Subsurface hydrological connectivity controls nitrate export flux in a hilly catchment.

Subsurface runoff represents the main pathway of nitrate transport in hilly catchments. The magnitude of nitrate export from a source area is closely related to subsurface hydrological connectivity, which refers to the linkage of separate regions of a catchment via subsurface runoff. However, understanding of how subsurface hydrological connectivity regulates catchment nitrate export remains insufficient. This study conducted high-frequency monitoring of shallow groundwater in a hilly catchment over 17 months. Subsurface hydrological connectivity of the catchment over 38 rainfall events was analyzed by combining topography-based upscaling of shallow groundwater and graph theory. Moreover, cross-correlation analysis was used to evaluate the time-series similarity between subsurface hydrological connectivity and nitrate flux during rainfall events. The results showed that the maximum subsurface hydrological connectivity during 32 out of 38 rainfall events was below 0.5. Although subsurface flow paths (i.e., the pathways of lateral subsurface runoff) exhibited clear dynamic extension and contraction during rainfall events, most areas in the catchment did not establish subsurface hydrological connectivity with the stream. The primary pattern of nitrate export was flushing (44.7%), followed by dilution (34.2%), and chemostatic behavior (21.1%). A threshold relationship between subsurface hydrological connectivity and nitrate flux was identified, with nitrate flux rapidly increasing after the subsurface connectivity strength exceeded 0.121. Moreover, the median value of cross-correlation coefficients reached 0.67, which indicated subsurface hydrological connectivity exerts a strong control on nitrate flux. However, this control effect is not constant and it increases with rainfall amount and intensity as a power function. The results of this study provide comprehensive insights into the subsurface hydrological control of catchment nitrate export.

[Reversal mechanism of hepatic fibrosis].

There is an increasing histological clinical evidence that both hepatic fibrosis and some degree of cirrhosis reversal can improve prognosis. Hepatic fibrosis involves a variety of cells and steps, and its reversal mechanism is also very complex, mainly including the reduction of hepatocyte necrosis and regeneration, the apoptosis and inactivation of activated hepatic stellate cells, and the reversal of hepatic sinusoidal endothelial cells and microvessels, restorative hepatic macrophages polarization and cell-to-cell interactions. Furthermore, the biochemical basis for reversal of hepatic fibrosis is decreased expression of matrix metalloproteinase inhibitors, up-regulation of matrix metalloproteinase activity, and increased degradation of extracellular matrix. However, at present, there are few studies on the clinicopathological mechanism of liver fibrosis reversal, and the key target groups of different etiologies with different degrees are still unclear, and the corresponding translational application research is lacking. Therefore, an in-depth and systematic understanding of the characteristics and mechanisms of hepatic fibrosis reversal can not only enrich the understanding of the natural history of hepatic fibrosis and cirrhosis, but also provide reference for the development and clinical application of anti-hepatic fibrotic drugs.

[The relationship between elevated antiphospholipid antibodies and thrombosis in hospitalized patients].

Objective: Assess the relationship between elevated antiphospholipid antibodies and thrombosis in hospitalized patients. Methods: Case control study. A total of 385 patients (149 males and 236 females, aged from 1 to 105 years, with a median age of 37 years) who were hospitalized in Peking University First Hospital from January 2015 to December 2019 and tested positive for any one of the anti-phospholipid antibodies were included in the study. All subjects were divided into thrombotic group and non-thrombotic group according to whether thrombus was detected by imaging examination during hospitalization. In thrombosis group, there were 66 males and 36 females, aged from 3 to 105 years, with a median age of 58 years. In non-thrombosis group, there were 83 males and 200 females, aged from 1 to 94 years, with a median age of 31 years. Clinical data and laboratory data of patients were recorded. ACL-IgM/IgG and anti-ß2GPI-IgM/IgG were detected by ELISA and LA was detected by dRVVT and SCT on automatic coagulation analyzer. The rates of age, gender, smoking, obesity, hypertension, hyperlipidemia, diabetes and the median level of antiphospholipid antibodies were compared between two groups. Logistic multivariate regression analysis was used to determine the risk factors for thrombotic events. The mid-to-high titer value of aCL was established by the χ2-trend test and verified by logistic regression. Results: The median age (58 years) and the rates of male (64.7%), smoking (16.7%), hypertension (63.7%) and diabetes (28.4%) in thrombus group were significantly higher than those in non-thrombus group (Z=7.685, χ²=38.077, 16.312, 37.769, 24.749 respectively; P<0.01). The positive rate of anti-ß2GPI-IgG and dRVVT in thrombosis group (11.8% and 78.4%) was significantly higher than that in non-thrombosis group (5.3% and 60.1%), as well as the median level of dRVVT (1.29 RU/ml vs 1.23 RU/ml) (χ²=3.864 and 10.309, Z=3.539; P<0.05). The median level of aCL-IgM was higher in non-thrombosis group (2.3 MPL vs 2.0 MPL). The positive rate of aCL-IgG was slightly higher in thrombosis group (18.6% vs 10.6%). Logistic regression analysis showed that men, hypertension, diabetes, advanced age, elevated dRVVT, and elevated anti-ß2GPI-IgG are risk factors for thrombosis. Taking 36 GPL as the medium-to-high titer value of aCL-IgG, the risk of thrombosis increased by 2.45 times. Conclusions: In the anti-phospholipid antibody profile, LA detected by dRVVT method, anti-ß2GPI-IgG and aCL-IgG may be valuable laboratory indicators for inpatient thrombotic events. The mid-to-high titer value of aCL-IgG is set at 36 GPL to distinguish the risk of thrombosis.

LINC00657 promotes malignant progression of oral squamous cell carcinoma via regulating microRNA-150.

OBJECTIVE: Previous studies have shown that LINC00657 is a cancer-promoting gene. However, the role of LINC00657 in oral squamous cell carcinoma (OSCC) has not been reported. This study was designed to investigate the role of LINC00657 in OSCC and its regulatory mechanism. PATIENTS AND METHODS: Quantitative Real Time-Polymerase Chain Reaction (qPCR) was used to detect the levels of LINC00657 and microRNA-150 in 32 pairs of OSCC tissues and normal ones, and the correlation between LINC00657 and clinical indicators and OSCC patient's prognosis was analyzed. qRT-PCR further verified the levels of LINC00657 and microRNA-150 in OSCC cells. In addition, LINC00657 overexpression and knockdown models were constructed using lentivirus in OSCC cell lines Fadu and Tca8113, and Cell Counting Kit-8 (CCK-8), plate clone experiment, and 5-Ethynyl-2'-deoxyuridine (EdU) assay were carried out to evaluate the influence of LINC00657 on the biological functions of OSCC cells. Further, Luciferase reporter gene and recovery experiments were used to explore its potential mechanism. RESULTS: qRT-PCR showed that LINC00657 expression in OSCC tissue specimens was increased in comparison to normal ones. Patients with high LINC00657 expression had higher pathological staging and lower overall survival. Besides, the cell proliferation ability of the LINC00657 silencing group was remarkably decreased, while the opposite result was observed in LINC00657 overexpression group. Subsequently, qRT-PCR demonstrated a significant decrease in microRNA-150 expression in OSCC cell lines and tissues and a negative correlation with LINC00657. Luciferase assay demonstrated that LINC00657 could be targeted by microRNA-150 in certain binding sites. In addition, cell reverse experiment also confirmed that LINC00657 and microRNA-150 can be mutually regulated, thereby jointly modulating the malignant progression of OSCC. CONCLUSIONS: LINC00657, remarkably upregulated in OSCC tissues, showed a close association with the poor prognosis of OSCC patients. Additionally, it may accelerate the malignant progression of OSCC via regulating microRNA-150.

[Clinical observation of autologous simple limbal epithelial transplantation for unilateral limbal stem cell deficiency].

Objective: To discuss effect of autologous simple limbal epithelial transplantation (SLET) performed for unilateral limbal stem cell deficiency (LSCD). Methods: Retrospective case study. In this retrospective study, records of 7 patients (7 eyes) who had undergone autologous SLET for unilateral LSCD, with a minimum of 6 months of follow-up, were reviewed. Demographic details, etiology of LSCD, duration between ocular burn and SLET, prior surgery performed, presence or absence of symblepharon, pre-and post-operative visual acuity, and complications were noted. Results: Seven eyes of 7 patients underwent autologous SLET. With a follow-up of 6 months, a completely epithelialised and stable corneal surface was obtained in all recipient eyes. Visual acuity improved in all patients, while none of the eyes developed any complications. Conclusions: Autologous SLET is an effective and safe modality for treatment of unilateral LSCD. Clinical success rates and visual acuity improvement are equal to or better than those reported with earlier techniques. (Chin J Ophthalmol, 2019, 55:923-927).

Adaptive velocity-dependent proportional-integral controller for high-speed atomic force microscopy.

High-speed atomic force microscopy (AFM) has been rapidly developed in recent years. To reduce the oscillation of the scanner, a single-tone sinusoidal wave is widely used as a scanning wave rather than a triangular wave in high-speed AFM. However, the sinusoidal wave is nonlinear, resulting in a nonconstant relative linear velocity between the sample and the tip while scanning in the x-direction. If a traditional proportional-integral controller is still used as a feedback controller in the z-direction, the control errors will be enormous. Therefore, the paper proposes a new adaptive velocity-dependent proportional-integral controller. The relationship between the proportional-integral parameters and the linear velocity is achieved by fitting the experimental results. The adaptive and traditional controllers are compared against each other in some examples. The experiments demonstrate that the adaptive controller decreases the control errors in the z-direction to a half, which provides more precise AFM images. LAY DESCRIPTION: Typically, the scanner follows a triangular waveform in fast axis (x-axis), and follows a very slow ramp signal in the slow axis (y-axis) of conventional AFM. This scanning mode can be called raster scan. However, the triangular waveform contains high-order Fourier harmonics, vibrating the scanner and distorting the image easily. In high-speed AFM, the effect of the high-order Fourier harmonics will be more severe. The above problems can be solved by replacing triangular waves with single-tone sinusoidal waveform. Therefore, the sinusoidal-raster scan and nonraster scan based on the sinusoidal waveform are widely used in high-speed atomic force microscopy. However, the nonlinearly scan path will cause a variable relative linear velocity between the sample and the tip. If a standard proportional-integral controller is still used as a feedback controller in Z direction, the control errors will be large, and this difference will be evident at high-speed scanning. Thus, the paper proposes a new adaptive velocity-dependent proportional-integral controller to solve this problem. Experiments show that the control errors obtained by using the adaptive controller is about a half of that without using it. These illustrate that the proposed method can improve the image quality of the AFM at both low and high scan speeds.

[An study on the incidence of heat stroke and explore it's prediction model in Pudong New Area of Shanghai from 2013-2017].

Objective: To know the incidence of heat stroke and explore it's prediction model in Pudong New Area of Shanghai. Methods: An epidemiological investigation was conducted on heat stroke cases in Pudong New Area of Shanghai from 2013 to 2017. Daily temperature data during this period were collected to explore it's influence. Results: 246 heat stroke cases were reported and investigated, 70.3% (173/246) of them were male. 170 cases are severe heat stroke, accounting for 69.1%. 28 patients died, accounting for 11.4% of all cases of heat stroke, and 16.5% (28/170) of severe heat stroke cases. Thermoplegia (56.5%, 96/170) was the most popular type among severe heat stroke cases. Heat prostration, heat cramps and mixed type account for 17.1% (29/170) , 12.4% (21/170) and 14.0% (24/170) respectively. Scatter plot and linear regression demonstrated that there was a significant linear relation between number of high temperature days and number of heat stroke cases (P<0.01) . And the prediction model is: Predictive number of annual heat stroke cases=ß×Number of annual high temperature days+Intercept. Leave-one-out cross validation result shows that the predictive number of annual heat stroke cases from 2013 to 2017 were 85.7%, 90.9%, 83.3%, 91.9 and 84.3% respectively. Conclusion: There was a significant linear relation between number of high temperature days and number of heat stroke cases in Pudong New Area. The related work arrangement for heat stroke prevention could be well planed according to the prediction model.

Improvement of wound healing by regulated oxygen-enriched negative pressure-assisted wound therapy in a rabbit model.

BACKGROUND: Development of drug therapies and other techniques for wound care have resulted in significant improvement of the cure rate and shortening of the healing time for wounds. A modified technique of regulated oxygen-enriched negative pressure-assisted wound therapy (RO-NPT) has been reported. AIM: To evaluate the efficacy and impact of RO-NPT on wound recovery and inflammation. METHODS: Infected wounds were established on 40 adult female white rabbits, which were then randomized to one of four groups: O2 group, regulated negative pressure-assisted wound therapy (RNPT) group, regulated oxygen-enriched negative pressure-assisted wound therapy (RO-NPT) group and healthy control (HC) group. Each day, the O2 group was treated with a constant oxygen supply (1 L/min) to the wound, while the RNPT group was treated with continuous regulated negative pressure (70 ± 5 mmHg) and the RNPT + O2 group was treated with both. The HC group was treated with gauze dressing alone, which was changed every day. Leucocyte count, colony count and wound-healing rate were calculated. Levels of tumour necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-8 were evaluated by ELISA. RESULTS: RO-RNPT significantly decreased bacterial count and TNF-α level, and increased the wound-healing rate. IL-1ß, IL-8 and leucocyte count had a tendency to increase in the early phase of inflammation and a tendency to decrease in the later phase of inflammation in the RO-RNPT group. CONCLUSIONS: RO-NPT therapy assisted wound recovery and inflammation control compared with the RNPT and oxygen-enriched therapies. RO-NPT therapy also increased levels of IL-1ß and IL-8 and attenuated expression of TNF-α in the early phase of inflammation.

[Textual research on P. t.1061, a Tibetan veterinary medical scroll fragment of Dunhuang literature preserved in France].

P. t.1061 preserved at the Manuscripts Department of the Bibiothèque Nationle de France, is a Dunhuang Tibetan veterinary medical manuscript that focuses on the surgical therapy of equine rhinopathy. The method of blowing a small quantity of ammonium chloride through a bamboo tube to treat horse nasal sinus diseases was described in the original scroll; if not effective, burn with a proper cauterization apparatus; if still ineffective, prick with a fine bloodletting needle; if still not effective, operate frontal trephination. The frontal trephination documented in P. t.1061 is by far the earliest record in Tibetan language of relative operation so far discovered.

Inhibition of α5 subunit-containing GABAA receptors facilitated spinal nociceptive transmission and plasticity.

BACKGROUND: γ-Aminobutyric acid (GABA) type A receptors (GABAA Rs) locate at both synaptic and extrasynaptic membrane, which generate phasic and tonic inhibition, respectively. In spinal cord dorsal horn, the phasic inhibition produced by transient activation of synaptic GABAA Rs plays an important role in the gating control over nociceptive conveyance. Although extrasynaptic GABAA Rs that contain α5 subunits (α5-GABAA Rs) are also detectable in spinal dorsal horn, much less is known about the function of these receptors. METHODS: The C fibre-evoked field potentials were recorded in superficial dorsal horn of spinal cord, and the effects of α5-GABAA R inverse agonist L-655708 on basal synaptic transmission and long-term potentiation (LTP) of C-fibre responses were examined. The possible changes of glutamate receptor function and pain sensitivity after α5-GABAA R inhibition were investigated by western blot and behavioural tests. RESULTS: Inhibition of α5-GABAA Rs by L-655708 boosted the basal synaptic transmission and facilitated the induction of N-methyl-d-aspartate subtype glutamate receptors (NMDARs)-dependent LTP. L-655708 was found to enhance the phosphorylation and synaptic accumulation of NMDARs and α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid receptors (AMPARs). Intrathecal L-655708 injection also decreased the pain thresholds of intact mice in a dose-dependent manner. CONCLUSIONS: α5-GABAA Rs were critical for the tonic inhibition of glutamatergic neurotransmission and plasticity in spinal dorsal horn. SIGNIFICANCE: Tonic inhibition generated by α5-GABAA Rs is important for information processing. However, whether and how α5-GABAA Rs regulate the conveyance of nociceptive signals in spinal cord is largely unknown. Here, we revealed a negative control by α5-GABAA Rs over nociceptive transmission and plasticity.

Noradrenergic α2 receptor attenuated inflammatory pain through STEP61/ERK signalling.

BACKGROUND: Activation of noradrenergic α2 receptor in spinal dorsal horn effectively alleviates the pathological pain. However, the precise mechanisms underlying noradrenergic pain suppression are not fully understood. Convincing evidence has indicated that extracellular signal-regulated kinases 1 and 2 (ERK1/2) play a key role in spinal sensitization. The present study investigated the potential influence of noradrenergic α2 receptor agonist clonidine on ERK1/2 activity. METHOD: Clonidine was intrathecally given after intraplantar injection of complete Freund's adjuvant (CFA) in mice. The possible changes of ERK1/2 signalling were detected by Western blot, immunohistochemistry, co-immunoprecipitation and behavioural tests. RESULTS: CFA significantly enhanced ERK1/2 activity in spinal dorsal horn, which was, however, greatly attenuated by clonidine application. Pretreatment with pertussis toxin abolished the inhibitory effect of clonidine on ERK1/2, suggesting the involvement of Giα subunit (Gi protein). Noradrenergic α2 receptor/Gi protein might repress ERK1/2 through cAMP-dependent protein kinase (PKA) pathway, because direct ERK1/2 activation by PKA agonist forskolin was also suppressed by clonidine. We found that 61 kD isoform of striatal-enriched protein phosphatase (STEP61) was a key intermediary for α2 receptor/Gi protein/PKA signalling to manipulate ERK1/2 activity. By reducing PKA-mediated phosphorylation of STEP61 at Ser221, clonidine significantly resumed the inhibition conferred by STEP61 on ERK1/2. Direct expression of STEP61 mutant devoid of Ser221 phosphorylation mimicked clonidine by inhibiting ERK1/2 and pain sensitization in CFA-injected mice. CONCLUSION: The analgesic action produced by noradrenergic α2 receptor agonist clonidine involved the reversal of ERK1/2 hyperactivity in spinal dorsal horn of inflamed mice.

Mitogen-activated protein kinase signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor-mediated amyloid-ß uptake in SH-SY5Y cells.

Intraneuronal accumulation of beta-amyloid protein (Aß) is an early pathological change in Alzheimer's disease (AD). Recent studies demonstrate that α7 nicotinic acetylcholine receptor (α7nAChR) binds to soluble Aß with a high affinity. In vitro and in vivo experiments also show that Aß activates p38 MAPK and ERK1/2 signaling pathways via the α7nAChR. Interestingly, it has been reported that p38 MAPK and ERK1/2 signaling pathways affect the regulation of receptor-mediated endocytosis. These data suggest that MAPK signaling pathways maybe involved in the regulation of α7nAChR-mediated Aß uptake. However, the evidence for this hypothesis is lacking. In the present study, we examined whether Aß1-42 oligomers activate MAPK signaling pathways via α7nAChR, and assessed the role of MAPK signaling pathways in the regulation of Aß1-42 uptake by α7nAChR. We confirm that undifferentiated SH-SY5Y cells are capable of taking up extracellular Aß1-42. The internalization of Aß1-42 accumulates in the endosomes/lysosomes and mitochondria. MAPK signaling pathways are activated by Aß1-42 via α7nAChR. Aß1-42 and α7nAChR are co-localized in SH-SY5Y cells and the expression of α7nAChR involves in Aß1-42 uptake and accumulation in SH-SY5Y cells. Our data demonstrate that Aß1-42 induces an α7nAChR-dependent pathway that relates to the activation of p38 MAPK and ERK1/2, resulting in internalization of Aß1-42. Our findings suggest that α7nAChR and MAPK signaling pathways play an important role in the uptake and accumulation of Aß1-42 in SH-SY5Y cells. Blockade of α7nAChR may have a beneficial effect by limiting intracellular accumulation of amyloid in AD brain and serves a potential therapeutic target for AD.

Fyn kinase-regulated NMDA receptor- and AMPA receptor-dependent pain sensitization in spinal dorsal horn of mice.

BACKGROUND: Inhibition of Src-family protein tyrosine kinases (SFKs) in spinal dorsal horn has been established as an effective strategy for the alleviation of chronic pathological pain. As one of the important SFKs members, Fyn kinase is critical for synaptic plasticity and many pathophysiological processes. However, whether Fyn is involved in spinal sensitization is far from being elucidated. METHOD: We manipulated Fyn activity by expressing a constitutively active Fyn mutant [Fyn(Y528F) ] or a catalytically null mutant [Fyn(K296M) ] in the spinal dorsal horn of mice, and performed behavioural and biochemical experiments to investigate the role of Fyn in regulating the nociceptive responses and the synaptic expression of ionotropic glutamate receptors. RESULTS: Spinal expression of Fyn(Y528F) alone in intact mice was sufficient to elicit persistent mechanical allodynia and thermal hyperalgesia, which lasted for at least 12 days. Fyn(Y528F) simultaneously enhanced the concentrations of N-methyl-D-aspartic acid (NMDA)-subtype and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-subtype glutamate receptors at synaptosomal membrane fraction. Pharmacological inhibition of NMDA receptors or AMPA receptors greatly alleviated Fyn(Y528F)-induced pain hypersensitivity. To evaluate the contribution of Fyn to inflammatory pain, we expressed Fyn(K296M) before intradermal injection of complete Freund's adjuvant (CFA), finding that Fyn(K296M) had no effect on the induction of inflammatory pain within 3 h post-CFA injection, which, however, repressed the synaptic accumulation of NMDA receptors and AMPA receptors to attenuate the maintenance of chronic pain states. CONCLUSION: Fyn played a key role in the sustained sensitization of nociceptive behaviours by up-regulating the functions of ionotropic glutamate receptors in spinal dorsal horn.

Expression of hypoxia inducible factor-1α and its significance in laryngeal carcinoma.

This study examined levels of hypoxia inducible factor-1α (HIF-1α) protein in 40 laryngeal squamous cell carcinoma specimens using immunohistochemistry. Correlations between HIF-1α immunoreactivity and patient age, tumour lymph node metastasis stage, histological grade (extent of differentiation), and alcohol and smoking history were evaluated. Of the tumour tissues obtained, 35 (87.5%) were located in the glottic area and five (12.5%) in the supraglottic area. All patients were male and aged between 35 and 71 years; 12 (30.0%) presented with lymph node metastases, 24 (60.0%) had cancer classified as T(1) or T(2), and 16 (40.0%) as high clinical stage (T(3) or T(4)). The pattern of HIF-1α protein localization in tumour tissues, when present, was mixed nuclear/cytoplasmic, with positive HIF-1α expression in 27 patients (67.5%). Differences in HIF-1α levels in samples from different tumour stages and in those with lymph node-positive versus lymph node-negative cancers were statistically significant.

Vasoactive intestinal peptide in rats with focal cerebral ischemia enhances angiogenesis.

We studied the effect of vasoactive intestinal peptide (VIP) on angiogenesis in the ischemic boundary area after focal cerebral ischemia. Adult male Sprague-Dawley rats underwent middle cerebral artery occlusion for 2 h. A single dose of VIP was given via i.c.v. injection at the beginning of reperfusion. Immunohistochemistry and Western blotting were performed to assay angiogenesis and brain levels of vascular endothelial growth factor (VEGF) protein, respectively. In addition, the expression of VEGF and its receptors (flt-1 and flk-1), as well as endothelial proliferation, was measured using rat brain microvascular endothelial cells. Immunohistochemical analyses revealed significant (P<0.05) increases in the numbers of bromodeoxyuridine (BrdU) positive endothelial cells and microvessels at the boundary of the ischemic lesion in rats treated with VIP compared with rats treated with saline. Western blotting analysis showed that treatment with VIP significantly (P<0.05) raised VEGF levels in the ischemic hemisphere. In addition, treatment with VIP increased flt-1 and flk-1 immunoreactivity in endothelial cells. In vitro, incubation with VIP significantly (P<0.01) increased the proliferation of endothelial cells and induced the expression of VEGF, flt-1 and flk-1 in endothelial cells. The stimulatory effect of VIP on the proliferation of endothelial cells was significantly (P<0.01) inhibited by SU5416, a selective inhibitor of VEGF receptor tyrosine kinase. Our data suggest that treatment with VIP enhances angiogenesis in the ischemic brain, and this effect may be mediated by increases in levels of VEGF and its receptors.

Efficient tuberculosis treatment in mice using chemotherapy and immunotherapy with the combined DNA vaccine encoding Ag85B, MPT-64 and MPT-83.

Although most cases of tuberculosis (TB) can be cured with antibiotics, relapse is common if patients do not continue chemotherapy for at least 6 months. Thus, improved therapeutic strategies are urgently needed. We previously found that the combined DNA vaccine encoding the Mycobacterium tuberculosis proteins Ag85B, MPT-64 and MPT-83 protected mice from TB following H37Rv challenge and considered whether this combined DNA vaccine has a therapeutic effect. In the present work, we demonstrate that boosting the efficiency of the immune system with the combined DNA vaccine may be a valuable adjunct to shorten the duration of antibacterial chemotherapy. Mice treated with the combined DNA vaccine along with isoniazid and pyrazinamide showed significantly higher interferon-gamma responses to a mixture of the three specific antigens (P<0.001), which were accompanied by a significant reduction in colony-forming unit in H37Rv-infected animals 3-5 months after treatment (P<0.001). These results suggest that the combined DNA vaccine along with conventional TB chemotherapy has strong potential for TB immunotherapy and may provide new alternatives to control the disease.

A combined DNA vaccine-prime, BCG-boost strategy results in better protection against Mycobacterium bovis challenge.

In this study, we demonstrated that calves vaccinated with a combined DNA vaccine encoding Ag85B, MPT- 64, and MPT-83 antigens from the Mycobacterium tuberculosis for the priming and subsequently boosting with BCG prior to experimental challenge with virulent Mycobacterium bovis (M. bovis) resulted in improved immune responses over immunizing. Vaccination with the combined DNA/BCG induced higher levels of antigen- specific gamma interferon (IFN-gamma) in whole-blood cultures 4 weeks after final vaccination and the level of antigen-specific IFN-gamma in response to Ag85, MPT-64, and MPT-83 were still higher 4 weeks after challenge when compared to the combined DNA group. There was a significant bias toward induction of CD4+ T cells rather than CD8+ T cells responses, and the mean percentage of CD4+ T cells was increased about 2.6-fold in peripheral blood mononuclear cells (PBMC) cultures in DNA prime-BCG boost vaccination when compared to the nonvaccinated group. In addition, DNA prime-BCG boost vaccination resulted in stronger humoral immune responses, and the levels of the specific antibodies to three antigens were increased two- to 32- fold when compared to the combined DNA group. Vaccination with the combined DNA/BCG induced a high level of protection against an intratracheal challenge with virulent M. bovis, based on a significant enhancement of six pathological and microbiological parameters of protection compared to the nonvaccinated group. Finally, the combined DNA/BCG increased the protective efficacy by more than 10-100-fold as measured by reduced CFU counts in the lungs from calves challenged with M. bovis compared to the combined DNA and BCG groups. These results suggest that use of the prime-boost strategy offers better protection against bovine tuberculosis than does the combined DNA vaccines and BCG.

The role of XPB in cell apoptosis and viability and its relationship with p53, p21(waf1/cip1) and c-myc in hepatoma cells.

BACKGROUND: Accumulation of DNA damage has been implicated in hepatocarcinogenesis. XPB plays a pivotal part in repairing damaged DNA. However, up to now, the biological effect of XPB on hepatoma cells remains elusive. MATERIALS AND METHODS: Here, we investigated the role of XPB in the apoptosis and the viability of hepatoma cells by using the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labelling and cell viability assay; we also investigated their relationship with p53, p21(waf1/cip1) and c-myc by using the RT-PCR and Western blot. RESULTS: Compared with the control cells HepG2/pcDNA3.1 or HepG2, XPB-transfected HepG2 cells (HepG2/pcDNA3.1-XPB) displayed lower viability, weaker activity and higher apoptosis index. At the same time, an increased expression of p21(waf1/cip1) mRNA, protein and p53 protein in addition to a decreased expression of c-myc mRNA and protein were detected in HepG2/pcDNA3.1-XPB cells. CONCLUSIONS: Our results indicated that XPB could inhibit the proliferation of hepatoma cells and had a positive effect on the expression of p53 and p21(waf1/cip1) but a negative effect on c-myc.

Combined DNA vaccines formulated either in DDA or in saline protect cattle from Mycobacterium bovis infection.

We tested the effectiveness of combined DNA vaccines encoding antigens Ag85B, MPT64 and MPT83 from Mycobacterium tuberculosis on cattle. Our results showed that calves treated with combined DNA vaccines in the presence of dimethyldioctyldecyl ammonium bromide (DDA) or saline elicited a strong gamma interferon (IFN-gamma) response 1 or 2 months after the third vaccination. All three antigens induced substantial levels of IFN-gamma production 1 month after the bacterial challenge, when the BCG-driven IFN-gamma levels dropped to less than one third of their peak values. Animals receiving combined DNA vaccines produced highest amounts of IgG antibody titer 2 months after the third vaccination. Steady state low IgG levels were found 2 months after bacterial challenge. A few small lung and lymph node lesions were detected in 1/5 animals treated with combined DNA vaccines, whereas 3/5 of BCG-treated and 5/5 of vector-control calves showed larger and significantly more lesions. About 70- to 100-fold fewer bacteria were found in the lungs and lymph nodes of combined DNA vaccine-treated animals compared to those of the control group. Histopathological analyses showed that vaccinated calves possessed substantially improved post-infection lung and lymph node pathology relative to the controls. Our data indicate that combined DNA vaccines may be used in cattle to combat bovine tuberculosis.