesis and development. Nevertheless, the precise role of survivin in initiation and progression of gliomas is still not completely clear. We constructed here three short hairpin RNA (shRNA) targeting survivin plasmid vectors and introduced them into glioma U251 cells. The three shRNAs were efficiently and specifically able to knockdown the survivin expression in transiently transfected U251 cells. The stable transfectants expressing the shRNA having the strongest inhibitory effect against survivin exhibited decreased cell growth, increased spontaneous apoptosis, mitotic catastrophe and cell cycle arrest. Furthermore, in nude mice xenografts, the stable transfectants presented decreased de novo glioma formation and reduced development of angiogenesis. Results from this study indicate that survivin plays an important role in malignant proliferation, antiapoptosis and angiogenesis of gliomas, which may become an attractive target for gene therapy of gliomas, while RNA interference (RNAi) mediated by shRNA may become a new promising strategy for cancer gene therapy.

The expression and genetic immunization of chimeric fragment of Hantaan virus M and S segments.

Hemorrhagic fever with renal syndrome (HFRS), which is characterized by severe symptoms and high mortality, is caused by hantavirus. There are still no effective prophylactic vaccines directed to HFRS until now. In this research, we fused expressed G2 fragment of M segment and 0.7kb fragment of S segment. We expect it could be a candidate vaccine. Chimeric gene G2S0.7 was first expressed in prokaryotic expression system pGEX-4T. After inducing expressed fusion proteins, GST-G2S0.7 was induced and its molecular weight was about 100kDa. Meanwhile, the fusion protein kept the activity of its parental proteins. Further, BALB/c mice were vaccinated by the chimeric gene. ELISA, cell microculture neutralization test in vitro were used to detect the humoral immune response in immunized BALB/c mice. Lymphocyte proliferation assay was used to detect the cellular immune response. The results showed that the chimeric gene could simultaneously evoke specific antibody against nucleocapsid protein (NP) and glycoprotein (GP). And the immunized mice of every group elicited neutralizing antibodies with different titers. But the titers were low. Lymphocyte proliferation assay results showed that the stimulation indexes of splenocytes of chimeric gene to NP and GP were significantly higher than that of control. It suggested that the chimeric gene of Hantaan virus containing G2 fragment of M segment and 0.7kb fragment of S segment could directly elicit specific anti-Hantaan virus humoral and cellular immune response in BALB/c mice.

[The effect of HCV core protein on the expression of cyclooxygenase 2 (COX-2) in HepG2 cells].

AIM: To investigate the effect of Hepatitis C virus (HCV) core protein on the expression of cyclooxygenase 2 (COX-2). METHODS: The genes encoded HCV core protein were amplified from plasmid containing full length genome of HCV strain H77 using PCR, and were cloned into eukaryotic expression vector pcDNA3.1. The recombinant HCV-C/pcDNA3.1 was transiently co-transfected into HepG2 cells with luciferase reporter vector containing COX-2 promotor (COX2pro1.5 kb/luc). The luciferase activity and COX-2 protein expression were detected. RESULTS: The recombinants HCV-C/pcDNA3.1 have been constructed successfully. The luciferase activity of COX-2 promotor was activated by the expressed HCV core, and the increased protein expression of COX-2 in transfected HepG2 cells was detected by Western blot. CONCLUSION: HCV core protein can activate the COX-2 promotor and induce its expression, which provides a new experimental basis for further research on relationship between COX-2 and HCV pathogenesis.

[Expression and genetic immunization of hantaan virus G2 recombinant adenovirus].

AIM: To express hantaan virus(HTNV) envelope glycoprotein G(2) recombinant adenovirus(Adeno-G(2)) in vero E6 cells and explore its property of inducing immune response. METHODS: Vero E6 cells were infected with the HTNV Adeno-G(2) (100 MOI). The expression of Adeno-G(2) in the infected Vero E6 cells was detected by IFA. BALB/c mice were immunized with HTNV Adeno-G(2), then the immune response to Adeno-G(2) was tested by ELISA, microcell-culture neutralizing experiment and lymphocyte proliferation test (MTT colorimetry). RESULTS: IFA detection showed the expression of Adeno-G(2) in the infected Vero E6 cells. The titer of specific antibody was 1:40; The low-titer neutralization antibody was also detected. But the lymphocyte proliferation reaction was not notable. CONCLUSION: The HTNV Adeno-G(2) can stimulate BALB/c mice to develop specific humoral immune response instead of specific cell-mediated immunity. This study provides the experimental basis for the development of gene engineering vaccine of HFRS.

[Transient expression and characterization of intracellular single chain Fv against the nucleocapsid protein of Hantavirus].

AIM: To transiently express an intracellular single chain Fv of monoclonal antibody 1A8 against nucleocapsid protein of Hantavirus and characterize the immunological activities of the expressed products. METHODS: COS-7 cells were transfected with mammalian expression vector 1A8-scFv-Ckappa/pCI-neo via lipofectin. The expressed product was identified by indirect immunofluorescence and immunoprecipitation. RESULTS: A diffuse pattern fluorescence was observed in less than 1% cytoplasm of transfected COS-7 cells. The binding of intracellular antibody fragments to NP antigen was confirmed by immunoprecipitation analysis. CONCLUSION: Transiently expressed single chain intrabodies can effectively target NP antigen in the cytoplasm. The present study may provide a new approach for treatment of Hantavirus.

[Expression of amino terminal of nucleoprotein and glycoprotein G2 of Hantaan virus in insect cells in the form of fusion protein].

AIM: To express the glycoprotein G2 and amino terminal of nucleoprotein (NP) of Hantaan virus in Bac-to-Bac baculovirus expression system in the form of fusion protein. METHODS: The recombinant baculovirus expression vector pFBDHTa-G2S 0.7 was constructed. The chimeric gene was inserted into bacmid in E.coli DH10Bac with the help of Tn7 transposition system. Then the recombinant baculovirus was screened and the fusion protein was expressed in insect cells. The expression product was detected by ELISA, immunofluorescence assay and Western blot analysis. RESULTS: The recombinant baculovirus containing the chimeric gene G2S 0.7 had been constructed successfully and the fusion protein could be expressed in insect cells. The expressed protein could be recognized by the Hantaan virus NP-specific mAb and glycoprotein G2-specific mAb. CONCLUSION: The successful expression of fusion protein G2S 0.7 with biological activity in insect cells lays the foundation for further research on its immunological characteristic.