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PloS One 2024Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum...
Itaconic acid inhibits nontuberculous mycobacterial growth in pH dependent manner while 4-octyl-itaconic acid enhances THP-1 clearance of nontuberculous mycobacteria in vitro.
Increasingly prevalent, nontuberculous mycobacteria (NTM) infections affect approximately 20% of people with cystic fibrosis (CF). Previous studies of CF sputum identified lower levels of the host metabolite itaconate in those infected with NTM. Itaconate can inhibit the growth of M. tuberculosis (MTB) in vitro via the inhibition of the glyoxylate cycle enzyme (ICL), but its impact on NTM is unclear. To test itaconic acid's (IA) effect on NTM growth, laboratory and CF clinical strains of Mycobacterium abscessus and Mycobacterium avium were cultured in 7H9 minimal media supplemented with 1-10 mM of IA and short-chain fatty acids (SCFA). M. avium and M. abscessus grew when supplemented with SCFAs, whereas the addition of IA (≥ 10 mM) completely inhibited NTM growth. NTM supplemented with acetate or propionate and 5 mM IA displayed slower growth than NTM cultured with SCFA and ≤ 1 mM of IA. However, IA's inhibition of NTM was pH dependent; as similar and higher quantities (100 mM) of pH adjusted IA (pH 7) did not inhibit growth in vitro, while in an acidic minimal media (pH 6.1), 1 to 5 mM of non-pH adjusted IA inhibited growth. None of the examined isolates displayed the ability to utilize IA as a carbon source, and IA added to M. abscessus isocitrate lyase (ICL) decreased enzymatic activity. Lastly, the addition of cell-permeable 4-octyl itaconate (4-OI) to THP-1 cells enhanced NTM clearance, demonstrating a potential role for IA/itaconate in host defense against NTM infections.
Topics: Succinates; Humans; Hydrogen-Ion Concentration; Nontuberculous Mycobacteria; THP-1 Cells; Mycobacterium Infections, Nontuberculous; Mycobacterium avium; Mycobacterium abscessus
PubMed: 38728330
DOI: 10.1371/journal.pone.0303516 -
The EMBO Journal Jun 2024The cellular consequences of activating FLT3 mutations in blood cancers are poorly understood. In this issue, Yan et al (2024) demonstrate that FLT3 activation promotes...
The cellular consequences of activating FLT3 mutations in blood cancers are poorly understood. In this issue, Yan et al (2024) demonstrate that FLT3 activation promotes mitobiogenesis and leukemia progression through a posttranslational control mechanism, linking cell metabolism with derepressed mitochondrial transcription and function.
Topics: Mitochondria; Humans; Transcription, Genetic; Leukemia; Animals; Succinic Acid; Mitochondrial Proteins
PubMed: 38724759
DOI: 10.1038/s44318-024-00116-2 -
Scientific Reports May 2024Individual theranostic agents with dual-mode MRI responses and therapeutic efficacy have attracted extensive interest due to the real-time monitor and high effective...
Individual theranostic agents with dual-mode MRI responses and therapeutic efficacy have attracted extensive interest due to the real-time monitor and high effective treatment, which endow the providential treatment and avoid the repeated medication with side effects. However, it is difficult to achieve the integrated strategy of MRI and therapeutic drug due to complicated synthesis route, low efficiency and potential biosafety issues. In this study, novel self-assembled ultrasmall FeO nanoclusters were developed for tumor-targeted dual-mode T/T-weighted magnetic resonance imaging (MRI) guided synergetic chemodynamic therapy (CDT) and chemotherapy. The self-assembled ultrasmall FeO nanoclusters synthesized by facilely modifying ultrasmall FeO nanoparticles with 2,3-dimercaptosuccinic acid (DMSA) molecule possess long-term stability and mass production ability. The proposed ultrasmall FeO nanoclusters shows excellent dual-mode T and T MRI capacities as well as favorable CDT ability due to the appropriate size effect and the abundant Fe ion on the surface of ultrasmall FeO nanoclusters. After conjugation with the tumor targeting ligand Arg-Gly-Asp (RGD) and chemotherapy drug doxorubicin (Dox), the functionalized FeO nanoclusters achieve enhanced tumor accumulation and retention effects and synergetic CDT and chemotherapy function, which serve as a powerful integrated theranostic platform for cancer treatment.
Topics: Magnetic Resonance Imaging; Theranostic Nanomedicine; Animals; Mice; Humans; Doxorubicin; Cell Line, Tumor; Neoplasms; Magnetite Nanoparticles; Succimer; Antineoplastic Agents
PubMed: 38724530
DOI: 10.1038/s41598-024-59525-2 -
Protein Science : a Publication of the... Jun 2024Recent studies have demonstrated that one can control the packing density, and in turn the filterability, of protein precipitates by changing the pH and buffer...
Recent studies have demonstrated that one can control the packing density, and in turn the filterability, of protein precipitates by changing the pH and buffer composition of the precipitating solution to increase the structure/order within the precipitate. The objective of this study was to examine the effect of sodium malonate, which is known to enhance protein crystallizability, on the morphology of immunoglobulin precipitates formed using a combination of ZnCl and polyethylene glycol. The addition of sodium malonate significantly stabilized the precipitate particles as shown by an increase in melting temperature, as determined by differential scanning calorimetry, and an increase in the enthalpy of interaction, as determined by isothermal titration calorimetry. The sodium malonate also increased the selectivity of the precipitation, significantly reducing the coprecipitation of DNA from a clarified cell culture fluid. The resulting precipitate had a greater packing density and improved filterability, enabling continuous tangential flow filtration with minimal membrane fouling relative to precipitates formed under otherwise identical conditions but in the absence of sodium malonate. These results provide important insights into strategies for controlling precipitate morphology to enhance the performance of precipitation-filtration processes for the purification of therapeutic proteins.
Topics: Malonates; Filtration; Chemical Precipitation; Immunoglobulins; Polyethylene Glycols; Chlorides; Calorimetry, Differential Scanning; Malates; Zinc Compounds
PubMed: 38723172
DOI: 10.1002/pro.5010 -
BMC Microbiology May 2024The production of succinic acid (SA) from biomass has attracted worldwide interest. Saccharomyces cerevisiae is preferred for SA production due to its strong tolerance...
BACKGROUND
The production of succinic acid (SA) from biomass has attracted worldwide interest. Saccharomyces cerevisiae is preferred for SA production due to its strong tolerance to low pH conditions, ease of genetic manipulation, and extensive application in industrial processes. However, when compared with bacterial producers, the SA titers and productivities achieved by engineered S. cerevisiae strains were relatively low. To develop efficient SA-producing strains, it's necessary to clearly understand how S. cerevisiae cells respond to SA.
RESULTS
In this study, we cultivated five S. cerevisiae strains with different genetic backgrounds under different concentrations of SA. Among them, KF7 and NBRC1958 demonstrated high tolerance to SA, whereas NBRC2018 displayed the least tolerance. Therefore, these three strains were chosen to study how S. cerevisiae responds to SA. Under a concentration of 20 g/L SA, only a few differentially expressed genes were observed in three strains. At the higher concentration of 60 g/L SA, the response mechanisms of the three strains diverged notably. For KF7, genes involved in the glyoxylate cycle were significantly downregulated, whereas genes involved in gluconeogenesis, the pentose phosphate pathway, protein folding, and meiosis were significantly upregulated. For NBRC1958, genes related to the biosynthesis of vitamin B6, thiamin, and purine were significantly downregulated, whereas genes related to protein folding, toxin efflux, and cell wall remodeling were significantly upregulated. For NBRC2018, there was a significant upregulation of genes connected to the pentose phosphate pathway, gluconeogenesis, fatty acid utilization, and protein folding, except for the small heat shock protein gene HSP26. Overexpression of HSP26 and HSP42 notably enhanced the cell growth of NBRC1958 both in the presence and absence of SA.
CONCLUSIONS
The inherent activities of small heat shock proteins, the levels of acetyl-CoA and the strains' potential capacity to consume SA all seem to affect the responses and tolerances of S. cerevisiae strains to SA. These factors should be taken into consideration when choosing host strains for SA production. This study provides a theoretical basis and identifies potential host strains for the development of robust and efficient SA-producing strains.
Topics: Saccharomyces cerevisiae; Succinic Acid; Gene Expression Regulation, Fungal; Saccharomyces cerevisiae Proteins; Fermentation
PubMed: 38720268
DOI: 10.1186/s12866-024-03314-4 -
Cellular & Molecular Biology Letters May 2024The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing...
The engineered clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas) system is currently widely applied in genetic editing and transcriptional regulation. The catalytically inactivated CasRx (dCasRx) has the ability to selectively focus on the mRNA coding region without disrupting transcription and translation, opening up new avenues for research on RNA modification and protein translation control. This research utilized dCasRx to create a translation-enhancement system for mammals called dCasRx-eIF4GI, which combined eukaryotic translation initiation factor 4G (eIF4GI) to boost translation levels of the target gene by recruiting ribosomes, without affecting mRNA levels, ultimately increasing translation levels of different endogenous proteins. Due to the small size of dCasRx, the dCasRx-eIF4GI translation enhancement system was integrated into a single viral vector, thus optimizing the delivery and transfection efficiency in subsequent applications. Previous studies reported that ferroptosis, mediated by calcium oxalate (CaOx) crystals, significantly promotes stone formation. In order to further validate its developmental potential, it was applied to a kidney stone model in vitro and in vivo. The manipulation of the ferroptosis regulatory gene FTH1 through single-guide RNA (sgRNA) resulted in a notable increase in FTH1 protein levels without affecting its mRNA levels. This ultimately prevented intracellular ferroptosis and protected against cell damage and renal impairment caused by CaOx crystals. Taken together, this study preliminarily validated the effectiveness and application prospects of the dCasRx-eIF4GI translation enhancement system in mammalian cell-based disease models, providing novel insights and a universal tool platform for protein translation research and future therapeutic approaches for nephrolithiasis.
Topics: Animals; Humans; Male; Mice; Calcium Oxalate; CRISPR-Cas Systems; Eukaryotic Initiation Factor-4G; Ferritins; Ferroptosis; Gene Editing; HEK293 Cells; Kidney; Kidney Calculi; Oxidoreductases; Protein Biosynthesis; RNA, Guide, CRISPR-Cas Systems
PubMed: 38714951
DOI: 10.1186/s11658-024-00582-w -
Anales de Pediatria May 2024. Neonatal screening of glutaric aciduria type 1 (GA-1) has brought radical changes in the course and outcomes of this disease. This study analyses the outcomes of the... (Observational Study)
Observational Study
INTRODUCTION
. Neonatal screening of glutaric aciduria type 1 (GA-1) has brought radical changes in the course and outcomes of this disease. This study analyses the outcomes of the first 5 years (2015-2019) of the AGA1 neonatal screening programme in our autonomous community.
MATERIAL
. We conducted an observational, descriptive and retrospective study. All neonates born between January 1, 2015 and December 31, 2019 that participated in the neonatal screening programme were included in the study. The glutarylcarnitine (C5DC) concentration in dry blood spot samples was measured by means of tandem mass spectrometry applying a cut-off point of 0.25 µmol/L.
RESULTS
. A total of 30 120 newborns underwent screening. We found differences in the C5DC concentration based on gestational age, type of feeding and hours of life at sample collection. These differences were not relevant for screening purposes. There were no differences between neonates with weights smaller and greater than 1500 g. Screening identified 2 affected patients and there were 3 false positives. There were no false negatives. The diagnosis was confirmed by genetic testing. Patients have been in treatment since diagnosis and have not developed encephalopathic crises in the first 4 years of life.
CONCLUSIONS
. Screening allowed early diagnosis of two cases of GA-1 in the first 5 years since its introduction in our autonomous community. Although there were differences in C5DC levels based on gestational age, type of feeding and hours of life at blood extraction, they were not relevant for screening.
Topics: Humans; Neonatal Screening; Infant, Newborn; Retrospective Studies; Glutaryl-CoA Dehydrogenase; Amino Acid Metabolism, Inborn Errors; Male; Female; Brain Diseases, Metabolic; Tandem Mass Spectrometry; Glutarates; Gestational Age; Carnitine
PubMed: 38714461
DOI: 10.1016/j.anpede.2024.04.011 -
Frontiers in Immunology 2024Allergic asthma is a widely prevalent inflammatory condition affecting people across the globe. T cells and their secretory cytokines are central to the pathogenesis of...
Allergic asthma is a widely prevalent inflammatory condition affecting people across the globe. T cells and their secretory cytokines are central to the pathogenesis of allergic asthma. Here, we have evaluated the anti-inflammatory impact of dimethyl fumarate (DMF) in allergic asthma with more focus on determining its effect on T cell responses in allergic asthma. By utilizing the ovalbumin (OVA)-induced allergic asthma model, we observed that DMF administration reduced the allergic asthma symptoms and IgE levels in the OVA-induced mice model. Histopathological analysis showed that DMF treatment in an OVA-induced animal model eased the inflammation in the nasal and bronchial tissues, with a particular decrease in the infiltration of immune cells. Additionally, RT-qPCR analysis exhibited that treatment of DMF in an OVA-induced model reduced the expression of inflammatory cytokine (IL4, IL13, and IL17) while augmenting anti-inflammatory IL10 and Foxp3 (forkhead box protein 3). Mechanistically, we found that DMF increased the expression of Foxp3 by exacerbating the expression of nuclear factor E2-related factor 2 (Nrf2), and the in-vitro activation of Foxp3+ Tregs leads to an escalated expression of Nrf2. Notably, CD4-specific Nrf2 deletion intensified the allergic asthma symptoms and reduced the in-vitro iTreg differentiation. Meanwhile, DMF failed to exert protective effects on OVA-induced allergic asthma in CD4-specific Nrf2 knock-out mice. Overall, our study illustrates that DMF enhances Nrf2 signaling in T cells to assist the differentiation of Tregs, which could improve the anti-inflammatory immune response in allergic asthma.
Topics: Animals; Female; Mice; Asthma; Cytokines; Dimethyl Fumarate; Mice, Inbred BALB C; Mice, Inbred C57BL; Mice, Knockout; NF-E2-Related Factor 2; Ovalbumin; Signal Transduction; T-Lymphocytes, Regulatory
PubMed: 38711519
DOI: 10.3389/fimmu.2024.1375340 -
Carbohydrate Polymers Aug 2024In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired...
In vivo, cells interact with the extracellular matrix (ECM), which provides a multitude of biophysical and biochemical signals that modulate cellular behavior. Inspired by this, we explored a new methodology to develop a more physiomimetic polysaccharide-based matrix for 3D cell culture. Maleimide-modified alginate (AlgM) derivatives were successfully synthesized using DMTMM to activate carboxylic groups. Thiol-terminated cell-adhesion peptides were tethered to the hydrogel network to promote integrin binding. Rapid and efficient in situ hydrogel formation was promoted by thiol-Michael addition "click" chemistry via maleimide reaction with thiol-flanked protease-sensitive peptides. Alginate derivatives were further ionically crosslinked by divalent ions present in the medium, which led to greater stability and allowed longer cell culture periods. By tailoring alginate's biofunctionality we improved cell-cell and cell-matrix interactions, providing an ECM-like 3D microenvironment. We were able to systematically and independently vary biochemical and biophysical parameters to elicit specific cell responses, creating custom-made 3D matrices. DMTMM-mediated maleimide incorporation is a promising approach to synthesizing AlgM derivatives that can be leveraged to produce ECM-like matrices for a broad range of applications, from in vitro tissue modeling to tissue regeneration.
Topics: Humans; Alginates; Cell Adhesion; Click Chemistry; Cross-Linking Reagents; Extracellular Matrix; Hydrogels; Maleimides; Sulfhydryl Compounds
PubMed: 38710569
DOI: 10.1016/j.carbpol.2024.122144 -
Synthetic and Systems Biotechnology Sep 2024The 3-ketoacyl-CoA thiolase is the rate-limiting enzyme for linear dicarboxylic acids production. However, the promiscuous substrate specificity and suboptimal catalytic...
The 3-ketoacyl-CoA thiolase is the rate-limiting enzyme for linear dicarboxylic acids production. However, the promiscuous substrate specificity and suboptimal catalytic performance have restricted its application. Here we present both biochemical and structural analyses of a high-efficiency 3-ketoacyl-CoA thiolase Tfu_0875. Notably, Tfu_0875 displayed heightened activity and substrate specificity for succinyl-CoA, a key precursor in adipic acid production. To enhance its performance, a deep learning approach (DLKcat) was employed to identify effective mutants, and a computational strategy, known as the greedy accumulated strategy for protein engineering (GRAPE), was used to accumulate these effective mutants. Among the mutants, Tfu_0875 exhibited the highest specific activity (320% of wild-type Tfu_0875), the greatest catalytic efficiency (/ = 1.00 minmM), the highest succinyl-CoA specificity ( = 0.59 mM, 28.1% of Tfu_0875) and dramatically reduced substrate binding energy (-30.25 kcal mol -15.94 kcal mol). A structural comparison between Tfu_0875 and the wild type Tfu_0875 revealed that the increased interaction between the enzyme and succinyl-CoA was the primary reason for the enhanced enzyme activity. This interaction facilitated rapid substrate anchoring and stabilization. Furthermore, a reduced binding pocket volume improved substrate specificity by enhancing the complementarity between the binding pocket and the substrate in stereo conformation. Finally, our rationally designed mutant, Tfu_0875, increased the adipic acid titer by 1.35-fold compared to the wild type Tfu_0875 in shake flask. The demonstrated enzymatic methods provide a promising enzyme variant for the adipic acid production. The above effective substrate binding pocket engineering strategy can be beneficial for the production of other industrially competitive biobased chemicals when be applied to other thiolases.
PubMed: 38694995
DOI: 10.1016/j.synbio.2024.04.014