TXNIP Regulates NLRP3 Inflammasome-Induced Pyroptosis Related to Aging via cAMP/PKA and PI3K/Akt Signaling Pathways.

Aging is an inevitable natural process with time-dependent dysfunction and the occurrence of various diseases, which impose heavy burdens on individuals, families, and society. It has been reported that NLRP3 inflammasome-induced pyroptosis contributes significantly to age-related diseases and aging, while TXNIP is suggested to be involved in regulating pyroptosis mediated by NLRP3. However, the mechanism between TXNIP and NLRP3 inflammasome is still unclear. In this study, we used HT-22 cells to explore the effect of TXNIP on pyroptosis and its potential association with the aging. Also, we delved into the underlying mechanisms. Our findings revealed that TXNIP significantly augmented pyroptosis in HT-22 cells, primarily by enhancing the activation of the NLRP3 inflammasome and promoting the release of proinflammatory cytokines. Remarkably, as TXNIP levels increased, we observed a corresponding rise in the number of p16-positive cells, which is indicative of aging. Furthermore, we conducted experiments to modulate the improvement of TXNIP on NLRP3 inflammasome-induced pyroptosis, that is, the PI3K activator 740 Y-P and the PKA activator DC2797 inhibited the effect, while the PI3K inhibitor LY294002 and the PKA inhibitor H89 enhanced the effect. In conclusion, our study demonstrated that TXNIP regulates NLRP3 inflammasome-induced pyroptosis in HT-22 cells related to aging via the PI3K/Akt and cAMP/PKA pathways.

Cognitive-exercise dual-task promotes cognitive function recovery in chronic cerebral ischemia male rats through regulating PI3K/Akt signaling pathway via inhibition of EphrinA3/EphA4.

Chronic cerebral ischemia (CCI) can lead to vascular cognitive impairment, but therapeutic options are limited. Cognitive-exercise dual-task (CEDT), as a potential rehabilitation intervention, can attenuate cognitive impairment. However, the related mechanisms remain unclear. In this study, 2-vessel occlusion (2-VO) in male SD rats was performed to establish the CCI model. The rats were treated with cognitive, exercise, or CEDT intervention for 21 days. The Morris water maze (MWM) test was used to assess cognitive ability. TUNEL staining was used to detect the neuronal apoptosis. Immunofluorescence, RT-qPCR and Western blot were used to detect the protein or mRNA levels of EphrinA3, EphA4, p-PI3K, and p-Akt. The results showed that CEDT could improve performance in the MWM test, reverse the increased expression of EphrinA3 and EphA4, and the reduced expression of p-PI3K and p-Akt in CCI rats, which was superior to exercise and cognitive interventions. In vitro, oxygenglucose deprivation (OGD) challenge of astrocytes and neuronal cells were used to mimic cerebral ischemia. Immunofluorescence assay revealed that the levels of MAP-2, p-PI3K, and p-Akt were reduced in EphrinA3 overexpressed cells after OGD stimulation. Finally, the knock-down of EphrinA3 by shRNA significantly promoted the recovery of cognitive function and activation of PI3K/Akt after CEDT treatment in CCI rats. In conclusion, our study suggests that CEDT promotes cognitive function recovery after CCI by regulating the signaling axis of EphrinA3/EphA4/PI3K/Akt.

Cognitive-exercise dual-task intervention ameliorates cognitive decline in natural aging rats via inhibiting the promotion of LncRNA NEAT1/miR-124-3p on caveolin-1-PI3K/Akt/GSK3ß Pathway.

Aging-related cognitive impairment (ARCI) is rapidly becoming a healthcare priority. However, there is currently no excellent cure for it. Cognitive-exercise dual-task intervention (CEDI) is a promising method to improve ARCI, while the underlying mechanisms remain unclear. Long non-coding RNAs (lncRNAs) and microRNAs (miRNAs) are involved in the onset, development, and rehabilitation of ARCI. This study aimed to investigate the effects of CEDI and the role of regulation of the lncRNA NEAT1/miR-124-3p on the caveolin-1-PI3K/Akt/GSK3ß pathway in CEDI improving cognitive function. Forty 18-month-old natural aging rats were randomly assigned to four groups: exercise training group, cognitive training group, CEDI group, and aging control group, and underwent 12 weeks of intervention. A novel object recognition test was performed to determine the cognitive function, and the hippocampus was separated three days after the behavioral tests for further molecular detection. In an in vitro study, the mouse hippocampal neuronal cell line HT22 was cultured. MiR-124-3p and lncRNA NEAT1 were over-expressed or down-expressed, respectively. The expressions of related proteins, lncRNA, and miRNA were examined by WB and/or qRT-PCR. The results showed that compared with the aging control group, the CEDI group had a higher discrimination index, and significantly decreased the expressions of lncRNA NEAT1, and the protein expressions of caveolin-1 and p-GSK3ß, while significantly increased the expressions of miR-124-3p, and the protein expressions of p-PI3K and p-Akt. Inhibition of the lncRNA NEAT1 could significantly increase the protein expressions of p-PI3K and p-Akt in HT22 cells. Upregulation of miR-124-3p decreased the protein expressions of caveolin-1 and p-GSK3ß, and increased the protein expressions of p-PI3K and p-Akt significantly. Inhibition of miR-124-3p had the opposite effects. Our study demonstrated that CEDI improved cognitive function in aging rats better than a single intervention. The mechanisms of cognitive improvement could be related to the regulation of the lncRNA NEAT1/miR-124-3p on the caveolin-1-PI3K/Akt/GSK3ß pathway.

Cell circuits between leukemic cells and mesenchymal stem cells block lymphopoiesis by activating lymphotoxin beta receptor signaling.

Acute lymphoblastic and myeloblastic leukemias (ALL and AML) have been known to modify the bone marrow microenvironment and disrupt non-malignant hematopoiesis. However, the molecular mechanisms driving these alterations remain poorly defined. Using mouse models of ALL and AML, here we show that leukemic cells turn off lymphopoiesis and erythropoiesis shortly after colonizing the bone marrow. ALL and AML cells express lymphotoxin α1ß2 and activate lymphotoxin beta receptor (LTßR) signaling in mesenchymal stem cells (MSCs), which turns off IL7 production and prevents non-malignant lymphopoiesis. We show that the DNA damage response pathway and CXCR4 signaling promote lymphotoxin α1ß2 expression in leukemic cells. Genetic or pharmacological disruption of LTßR signaling in MSCs restores lymphopoiesis but not erythropoiesis, reduces leukemic cell growth, and significantly extends the survival of transplant recipients. Similarly, CXCR4 blocking also prevents leukemia-induced IL7 downregulation and inhibits leukemia growth. These studies demonstrate that acute leukemias exploit physiological mechanisms governing hematopoietic output as a strategy for gaining competitive advantage.

Dynamic phosphatase-recruitment controls B-cell selection and oncogenic signaling.

Initiation of B-cell receptor (BCR) 1 signaling, and subsequent antigen-encounter in germinal centers 2,3 represent milestones of B-lymphocyte development that are both marked by sharp increases of CD25 surface-expression. Oncogenic signaling in B-cell leukemia (B-ALL) 4 and lymphoma 5 also induced CD25-surface expression. While CD25 is known as an IL2-receptor chain on T- and NK-cells 6-9 , the significance of its expression on B-cells was unclear. Our experiments based on genetic mouse models and engineered patient-derived xenografts revealed that, rather than functioning as an IL2-receptor chain, CD25 expressed on B-cells assembled an inhibitory complex including PKCδ and SHIP1 and SHP1 phosphatases for feedback control of BCR-signaling or its oncogenic mimics. Recapitulating phenotypes of genetic ablation of PKCδ 10 - 12 , SHIP1 13,14 and SHP1 14, 15,16 , conditional CD25-deletion decimated early B-cell subsets but expanded mature B-cell populations and induced autoimmunity. In B-cell malignancies arising from early (B-ALL) and late (lymphoma) stages of B-cell development, CD25-loss induced cell death in the former and accelerated proliferation in the latter. Clinical outcome annotations mirrored opposite effects of CD25-deletion: high CD25 expression levels predicted poor clinical outcomes for patients with B-ALL, in contrast to favorable outcomes for lymphoma-patients. Biochemical and interactome studies revealed a critical role of CD25 in BCR-feedback regulation: BCR-signaling induced PKCδ-mediated phosphorylation of CD25 on its cytoplasmic tail (S 268 ). Genetic rescue experiments identified CD25-S 268 tail-phosphorylation as central structural requirement to recruit SHIP1 and SHP1 phosphatases to curb BCR-signaling. A single point mutation CD25 S268A abolished recruitment and activation of SHIP1 and SHP1 to limit duration and strength of BCR-signaling. Loss of phosphatase-function, autonomous BCR-signaling and Ca 2+ -oscillations induced anergy and negative selection during early B-cell development, as opposed to excessive proliferation and autoantibody production in mature B-cells. These findings highlight the previously unrecognized role of CD25 in assembling inhibitory phosphatases to control oncogenic signaling in B-cell malignancies and negative selection to prevent autoimmune disease.

Multimodal fusion and human-robot interaction control of an intelligent robot.

Introduction: Small-scaled robotic walkers play an increasingly important role in Activity of Daily Living (ADL) assistance in the face of ever-increasing rehab requirements and existing equipment drawbacks. This paper proposes a Rehabilitation Robotic Walker (RRW) for walking assistance and body weight support (BWS) during gait rehabilitation. Methods: The walker provides the patients with weight offloading and guiding force to mimic a series of the physiotherapist's (PT's) movements, and creates a natural, comfortable, and safe environment. This system consists of an omnidirectional mobile platform, a BWS mechanism, and a pelvic brace to smooth the motions of the pelvis. To recognize the human intentions, four force sensors, two joysticks, and one depth-sensing camera were used to monitor the human-machine information, and a multimodal fusion algorithm for intention recognition was proposed to improve the accuracy. Then the system obtained the heading angle E, the pelvic pose F, and the motion vector H via the camera, the force sensors, and the joysticks respectively, classified the intentions with feature extraction and information fusion, and finally outputted the motor speed control through the robot's kinematics. Results: To validate the validity of the algorithm above, a preliminary test with three volunteers was conducted to study the motion control. The results showed that the average error of the integral square error (ISE) was 2.90 and the minimum error was 1.96. Discussion: The results demonstrated the efficiency of the proposed method, and that the system is capable of providing walking assistance.

Effects of dual-task training in patients with post-stroke cognitive impairment: A randomized controlled trial.

Background: Evidence for the efficacy of cognitive-motor dual-task (CMDT) training in patients with post-stroke cognitive impairment (PSCI) and no dementia is still lacking. More importantly, although some studies on the cognitive effect of CMDT training show an improvement in cognitive performance, the results are still controversial, and the intervention mechanism of CMDT training on cognitive function improvement is not clear. The main purpose of this study was to analyze the effects of CMDT training on cognitive function, neuron electrophysiology, and frontal lobe hemodynamics in patients with PSCI. Methods: Here we tested the effects of CMDT training on cognitive function in PSCI patients. Forty subjects who met the criteria of PSCI were randomly assigned to control and experimental groups. CMDT training or cognitive task (CT) training was administered to each patient in the experimental and control groups, respectively. All subjects performed Mini-mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) scale before and after the intervention, and the event-related potentials (ERP) and functional near-infrared spectroscopy (fNIRS) were used to evaluate the changes in neuron electrophysiology and hemodynamics. Results: Forty patients were randomized across Beijing Rehabilitation Hospital Capital Medical University in Beijing. At the end of the intervention, 33 subjects completed the experimental process. The CMDT group showed significant improvement in the MMSE (P = 0.01) and MoCA (P = 0.024) relative to the CT group. The results of ERP and fNIRS showed that CMDT training could shorten the latency of P300 (P = 0.001) and the peak time of oxygenated hemoglobin (P = 0.004). The results showed that CMDT training shortened the response time of central neurons and significantly increased the rate of oxygen supply to the frontal lobe. Conclusion: CMDT training in patients with PSCI improved global cognitive function, which was supported by the improved neural efficiency of associated brain areas. Clinical trial registration: http://www.chictr.org.cn, identifier ChiCTR2000034862.

Risk Factors for Poor Pain Control in Zoster-Associated Pain: A Retrospective Study.

INTRODUCTION: The objective was to investigate the risk factors for poor pain control in patients with herpes zoster (HZ)-associated neuropathic pain treated with drugs combined with nerve block therapy. Neuropathic pain commonly follows HZ. Nerve block therapy is the most commonly used clinical treatment for such pain, combining anti-inflammation and analgesia to prevent peripheral sensitization of nerve. METHODS: Using clinical practice data from a cohort study at our research center, we established a multivariate logistic regression model to investigate potential risk factors for poor control of zoster-associated pain (ZAP) treated with drugs plus nerve block therapy, including demographic characteristics, complications, laboratory tests, and characteristics of HZ attacks. RESULTS: Of the 429 patients with ZAP who received drugs plus nerve block therapy, 95 (22.14%) had poor pain control after treatment. The risk of poor pain control was closely related to presence of cancer (odds ratio (OR) 4.173, 95% confidence interval (CI) 1.342-12.970), numerical rating scale score on admission (OR 1.929, 95% CI 1.528-2.434), and red blood cell count (OR 0.560, 95% CI 0.328-0.954). Area under the receiver operator characteristic curve was 0.730. Goodness of fit (Hosmer-Lemeshow) was 0.874. CONCLUSIONS: The risk of poor pain control in patients with ZAP increased as a result of certain patient characteristics and complications, especially severe pain before treatment and cancer.

A Novel Long-Noncoding RNA LncZFAS1 Prevents MPP+-Induced Neuroinflammation Through MIB1 Activation.

Parkinson's disease remains one of the leading neurodegenerative diseases in developed countries. Despite well-defined symptomology and pathology, the complexity of Parkinson's disease prevents a full understanding of its etiological mechanism. Mechanistically, α-synuclein misfolding and aggregation appear to be central for disease progression, but mitochondrial dysfunction, dysfunctional protein clearance and ubiquitin/proteasome systems, and neuroinflammation have also been associated with Parkinson's disease. Particularly, neuroinflammation, which was initially thought to be a side effect of Parkinson's disease pathogenesis, has now been recognized as driver of Parkinson's disease exacerbation. Next-generation sequencing has been used to identify a plethora of long noncoding RNAs (lncRNA) with important transcriptional regulatory functions. Moreover, a myriad of lncRNAs are known to be regulators of inflammatory signaling and neurodegenerative diseases, including IL-1ß secretion and Parkinson's disease. Here, LncZFAS1 was identified as a regulator of inflammasome activation, and pyroptosis in human neuroblast SH-SY5Y cells following MPP+ treatment, a common in vitro Parkinson's disease cell model. Mechanistically, TXNIP ubiquitination through MIB1 E3 ubiquitin ligase regulates NLRP3 inflammasome activation in neuroblasts. In contrast, MPP+ activates the NLPR3 inflammasome through miR590-3p upregulation and direct interference with MIB1-dependent TXNIP ubiquitination. LncZFAS overexpression inhibits this entire pathway through direct interference with miR590-3p, exposing a novel research idea regarding the mechanism of Parkinson's disease.

Increased activation of the caudate nucleus and parahippocampal gyrus in Parkinson's disease patients with dysphagia after repetitive transcranial magnetic stimulation: a case-control study.

Repetitive transcranial magnetic stimulation (rTMS) has been shown to effectively improve impaired swallowing in Parkinson's disease (PD) patients with dysphagia. However, little is known about how rTMS affects the corresponding brain regions in this patient group. In this case-control study, we examined data from 38 PD patients with dysphagia who received treatment at Beijing Rehabilitation Medicine Academy, Capital Medical University. The patients received high-frequency rTMS of the motor cortex once per day for 10 successive days. Changes in brain activation were compared via functional magnetic resonance imaging in PD patients with dysphagia and healthy controls. The results revealed that before treatment, PD patients with dysphagia showed greater activation in the precentral gyrus, supplementary motor area, and cerebellum compared with healthy controls, and this enhanced activation was weakened after treatment. Furthermore, before treatment, PD patients with dysphagia exhibited decreased activation in the parahippocampal gyrus, caudate nucleus, and left thalamus compared with healthy controls, and this activation increased after treatment. In addition, PD patients with dysphagia reported improved subjective swallowing sensations after rTMS. These findings suggest that swallowing function in PD patients with dysphagia improved after rTMS of the motor cortex. This may have been due to enhanced activation of the caudate nucleus and parahippocampal gyrus. The study protocol was approved by the Ethics Committee of Beijing Rehabilitation Hospital of Capital Medical University (approval No. 2018bkky017) on March 6, 2018 and was registered with Chinese Clinical Trial Registry (registration No. ChiCTR 1800017207) on July 18, 2018.

Effects of Combined Cognitive and Exercise Interventions on Poststroke Cognitive Function: A Systematic Review and Meta-Analysis.

OBJECTIVE: We investigated combined cognitive and exercise interventions in the literature and summarized their effectiveness in improving poststroke cognitive impairment (PSCI). Data Sources. Electronic databases and trial registries were searched from their inception until July 2020. Study Selection. Trials were collected with the following study inclusion criteria: (1) patients over 18 years of age who were diagnosed with PSCI; (2) combined cognitive-exercise interventions, regardless of the order of the two types of interventions or whether they were administered simultaneously; (3) any control group studied at the same time that was deemed acceptable, including no intervention/routine care, delayed intervention, sham intervention, and passive training; (4) the use of any validated cognitive neuropsychological test to evaluate cognitive function; and (5) clinically administered random trials with controls. Data Extraction. Five randomized controlled trials met the inclusion criteria. Two reviewers independently assessed the eligibility of the full texts and methodological quality of the included studies using the Cochrane risk of bias tool. Inconsistent results were resolved by additional discussion or decided by a third examiner, if necessary. Data Analysis. Meta-analysis demonstrated that the combined interventions had a significant effect on executive function and working memory [Stroop test (time), standardized mean difference (SMD) = 0.42, 95% confidence interval (CI): 0.80-0.04, p = 0.02; Trail Making Test, SMD = 0.49, 95% CI: 0.82-0.16, p = 0.004; Forward Digit Span Test, SMD = 0.91, 95% CI: 0.54-1.29, p ≤ 0.001]. While it was impossible to conduct a meta-analysis of global cognitive function and other cognitive domains, individual experiments demonstrated that the combined interventions played a significant role in global cognition, reasoning ability, logical thinking, and visual-spatial memory function. CONCLUSIONS: Our analyses demonstrated that the combined interventions had a significant effect on the improvement of PSCI, particularly in terms of executive function. However, the moderate risk of bias in the included trials and the small number of relevant studies indicated a need for more uniform diagnostic and evaluation criteria, and larger trials would provide stronger evidence to better understand the effectiveness of the combined interventions. This trial is registered with trial registration number INPLASY202160090.

Hematopoietic Stem Cell Niches and Signals Controlling Immune Cell Development and Maintenance of Immunological Memory.

Studies over the last couple of decades have shown that hematopoietic stem cells (HSCs) are critically dependent on cytokines such as Stem Cell Factor and other signals provided by bone marrow niches comprising of mesenchymal stem and progenitor cells (MSPCs) and endothelial cells (ECs). Because of their critical roles in HSC maintenance the niches formed by MSPCs and ECs are commonly referred to as HSC niches. For the most part, the signals required for HSC maintenance act in a short-range manner, which imposes the necessity for directional and positional cues in order for HSCs to localize and be retained properly in stem cell niches. The chemokine CXCL12 and its Gαi protein coupled receptor CXCR4, besides promoting HSC quiescence directly, also play instrumental roles in enabling HSCs to access bone marrow stem cell niches. Recent studies have revealed, however, that HSC niches also provide a constellation of hematopoietic cytokines that are critical for the production of most, if not all, blood cell types. Some hematopoietic cytokines, namely IL-7 and IL-15 produced by HSC niches, are not only required for lymphopoiesis but are also essential for memory T cell maintenance. Consequently, hematopoietic progenitors and differentiated immune cells, such as memory T cell subsets, also depend on the CXCL12/CXCR4 axis for migration into bone marrow and interactions with MSPCs and ECs. Similarly, subsets of antibody-secreting plasma cells also reside in close association with CXCL12-producing MSPCs in the bone marrow and require the CXCR4/CXCL12 axis for survival and long-term maintenance. Collectively, these studies demonstrate a broad range of key physiological roles, spanning blood cell production and maintenance of immunological memory, that are orchestrated by stem cell niches through a common and simple mechanism: CXCL12/CXCR4-mediated cell recruitment followed by receipt of a maintenance and/or instructive signal. A fundamental flaw of this type of cellular organization is revealed by myeloid and lymphoid leukemias, which target stem cell niches and induce profound transcriptomic changes that result in reduced hematopoietic activity and altered mesenchymal cell differentiation.

[Spatial and temporal responses of habitat quality to urbanization: A case study of Changchun City, Jilin Province, China]. / 生境质量对城镇化的时空响应­­ä»¥é•¿æ˜¥å¸‚为例.

Habitat quality is an important index to evaluate regional ecological security. Revealing its spatial and temporal responses to urbanization is conducive to the in-depth implementation of new urbanization. Based on land use data, we analyzed the spatio-temporal characteristics of Changchun's landscape pattern, habitat quality and its sample zone from the grid scale with comprehensive utilization of spatial analysis and ecological model analysis. We further discussed the responses of habitat quality during urbanization. The results showed that the low values of patch density (PD), edge density (ED) and Shannon diversity index (SHDI) were distributed in the western plains, while the high aggregation index (AI) showed a patchy distribution in eastern and southern of the city. During 2000-2015, the habitat quality of Changchun showed a trend of degradation and significant spatial heterogeneity, showing a distribution of "high in the east, and low in the west". The expansion of construction land and the transportation infrastructure played a leading role in the degradation of regional habitat quality. The changes of habitat quality differed significantly in different zones. The overall variation of water belt was relatively small, while the variation frequency and amplitude of mountain, urban expansion, and traffic belt were relatively high. Natural factors including slope and elevation basically shaped the overall distribution pattern of habitat quality in Changchun, while urbanization factors including population density, GDP and night light index showed significant negative correlation with habitat quality. To alleviate the ecological pressure of urbanization and promote habitat quality, we proposed differentiated development strategies, such as preventing deforestation in the Dahei Mountains, using ecological strategies to restore habitat degradation areas, improving land use efficiency in built-up urban areas, promoting "smart growth" in urban areas, setting red line of farmland in hilly areas, and strengthening ecological infrastructure construction.

Prevalence of dermatologic manifestations and metabolic biomarkers in women with polycystic ovary syndrome in north China.

BACKGROUND: Cutaneous features of hyperandrogenism in polycystic ovary syndrome (PCOS) include acne, hirsutism, seborrhea, androgenic alopecia (AGA), and acanthosis nigricans (AN). However, the relationships have not been well known broadly in terms of clinical hyperandrogenism and biochemical markers. OBJECTIVES: The aim of this study was to investigate biochemical and metabolic parameters in relation to cutaneous characters women in with and without PCOS. METHODS: This was a cross-sectional retrospective study including 186 women with PCOS and 113 age-matched without PCOS women. Acne grade, hirsutism, seborrhea, AGA, and AN were recorded. Hormonal and metabolic parameters were measured. RESULTS: The most common finding was acne, and AN was the least dermatological manifestations between PCOS and non-PCOS groups. The severity location and type of acne did not differ in PCOS women compared to non-PCOS women. Significant differences were found with respect to free androgen index (FAI) (P = .036), sex hormone-binding globulin (SHBG) (P = .023), and body mass index (BMI) (P = .001) between PCOS with acne and PCOS without acne groups. Overall, age (P = .005) was significantly decreased, while BMI (P = .004) was significantly higher in PCOS with hirsutism. The mean serum total testosterone (TT), dehydroepiandrosterone sulfate, and FAI were significantly elevated, but SHBG was decreased between PCOS with and without hirsutism groups. There were significantly different BMI (P = .018) and triglyceride (P = .024) except other hormonal parameter of without AGA group. CONCLUSION: This study indicated a strong correlation between hirsutism and metabolic abnormalities. Hirsutism is the most common cutaneous finding in PCOS women. Acne and AGA are associated with other manifestations of clinical hyperandrogenism, but not obvious markers of biochemical hyperandrogenemia and metabolic dysfunction.

The Impact of Genome Region of Difference 4 (RD4) on Mycobacterial Virulence and BCG Efficacy.

Comparative genome analyses have revealed a number of regions of difference (RD) among mycobacterial species. The functional consequences of most of these genome variations have not been studied. RD4, which encompasses Rv1506c-Rv1516c of Mycobacterium tuberculosis (M. tb) H37Rv, is absent in the closely related Mycobacterium bovis and M. bovis Bacille Calmette-Guérin (BCG). On the other hand, we previously found that Mycobacterium marinum has an extended RD4 which includes a number of genes involved in the biosynthesis of lipooligosaccharides (LOSs). As such, there appears to be a gradual decay of RD4 in mycobacterial genomes in the order of M. marinum, M. tb, and M. bovis (including BCG). To understand the potential effect of RD4 on mycobacterial virulence, in this study, we cloned the entire (Rv1501-1516c) and partial (Rv1501-1508c) RD4 into an integrating vector. These constructs were introduced to M. bovis BCG and M. marinum and the virulence of the RD4 knock-in strains were evaluated in the SCID mice and zebrafish infection models, respectively. BCG containing the entire RD4 exhibited similar levels of virulence to the parental strain but BCG containing partial RD4 (Rv1501-Rv1508c) was more attenuated. Similarly, zebrafish infection experiments showed that addition of partial RD4 also appeared to attenuate the virulence of M. marinum. However, M. marinum containing entire RD4 was more virulent than the wild type strain. Interestingly, BCG strains containing the entire or partial RD4 exhibited better protection of zebrafish against M. marinum challenge than the parental BCG. Taken together, our data suggest that RD4 plays a role in mycobacterial virulence and that RD4 knock-in BCG strains confer improved protection. Our study has provided new insights into the biological function of RD4 and evolution of mycobacterial genomes.

Immunogenicity and Protective Efficacy of a Fusion Protein Tuberculosis Vaccine Combining Five Esx Family Proteins.

One strategy to develop the next generation of tuberculosis vaccines is to construct subunit vaccines based on T cell antigens. In this study, we have evaluated the vaccine potential of a fusion protein combining EsxB, EsxD, EsxG, EsxU, and EsxM of Mycobacterium tuberculosis (M. tb). This recombinant protein, named BM, was expressed in and purified from Escherichia coli. Immunization of C57BL/6 mice with purified BM protein formulated in Freund's incomplete adjuvant induced the production of Th1 cytokines (IFN-γ, TNF, and IL-2) and multifunctional CD4+ T cells. Vaccination of BALB/c mice with BM protein followed by intravenous challenge with Mycobacterium bovis BCG resulted in better levels of protection than the two leading antigens, Ag85A and PPE18. Taken together, these results indicate that BM is a protective antigen. Future studies to combine BM with other antigens and evaluate its effectiveness as a booster of BCG or as a therapeutic vaccine are warranted.