Method to Improve DNA Condensation Efficiency by Alkali Treatment

Hou, Sen; Li, Xinxin; Feng, Xisheng

doi:10.1080/15257770903155493

Cited by 2 publications

(2 citation statements)

References 25 publications

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“…The Kanamycin resistance gene was excised out from pSD5 as an Nhe I- Bst EII fragment, end-repaired and cloned into Nde I digested, end-repaired pLITΔA to generate pLIT38ΔAK. The vector pLIT38ΔAK was pretreated with alkali [28] and 2 μg of DNA was electroporated into Mtb∆ mptpB electrocompetent cells to generate a double gene mutant, namely, Mtb∆ mptpB ∆ mptpA . …”

Section: Methodsmentioning

confidence: 99%

Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs

et al. 2013

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BackgroundThe failure of Mycobacterium bovis Bacille Calmette-Guérin to impart satisfactory protection against adult pulmonary tuberculosis has necessitated the development of more effective TB vaccines. The assumption that the vaccine strain should be antigenically as similar as possible to the disease causing pathogen has led to the evaluation of M.tuberculosis mutants as candidate tuberculosis vaccines.Methods/Principal FindingsIn this study, we have generated a mutant of M.tuberculosis (Mtb∆mms) by disrupting 3 virulence genes encoding a mycobacterial secretory acid phosphatase (sapM) and two phosphotyrosine protein phosphatases (mptpA and mptpB) and have evaluated its protective efficacy in guinea pigs. We observed that Mtb∆mms was highly attenuated in THP-1 macrophages. Moreover, no bacilli were recovered from the lungs and spleens of guinea pigs after 10 weeks of Mtb∆mms inoculation, although, initially, the mutant exhibited some growth in the spleens. Subsequently, when Mtb∆mms was evaluated for its protective efficacy, we observed that similar to BCG vaccination, Mtb∆mms exhibited a significantly reduced CFU in the lungs of guinea pigs when compared with the unvaccinated animals at 4 weeks after challenge. In addition, our observations at 12 weeks post challenge demonstrated that Mtb∆mms exhibited a more sustainable and superior protection in lungs as compared to BCG. However, the mutant failed to control the hematogenous spread as the splenic bacillary load between Mtb∆mms vaccinated and sham immunized animals was not significantly different. The gross pathological observations and histopathological observations corroborated the bacterial findings. Inspite of disruption of phosphatase genes in MtbΔmms, the lipid profiles of M.tuberculosis and MtbΔmms were identical indicating thereby that the phenotype of the mutant was ascribed to the loss of phosphatase genes and the influence was not related to any alteration in the lipid composition.Conclusions/SignificanceThis study highlights the importance of M.tuberculosis mutants in imparting protection against pulmonary TB.

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Section: Methodsmentioning

confidence: 99%

Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs

et al. 2013

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“…Pulmonary hypertension (PH) is a heterogeneous group of diseases characterized by elevated pulmonary arterial pressures as a result of pulmonary vascular inflammation and remodeling, eventually leading to right heart failure and death (1)(2)(3)(4). In PH, hypoxia and immune dysfunction are central contributors to disease pathogenesis (5)(6)(7)(8)(9)(10). The mechanisms through which hypoxia and immune dysfunction contribute to PH have been examined in animal models, including hypoxia-induced PH (11,12).…”

Section: Introductionmentioning

confidence: 99%

Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension

Kumar,

Mickael,

Kumar

et al. 2024

Front. Immunol.

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IntroductionHypoxia is a common pathological driver contributing to various forms of pulmonary vascular diseases leading to pulmonary hypertension (PH). Pulmonary interstitial macrophages (IMs) play pivotal roles in immune and vascular dysfunction, leading to inflammation, abnormal remodeling, and fibrosis in PH. However, IMs’ response to hypoxia and their role in PH progression remain largely unknown. We utilized a murine model of hypoxia-induced PH to investigate the repertoire and functional profiles of IMs in response to acute and prolonged hypoxia, aiming to elucidate their contributions to PH development.MethodsWe conducted single-cell transcriptomic analyses to characterize the repertoire and functional profiles of murine pulmonary IMs following exposure to hypobaric hypoxia for varying durations (0, 1, 3, 7, and 21 days). Hallmark pathways from the mouse Molecular Signatures Database were utilized to characterize the molecular function of the IM subpopulation in response to hypoxia.ResultsOur analysis revealed an early acute inflammatory phase during acute hypoxia exposure (Days 1-3), which was resolved by Day 7, followed by a pro-remodeling phase during prolonged hypoxia (Days 7-21). These phases were marked by distinct subpopulations of IMs: MHCIIhiCCR2+EAR2+ cells characterized the acute inflammatory phase, while TLF+VCAM1hi cells dominated the pro-remodeling phase. The acute inflammatory phase exhibited enrichment in interferon-gamma, IL-2, and IL-6 pathways, while the pro-remodeling phase showed dysregulated chemokine production, hemoglobin clearance, and tissue repair profiles, along with activation of distinct complement pathways.DiscussionOur findings demonstrate the existence of distinct populations of pulmonary interstitial macrophages corresponding to acute and prolonged hypoxia exposure, pivotal in regulating the inflammatory and remodeling phases of PH pathogenesis. This understanding offers potential avenues for targeted interventions, tailored to specific populations and distinct phases of the disease. Moreover, further identification of triggers for pro-remodeling IMs holds promise in unveiling novel therapeutic strategies for pulmonary hypertension.

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Method to Improve DNA Condensation Efficiency by Alkali Treatment

Cited by 2 publications

References 25 publications

Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs

Secretory Phosphatases Deficient Mutant of Mycobacterium tuberculosis Imparts Protection at the Primary Site of Infection in Guinea Pigs

Single cell transcriptomic analyses reveal diverse and dynamic changes of distinct populations of lung interstitial macrophages in hypoxia-induced pulmonary hypertension

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