Statistical and network analyses reveal mechanisms for the enhancement of macrophage immunity by manganese in Mycobacterium tuberculosis infection.

Tuberculosis is a significant infectious disease that poses a serious risk to human health. Our previous research has indicated that manganese ions reduce the bacterial load of Mycobacterium tuberculosis in macrophages, but the exact immune defense mechanism remains unknown. Several critical proteins and pathways involved in the host's immune response during this process are still unidentified. Our research aims to identify these proteins and pathways and provide a rationale for the use of manganese ions in the adjuvant treatment of tuberculosis. We downloaded GSE211666 data from the GEO database and selected the RM (Post-infection manganese ion treatment group) and Ra (single-infection group) groups for comparison and analysis to identify differential genes. These differential genes were then enriched and analyzed using STRING, Cytoscape, and NDEx tools to identify the two most relevant pathways of the "Host Response Signature Network." After conducting an in-depth analysis of these two pathways, we found that manganese ions mainly mediate (1) the interferon -gamma (IFN-γ) and its receptor IFNGR and the downstream JAK-STAT pathway and (2) the NFκB pathway to enhance macrophage response to interferon, autophagy, polarization, and cytokine release. Using qPCR experiments, we verified the increased expression of CXCL10, MHCII, IFNγ, CSF2, and IL12, all of which are cytokines that play a key role in resistance to Mycobacterium tuberculosis infection, suggesting that macrophages enter a state of pro-inflammatory and activation after the addition of manganese ions, which enhances their immunosuppressive effect against Mycobacterium tuberculosis. We conclude that our study provides evidence of manganese ion's ability to treat tuberculosis adjuvantly.

DUSP2 deletion with CRISPR/Cas9 promotes Mauthner cell axonal regeneration at the early stage of zebrafish.

Axon regeneration of central neurons is a complex process that is tightly regulated by multiple extrinsic and intrinsic factors. The expression levels of distinct genes are changed after central neural system (CNS) injury and affect axon regeneration. A previous study identified dusp2 as an upregulated gene in zebrafish with spinal cord injury. Here, we found that dual specificity phosphatase 2 (DUSP2) is a negative regulator of axon regeneration of the Mauthner cell (M-cell). DUSP2 is a phosphatase that mediates the dephosphorylation of JNK. In this study, we knocked out dusp2 by CRISPR/Cas9 and found that M-cell axons of dusp2-/- zebrafish had a better regeneration at the early stage after birth (within 8 days after birth), while those of dusp2+/- zebrafish did not. Overexpression of DUSP2 in Tg (Tol 056) zebrafish by single-cell electroporation retarded the regeneration of M-cell axons. Western blotting results showed that DUSP2 knockout slightly increased the levels of phosphorylated JNK. These findings suggest that knocking out DUSP2 promoted the regeneration of zebrafish M-cell axons, possibly through enhancing JNK phosphorylation.

Manganese enhances macrophage defense against Mycobacterium tuberculosis via the STING-TNF signaling pathway.

The host cell antiviral response pathway depends heavily on manganese (Mn), but its role in defense against Mycobacterium tuberculosis (M. tuberculosis) infection is rarely reported. In this study, we found that, in H37Ra-infected macrophages, Mn2+ increases the phosphorylation of stimulator of interferon genes (STING) and P65, as well as triggers the phosphorylation cascade of tumor necrosis factor (TNF) signaling pathway proteins, signal-regulated kinase (ERK), P38, and c-Jun N-terminal kinase (JNK). The activation of the TNF signaling pathway stimulated the expression of downstream inflammatory factors TNF-α, C-X-C Motif Chemokine Ligand 10(CXCL10), CC Motif Chemokine Ligand 20(CCL20), Colony Stimulating Factor 1(CSF1), Colony Stimulating Factor 2(CSF2), and Jagged Canonical Notch Ligand 1(JAG1), thereby triggering a strong inflammatory response in the cells. The excessive accumulation of TNF-α in macrophages induces necroptosis and inhibits the survival of M. tuberculosis in macrophages. When we treated macrophages with the STING inhibitor H-151, the phosphorylation of P38 was reduced, and the secretion of downstream inflammatory factors TNF-α and CXCL10, CCL20, CSF1, and CSF2 were also inhibited. Overall, this study reveals that Mn2+ plays a crucial role in host cell defense against M. tuberculosis infection, contributes to a deeper understanding of pathogen-host interactions, and offers theoretical support for the use of Mn2+ as a drug cofactor for the treatment of tuberculosis and the development of a new generation of drugs and vaccine adjuvants.

Diagnosis of patients with mucopolysaccharidosis type II via RNA sequencing.

BACKGROUND: Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disorder caused by various variants in the IDS gene. It is known that genomic recombinants between IDS and its homologous pseudogene IDSP1 account for a small number of patients, for whom genetic diagnosis usually relies on restriction enzyme digestion at specific loci. Nevertheless, such approach cannot reveal the impact of rearrangements on IDS transcription, which is crucial for the interpretation of the pathogenicity of rearrangement variants. METHODS: RNA sequencing (RNA-seq) was explored to analyze transcriptional alterations in four male MPS II patients who were negative for Sanger sequencing of the IDS gene. Reverse transcription-polymerase chain reaction and TA clone sequencing were used to validate RNA-seq analysis results. The IDS-IDSP1 recombinant was determined by sequencing the indicated loci in genome. RESULTS: Differential expression analysis showed the expression levels of IDS gene in patients were largely reduced compared to the healthy individuals. Differential splicing analysis revealed skipping of exons 8 and 9 of IDS, without any splice-junction defects at the genomic level. In addition, two types of fusion transcripts, IDS_EOLA1 and IDS_EOLA1-DT_EOLA1 were identified by gene fusion analysis. Sequencing of the known rearrangement alleles showed these four patients have the same type of IDS-IDSP1 recombinant. CONCLUSION: We establish an RNA-seq workflow to analyze transcriptional characteristics of IDS gene from multiple perspectives. Our study validates the diagnostic value of RNA-seq in MPS II, including the discovery of transcriptional alterations and the potential to suggest genome-level rearrangements in IDS.

Sulforaphane kills Mycobacterium tuberculosis H37Ra and Mycobacterium smegmatis mc2155 through a reactive oxygen species dependent mechanism.

Mycobacterium tuberculosis (M. tuberculosis) is a highly pathogenic intracellular pathogen that causes tuberculosis (TB), the leading cause of mortality from single infections. Redox homeostasis plays a very important role in the resistance of M. tuberculosis to antibiotic damage and various environmental stresses. The antioxidant sulforaphane (SFN) has been reported to exhibit anticancer activity and inhibit the growth of a variety of bacteria and fungi. Nonetheless, it remains unclear whether SFN exhibits anti-mycobacterial activity. Our results showed that the SFN against M. tuberculosis H37Ra exhibited bactericidal activity in a time and dose-dependent manner. The anti-tubercular activity of SFN was significantly correlated with bacterial reactive oxygen species (ROS) levels. In addition, SFN promoted the bactericidal effect of macrophages on intracellular bacteria in a dose-dependent manner, mediated by increasing intracellular mitochondrial ROS levels and decreasing cytoplasmic ROS levels. Taken together, our data revealed the previously unrecognized antimicrobial functions of SFN. Future studies focusing on the mechanism of SFN in macrophages against M. tuberculosis are essential for developing new host-directed therapeutic approaches against TB.

Fluoxetine modifies circadian rhythm by reducing melatonin content in zebrafish.

Fluoxetine (FLX), a selective serotonin reuptake inhibitor (SSRI), increases the serotonin levels in the brain to treat depression. Antidepressants have been demonstrated to modulate circadian rhythm, but the underlying mechanisms by which antidepressants regulate circadian rhythm require more research. This study aimed to investigate the role of FLX on circadian rhythm by analyzing the movement behavior and internal circadian oscillations in zebrafish. The results showed that the expression of clock genes clock1a and bmal1b was significantly down-regulated, and the amplitude reduction and phase shift were observed after FLX treatment. Furthermore, FLX exposure inhibited the expression of aanat2, which led to a decrease in nocturnal melatonin secretion. aanat2-/- larvae showed disrupted circadian rhythm. These findings may help reveal the effect of FLX exposure on the circadian rhythm and locomotor activity. It may provide theoretical data for the clinical application of FLX.

Class â histone deacetylase inhibitor regulate of Mycobacteria-Driven guanylate-binding protein 1 gene expression.

Guanylate-binding proteins (GBPs) are a class of interferon (IFN)-stimulated genes with well-established activity against viruses, intracellular bacteria, and parasites. The effect of epigenetic modification on GBP activity upon Mycobacterium tuberculosis (Mtb) infection is poorly understood. In this study, we found that Mtb infection can significantly increase the expression of GBPs. Class Ⅰ histone deacetylase inhibitor (HDACi) MS-275 can selectively inhibit GBP1 expression, ultimately affecting the release of inflammatory cytokines IL-1ß and suppressing Mtb intracellular survival. Moreover, interfering with GBP1 expression could reduce the production of IL-1ß and the level of cleaved-caspase-3 in response to Mtb infection. GBP1 silencing did not affect Mtb survival. Besides, using the bisulfite sequencing PCR, we showed that the CpG site of the GBP1 promoter was hypermethylated, and the methylation status of the GBP1 promoter did not change significantly upon Mtb infection. Overall, this study sheds light on the role of GBP in Mtb infection and provides a link between epigenetics and GBP1 activity.

Hypoxia regulates cytokines expression and neutrophils migration by ERK signaling in zebrafish.

Normal dissolved oxygen in water is essential for maintaining the physiological functions of fish, but environmental pollution, such as eutrophication can lead to a decrease in oxygen content in water. How this reduction of dissolved oxygen in water affects the immune functions of fish and the potential regulatory mechanisms have not been thoroughly elucidated. In this study, we made full use of the aquatic model animal zebrafish to explore this question. In a model of LPS-induced inflammation, we found that hypoxia induced by infusing nitrogen into water increased the expression of pro-inflammatory cytokines, such as il-1ß, il-6, and il-8. In vivo imaging also showed that hypoxia significantly increased neutrophil migration to the site of caudal fin injury in the transgenic line. Subsequently, we found that the phosphorylation level of ERK protein was significantly activated upon hypoxia and proved the roles of ERK signaling in the expression of pro-inflammatory cytokines and neutrophil migration in zebrafish. This study indicated that reduced water oxygen significantly increases the inflammatory response of the zebrafish.

The role of neoantigens in tumor immunotherapy.

Tumor neoantigens are aberrant polypeptides produced by tumor cells as a result of genomic mutations. They are also tumor-specific antigens (TSA). Neoantigens are more immunogenic than tumor-related antigens and do not induce autoimmunity. Based on the rapid development of bioinformatics and the continuous update of sequencing technology, cancer immunotherapy with tumor neoantigens has made promising breakthroughs and progress. In this review, the generation, prediction, and identification of novel antigens, as well as the individualized treatments of neoantigens, were first introduced. Secondly, the mechanism of Chimeric Antigen Receptor T-Cell Immunotherapy (CAR-T) therapy and immune checkpoint blockade therapy in the treatment of tumors were outlined, and the three treatment methods were compared. Thirdly, the application of neoantigens in CAR-T therapy and PD-1/PD-L1 blockade therapy was briefly described. The benefits of the neoantigen vaccines over common vaccines were summarized as well. Finally, the prospect of neoantigen therapy was presented.

AZI2 positively regulates the induction of type I interferon in influenza-trigger pediatric pneumonia.

5-azacytidine-induced protein 2 (AZI2) is known to have a crucial role in antiviral innate immunity. This study aims to explore the roles of AZI2 in influenza-trigger pediatric pneumonia and its molecular mechanism. qPCR and immunoblotting assays were used to determine the levels of target genes and proteins. The lung infection mouse model was established by using PR8 H1N1 virus in AZI2 germline knockout (AZI2-/-) and wild-type (WT) mice. In addition, HEK293T cell-based luciferase reporter assays were used to investigate the regulatory effects of AZI2 on type I interferon. Immune precipitation and immunofluorescence staining were used to evaluate the interactions between AZI2 and TANK binding kinase 1 (TBK1). We observed an elevation in the expressions of IFN-I and AZI2 in peripheral blood mononuclear cells from the pneumonia patients with mild symptoms. Interestingly, AZI2 deficiency deteriorated the influenza-induced pathological symptoms in the lung as well as reduced the survival rate. It was further showed that AZI2 positively regulated the expressions of type I interferon, inflammatory cytokines, and IFN production-related genes. The molecular mechanism data revealed that AZI2 regulated the interactions between TBK1 and TANK. In summary, AZI2 positively regulates type I interferon production in influenza-induced pediatric pneumonia by promoting the interactions between TBK1 and TANK.

N-acetylcholine receptors regulate cytokines expression and neutrophils recruitment via MAPK/ERK signaling in zebrafish.

N-acetylcholine receptors (AChRs) are mainly distributed in the postsynaptic membrane and have been widely studied for their control of muscle contraction by regulating neural action potentials. However, the influences of AChRs on immune responses and potential mechanisms remain unclear. Here, we used the advantages of live imaging of zebrafish to explore the regulation process of AChRs on inflammatory responses. Pharmacologically activating of the receptor, we found that the expression of pro-inflammatory cytokines il-1ß, il-6, tnf-α and il-8 was significantly up-regulated and neutrophil migration to injury sites was also significantly increased. However, these phenomena were reversed under antagonism of the receptor activity. Results showed that interfering with nAChRs functions did not significantly affect zebrafish motion behavior. Results also showed that activation and antagonism of nAChRs function could regulate the phosphorylation of ERK protein respectively. We further demonstrated that ERK participated in the regulation of AChRs in cytokines expression and neutrophils migration in zebrafish. This study preliminarily revealed the roles of AChRs in inflammatory processes and their potential mechanism, providing additional evidence of peripheral immune regulation by cholinergic receptors.

Tacrolimus versus cyclophosphamide for patients with idiopathic membranous nephropathy and treated with steroids: a systematic review and meta-analysis of randomized controlled trials.

BACKGROUND: The therapeutic effects of tacrolimus (TAC) versus cyclophosphamide (CTX) were not fully illustrated for patients with idiopathic membranous nephropathy (IMN). METHODS: The PubMed, EmBase, Cochrane library, and CNKI were systematically searched throughout March 2020 for randomized controlled trials evaluating the therapeutic effects of TAC versus CTX for IMN patients treated with steroids. The pooled relative risks (RRs) and weighted mean differences (WMDs) with 95% confidence intervals (CIs) were calculated using the random-effects model. RESULTS: Twelve trials recruited a total of 868 IMN patients were identified and contained in final meta-analysis. Patients in TAC group was associated with an increased incidence of overall remission (12 trials: 868 patients; RR: 1.21; 95% CI: 1.11-1.31; p < 0.001) and complete remission (12 trials: 868 patients; RR: 1.50; 95% CI: 1.25-1.80; p < 0.001). Moreover, we noted TAC therapy significantly reduced urinary protein excretion (9 trials: 567 patients; WMD: -1.06; 95%CI: -1.41 to -0.71; p < 0.001), and increased serum albumin (9 trials: 567 patients; WMD: 5.37; 95%CI: 2.97 to 7.77; p < 0.001) than CTX therapy. Furthermore, no significant difference between TAC and CTX for serum creatinine was detected (6 trials: 378 patients; WMD: 0.15; 95%CI: -3.46 to 3.75; p = 0.936). Finally, the risk of alopecia (p = 0.008), infection (p = 0.045), leukocytosis (p = 0.002), and elevated ALT/AST (p = 0.011) in TAC group was significantly lower than CTX group, whereas TAC was associated with an increased risk of tremor than CTX (p = 0.010). CONCLUSIONS: This study found IMN patients treated with TAC combined with steroids provides a better therapeutic effect and less adverse events than those treated with CTX combined with steroids, with moderate-certainty evidence.

Efficacy of low-dose insulin combined with electrolyte in the treatment of pediatric diabetic ketoacidosis and its effect on serum inflammatory factors.

Diabetic ketoacidosis (DKA) is a very serious disease that can occur in both types of diabetes (type 1 and 2). It is caused by a combination of high blood sugar and low insulin levels, which can cause the body to produce too much ketone. Ketones are toxic to human organs. This research aimed to investigate the clinical efficacy of low-dose insulin combined with electrolyte in the treatment of pediatric DKA and its effect on serum inflammatory factors. For this purpose, a total of 122 children with DKA admitted to our hospital from April 2013 to May 2016 were selected as research objects. They were divided into group A with 60 cases and group B with 62 cases. Group B was treated with supplemental electrolytes, and group A was treated with low-dose insulin based on group B. The serum levels of TNF-α, IL-6, and IL-18 were measured by enzyme-linked immunosorbent assay (ELISA) before and after treatment, and the blood sugar, sodium, and potassium levels were measured by an automatic biochemical analyzer. The time when blood sugar reached the standard level when acidosis was corrected and hospitalization time was compared between the two groups. The total effective rate of group A was significantly higher than that of group B (p< 0.05). There was no significant difference in blood glucose, sodium, potassium, TNF-α, IL-6, and IL-18 levels between the two groups before treatment. (all p > 0.05). But the blood glucose, sodium and potassium levels in group A were significantly better than those in group B (all p< 0.001). The levels of serum TNF-α, IL-6, and IL-18 in group A were significantly lower than those in group B after treatment (all p< 0.001). After treatment, the time when blood sugar reached the standard level when acidosis was corrected and hospitalization time in group A were significantly shorter than those in group B (all p< 0.001). Low-dose insulin combined with electrolyte supplementation is effective in the treatment of DKA in children, which can effectively control blood sugar, sodium, potassium level, and inflammatory factor concentration.

Pneumonia caused by Mycobacterium tuberculosis.

Tuberculosis (TB) is one of the top ten leading causes of death worldwide. The causative agent of TB is Mycobacterium tuberculosis. Acute tuberculous pneumonia (TP) is an acute form of pulmonary TB. However, acute TP and non-tuberculous community-acquired pneumonia can be easily confused, resulting in deterioration of TP due to delayed treatment. Therefore, rapid and accurate diagnosis of acute TP is crucial in order to stop the transmission of TB. Moreover, development of new diagnostic tools (technologies and approaches), and flexible application of different therapy schemes will help to reduce the incidence of TP and promote the goal of ending the TB epidemic.

A Term Neonatal Case With Lethal Respiratory Failure Associated With a Novel Homozygous Mutation in ABCA3 Gene.

The mutations in the ABCA3 (ATP-binding cassette transporter subfamily A member 3) gene could result in lethal respiratory distress syndrome (RDS) in neonates and interstitial lung disease (ILD) in infants and children. Here, we describe a full-term newborn who manifested respiratory distress 20 min after birth and then gradually developed hypoxemic respiratory failure and died on 53 days of life. A homozygous missense mutation (c.746C >T) was identified in exon 8 of ABCA3 gene in the neonate by next-generation sequencing, and the mutations were inherited from parents, respectively. This homozygous mutation is the first reported to date.

A Neonatal Case With Perinatal Lethal Gaucher Disease Associated With Missense G234E and H413P Heterozygous Mutations.

Perinatal lethal Gaucher disease (PLGD), a particular and serious form of type 2 Gaucher disease (GD), often causes lethality in utero or death within hours after birth. The typical clinical manifestations include non-immune hydrops fetalis (NIHF), premature birth, fetal growth restriction, fetal intrauterine death, or neonatal distress and rapid death after birth. Here, we present a premature neonate with GD whose main clinical manifestations included intrauterine growth retardation, anasarca, facial dysmorphia, ichthyosis, respiratory distress, hepatosplenomegaly, joint contractures, myoclonus, refractory thrombocytopenia, anemia, elevated levels of liver enzymes, bile acid and direct bilirubin, cholestasis, pulmonary hypoplasia, intracranial hemorrhage, and abnormal electroencephalogram. The activity of ß- glucocerebrosidase was 0 in the peripheral white blood cells of the neonate. The sequencing analysis identified the presence of missense G234E and H413P heterozygous mutations in glucerebrosidase (GBA) exon 7 and 10, with the latter first observed to be associated with PLGD. This infant died at 73 days of age.

Oxidation of dCTP contributes to antibiotic lethality in stationary-phase mycobacteria.

Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of mazG-encoded 5-OH-dCTP-specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of mazG deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the ΔmazG mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria.

Differences in weathering pattern, stress resistance and community structure of culturable rock-weathering bacteria between altered rocks and soils.

In this study, we isolated and characterized rock-weathering bacteria from the surfaces of less and more altered tuffs, along with the adjacent soils, with respect to their rock weathering pattern, stress resistance, community structure, and the changes in these rocks and soils. Using a moderate-nutrition medium, we obtained 150 isolates from the rocks and soils. The rock-weathering patterns of the isolates were characterized using batch cultures that measure the quantity of Si, Al, and Fe released from tuff under aerobic conditions. Based on the potential of the bacterial influence on the element releases, the isolates could be grouped into highly, moderately, and least effective element solubilizers, respectively. Significantly more highly effective Al and Fe solubilizers were observed in the altered rocks, while the soils had more highly effective Si solubilizers. Furthermore, more isolates from the altered rocks significantly acidified the culture medium in the rock weathering process. Dynamic changes in the element release showed the distinct element releasing patterns of three selected isolates. More isolates from the altered rocks could grow at 4 °C or at 55 °C or at pH 4. Some isolates from the altered rocks could grow at pH 10 and with 10-15% (w/v) NaCl. The altered rocks and the soils existed in diverse and different highly weathering-specific culturable rock-weathering community structures. The changes in the culturable weathering communities between the altered rocks and the soils were attributable not only to major bacterial groups but also to a change in the minor population structure.

Antimicrobial Activity of Quinazolin Derivatives of 1,2-Di(quinazolin-4-yl)diselane against Mycobacteria.

Mycobacterium tuberculosis (M. tuberculosis) is one of the leading causes of morbidity and mortality. Currently, the emergence of drug resistance has an urgent need for new drugs. In previous study, we found that 1,2-di(quinazolin-4-yl)diselane (DQYD), a quinazoline derivative, has anticancer activities against many cancers. However, whether DQYD has the activity of antimycobacterium is still little known. Here our results show that DQYD has a similar value of the minimum inhibitory concentration with clinical drugs against mycobacteria and also has the ability of bacteriostatic activity with dose-dependent and time-dependent manner. Furthermore, the activities of DQYD against M. tuberculosis are associated with intracellular ATP homeostasis. Meanwhile, mycobacterium DNA damage level was increased after DQYD treatment. But there was no correlation between survival of mycobacteria in the presence of DQYD and intercellular reactive oxygen species. This study enlightens the possible benefits of quinazoline derivatives as potential antimycobacterium compounds and furtherly suggests a new strategy to develop new methods for searching antituberculosis drugs.

Cloning identification and functional analysis of human IL-17A promoter.

OBJECTIVE: To conduct the cloning identification and characterization of the sequence of human IL-17A promoter so as to analyze the regulatory mechanism of the gene expression of IL-17. METHODS: First of all, the potential promoter region of IL-17A was found by means of the bioinformatics methods. Then, it was cloned into the reporter vector with PCR technique. Finally, the activity of the test promoter was determined by dual luciferase reporter system. RESULTS: Two transcriptional start points of the upper region, 600 bp and 1000 bp, of IL-17A were obtained by PCR clone and proved to have certain activities by dual luciferase reporter system. Also, they could be activated by IL-17A activator STAT3, which could start the expression of the reported gene. CONCLUSIONS: Clone established the regulatory region of human IL-17A promoter, which provided bases to the subsequent function research.