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ACS Infectious Diseases Apr 2024Tuberculosis (TB), caused by (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple...
Tuberculosis (TB), caused by (Mtb), is the leading cause of death worldwide by infectious disease. Treatment of Mtb infection requires a six-month course of multiple antibiotics, an extremely challenging regimen necessitated by Mtb's ability to form drug-tolerant persister cells. Mtb persister formation is dependent on the trehalose catalytic shift, a stress-responsive metabolic remodeling mechanism in which the disaccharide trehalose is liberated from cell surface glycolipids and repurposed as an internal carbon source to meet energy and redox demands. Here, using a biofilm-persister model, metabolomics, and cryo-electron microscopy (EM), we found that azidodeoxy- and aminodeoxy-d-trehalose analogues block the Mtb trehalose catalytic shift through inhibition of trehalose synthase TreS (Rv0126), which catalyzes the isomerization of trehalose to maltose. Out of a focused eight-member compound panel constructed by chemoenzymatic synthesis, the natural product 2-trehalosamine exhibited the highest potency and significantly potentiated first- and second-line TB drugs in broth culture and macrophage infection assays. We also report the first structure of TreS bound to a substrate analogue inhibitor, obtained via cryo-EM, which revealed conformational changes likely essential for catalysis and inhibitor binding that can potentially be exploited for future therapeutic development. Our results demonstrate that inhibition of the trehalose catalytic shift is a viable strategy to target Mtb persisters and advance trehalose analogues as tools and potential adjunctive therapeutics for investigating and targeting mycobacterial persistence.
Topics: Humans; Mycobacterium tuberculosis; Trehalose; Cryoelectron Microscopy; Tuberculosis; Catalysis
PubMed: 38485491
DOI: 10.1021/acsinfecdis.4c00138 -
STAR Protocols Jun 2024Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent...
Polyphosphate exhibits a unique post-translational modification-like function, known as histidine polyphosphate modification (HPM), marked by a robust non-covalent interaction with histidine repeat proteins. Here, we present a protocol for detecting HPM of human proteins via maltose-binding protein-tagged expression in E. coli. We describe steps for detecting HPM by observing electrophoretic mobility shifts on NuPAGE gels followed by western blot. We then detail procedures for analyzing the influence of ionic strength and pH on HPM. For complete details on the use and execution of this protocol, please refer to Neville et al..
Topics: Escherichia coli; Humans; Polyphosphates; Maltose-Binding Proteins; Histidine; Protein Processing, Post-Translational; Blotting, Western
PubMed: 38470910
DOI: 10.1016/j.xpro.2024.102947 -
Veterinary Research Forum : An... 2024The non-structural protein (nsp) 8 of the porcine epidemic diarrhea virus (PEDV) is highly stable across different PEDV strains and plays an important role in PEDV...
The non-structural protein (nsp) 8 of the porcine epidemic diarrhea virus (PEDV) is highly stable across different PEDV strains and plays an important role in PEDV virulence. In current study, nsp8 prokaryotic expression vectors were constructed based on parental vectors pMAL-c2x-maltose binding protein (MBP) and pET-28a (+). Subsequently, the optimization of expression conditions in , including induced temperature, time and isopropyl β-D-thiogalactopyranoside concentration were performed to obtain a stable expression of MBP-nsp8 and nsp8. The nsp8 fused with MBP increased the water solubility of the expressed products. Target proteins were further purified from culture and their immunogenicities were evaluated by mice. The antibody titers of serum from nsp8 immunized mice were up to 1:7,750,000 when measured by indirect enzyme-linked immunosorbent assay; meanwhile, the mice immunized with MBP-nsp8 gave an antibody titer reaching 1:1,000,000. In all, the expression and purification system of PEDV nsp8 and MBP-nsp8 were successfully established in this work and a strong immune response was elicited in mice by both purified nsp8 and MBP-nsp8, providing a basis for the study of the structure and function of PEDV nsp8.
PubMed: 38465319
DOI: 10.30466/vrf.2023.2009322.3977 -
Food Science & Nutrition Mar 2024There is a dearth of information on the comparative studies of the physicochemical, proximate, and antioxidant properties as well as quality standards of stingless bee...
There is a dearth of information on the comparative studies of the physicochemical, proximate, and antioxidant properties as well as quality standards of stingless bee honey (SBH) in Ethiopia. Hence, this study was designed to assess and compare the physicochemical, proximate, and antioxidant properties of SBH, specifically sourced from , and produced under both wild and modern apiary conditions at two distinct geographical locations. A total of forty-six honey samples were meticulously collected from domesticated stingless bee colonies and naturally occurring wild nests at Wolmera and Cheliya districts. Pollen analysis unveiled eleven distinct bee plant species distributed across six families, with being the most prevalent, primarily represented by and . Notably, the physicochemical, proximate, and antioxidant properties of SBH collected from modern pot hives exhibited significant variances ( < .05) when compared to SBH from wild nests. Principal component analysis (PCA) delineated the differentiation of SBH sources based on both geographical location and the type of beehive. One-way ANOVA corroborated these distinctions, underscoring significantly higher levels ( < .05) of ash, electrical conductivity, free acidity, hydroxymethylfurfural, sucrose, total phenolic content, total flavonoid content, and radical scavenging activities of SBH from modern pot hives in Wolmera. Whereas, Cheliya modern pot hives recorded higher values in pH, hydroxymethylfurfural and maltose contents compared to the wild nest SBH. Further analysis through Pearson correlation highlighted a strong positive association between total phenolic content and total flavonoid content with the antioxidant capacity of SBH. These findings underscore the significance of integrating modern pot hives to enhance the quality of SBH within Ethiopia's beekeeping sector.
PubMed: 38455209
DOI: 10.1002/fsn3.3861 -
Food Science & Nutrition Mar 2024The kefir-based smoothies with kale and spinach were designed as a ready-to-drink and innovative functional snack. Microbiological, physicochemical, as well as pre- and...
The kefir-based smoothies with kale and spinach were designed as a ready-to-drink and innovative functional snack. Microbiological, physicochemical, as well as pre- and postgastrointestinal total antioxidant capacity (TAC; CUPRAC, DPPH, and FRAP) analyses were conducted. It was determined that the kefir-based smoothies with vegetables had higher ash, carbohydrate, and dietary fiber values. Fructose and glucose contents of smoothy with kale were high, while smoothy with spinach included high sucrose and maltose. The microbiology results revealed that kefir-based vegetable smoothies had minimum Lactobacillaceae viability (>log 7 cfu g) for the required functional effect after 14-day storage. Moreover, the addition of kale significantly increased ( < .01) the level of initial TAC (CUPRAC, DPPH, and FRAP) and total phenolic content (TPC) values. After in vitro gastric digestion analysis, smoothie with spinach demonstrated higher TAC and TPC values and the control sample had higher TAC and TPC values compared with a predigestion step. It was found that in vitro intestinal DPPH values were higher for the sample with spinach samples, while the sample with kale had the highest FRAP values. It was also found that the bioaccessibility indexes of plain kefir were determined to be the highest in both in vitro gastric and intestinal procedures. The present study provided novel insights into the in vitro digestion properties of kefir fortified with vegetables. Nevertheless, further studies are needed to identify the functional properties of the milk and plant matrices mixture using in vitro and in vivo trials.
PubMed: 38455206
DOI: 10.1002/fsn3.3917 -
Scientific Reports Mar 2024Byproducts from the sugarcane manufacturing process, specifically sugarcane molasses (SM) and sugarcane bagasse (SB), can be used as alternative raw materials for...
Byproducts from the sugarcane manufacturing process, specifically sugarcane molasses (SM) and sugarcane bagasse (SB), can be used as alternative raw materials for sorbitol production via the biological fermentation process. This study investigated the production of sorbitol from SM and sugarcane bagasse hydrolysate (SBH) using a thermally adapted Zymomonas mobilis ZM AD41. Various combinations of SM and SBH on sorbitol production using batch fermentation process were tested. The results revealed that SM alone (FM1) or a mixture of SM and SBH at a ratio of 3:1 (FM2) based on the sugar mass in the raw material proved to be the best condition for sorbitol production by ZM AD41 at 37 °C. Further optimization conditions for sorbitol production revealed that a sugar concentration of 200 g/L and a CaCl concentration of 5.0 g/L yielded the highest sorbitol content. The maximum sorbitol concentrations produced by ZM AD41 in the fermentation medium containing SM (FM1) or a mixture of SM and SBH (FM2) were 31.23 and 30.45 g/L, respectively, comparable to those reported in the literature using sucrose or a mixture of sucrose and maltose as feedstock. These results suggested that SBH could be used as an alternative feedstock to supplement or blend with SM for sustainable sorbitol production. In addition, the fermentation conditions established in this study could also be applied to large-scale sorbitol production. Moreover, the thermally adapted Z. mobilis ZM AD41 is also a promising sorbitol-producing bacterium for large-scale production at a relatively high fermentation temperature using agricultural byproducts, specifically SM and SB, as feedstock, which could reduce the operating cost due to minimizing the energy required for the cooling system.
Topics: Cellulose; Sorbitol; Zymomonas; Molasses; Saccharum; Maltose; Sucrose
PubMed: 38448501
DOI: 10.1038/s41598-024-56307-8 -
Molecular & Cellular Proteomics : MCP Apr 2024In recent years, there has been a growing demand for low-input proteomics, particularly in the context of single-cell proteomics (SCP). In this study, we have developed...
In recent years, there has been a growing demand for low-input proteomics, particularly in the context of single-cell proteomics (SCP). In this study, we have developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method. This method effectively reduces protein and peptide loss in samples by incorporating LMNG, a surfactant, into the digestion solution and subsequently removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is the prevention of mass spectrometry (MS) contamination. When we applied the LASP method to the low-input SP3 method and on-bead digestion in coimmunoprecipitation-MS, we observed a significant improvement in the recovery of the digested peptides. Furthermore, we have established a simple and easy sample preparation method for SCP based on the LASP method and identified a median of 1175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.
Topics: Maltose; Glycols; Single-Cell Analysis; Proteomics; Analytic Sample Preparation Methods; Humans; HEK293 Cells; Liquid Chromatography-Mass Spectrometry; Immunoprecipitation
PubMed: 38447790
DOI: 10.1016/j.mcpro.2024.100745 -
The Indian Journal of Medical Research Jan 2024Iron deficiency anaemia (IDA) during pregnancy is treated with oral and parenteral iron. The objective of this review was to compare the clinical effectiveness, safety,... (Meta-Analysis)
Meta-Analysis
BACKGROUND OBJECTIVES
Iron deficiency anaemia (IDA) during pregnancy is treated with oral and parenteral iron. The objective of this review was to compare the clinical effectiveness, safety, pregnancy and neonatal outcomes of intravenous (iv) ferric carboxymaltose (FCM) and iv iron sucrose (IS) in treating IDA in pregnancy.
METHODS
The Department of Health Research funded this study. PubMed, Cochrane Library, EMBASE and Scopus were searched to include studies published till November 2022. The protocol was registered in PROSPERO (CRD42022306092). Pregnant women (15-49 yr) in second and third trimesters, diagnosed with moderate-to-severe iron deficiency anaemia, treated with either of the drugs were included. The included studies were critically assessed using appropriate tools. We conducted a qualitative synthesis of the studies and meta-analysis for improvement in haematological parameters and incidence of adverse events.
RESULTS
A total of 18 studies were included. The risk of bias was low to moderate. A rise in haemoglobin up to four weeks was higher with FCM than IS by 0.57 (0.24, 0.9) g/dl. Intravenous FCM is associated with fewer adverse events than IS [pooled odds ratio: 0.5 (0.32, 0.79)]. The included studies had limited evidence on pregnancy and neonatal outcomes after iv iron treatment.
INTERPRETATION CONCLUSIONS
Intravenous FCM is effective and safer than intravenous IS in terms of haematological parameters, in treating IDA in pregnancy. Further research is required on the effects of iv FCM and iv IS on the pregnancy and neonatal outcomes when used for treating IDA in pregnancy.
Topics: Pregnancy; Infant, Newborn; Female; Humans; Ferric Oxide, Saccharated; Anemia, Iron-Deficiency; Treatment Outcome; Iron; Ferric Compounds; Maltose
PubMed: 38439125
DOI: 10.4103/ijmr.ijmr_246_23 -
Journal of Controlled Release :... Apr 2024Intravenous (IV) iron-carbohydrate complexes are widely used nanoparticles (NPs) to treat iron deficiency anaemia, often associated with medical conditions such as...
Intravenous (IV) iron-carbohydrate complexes are widely used nanoparticles (NPs) to treat iron deficiency anaemia, often associated with medical conditions such as chronic kidney disease, heart failure and various inflammatory conditions. Even though a plethora of physicochemical characterisation data and clinical studies are available for these products, evidence-based correlation between physicochemical properties of iron-carbohydrate complexes and clinical outcome has not fully been elucidated yet. Studies on other metal oxide NPs suggest that early interactions between NPs and blood upon IV injection are key to understanding how differences in physicochemical characteristics of iron-carbohydrate complexes cause variance in clinical outcomes. We therefore investigated the core-ligand structure of two clinically relevant iron-carbohydrate complexes, iron sucrose (IS) and ferric carboxymaltose (FCM), and their interactions with two structurally different human plasma proteins, human serum albumin (HSA) and fibrinogen, using a combination of cryo-scanning transmission electron microscopy (cryo-STEM), x-ray diffraction (XRD), small-angle x-ray scattering (SAXS) and small-angle neutron scattering (SANS). Using this orthogonal approach, we defined the nano-structure, individual building blocks and surface morphology for IS and FCM. Importantly, we revealed significant differences in the surface morphology of the iron-carbohydrate complexes. FCM shows a localised carbohydrate shell around its core, in contrast to IS, which is characterised by a diffuse and dynamic layer of carbohydrate ligand surrounding its core. We hypothesised that such differences in carbohydrate morphology determine the interaction between iron-carbohydrate complexes and proteins and therefore investigated the NPs in the presence of HSA and fibrinogen. Intriguingly, IS showed significant interaction with HSA and fibrinogen, forming NP-protein clusters, while FCM only showed significant interaction with fibrinogen. We postulate that these differences could influence bio-response of the two formulations and their clinical outcome. In conclusion, our study provides orthogonal characterisation of two clinically relevant iron-carbohydrate complexes and first hints at their interaction behaviour with proteins in the human bloodstream, setting a prerequisite towards complete understanding of the correlation between physicochemical properties and clinical outcome.
Topics: Humans; Iron; Scattering, Small Angle; Ligands; X-Ray Diffraction; Ferric Compounds; Ferric Oxide, Saccharated; Anemia, Iron-Deficiency; Metal Nanoparticles; Fibrinogen; Maltose
PubMed: 38438093
DOI: 10.1016/j.jconrel.2024.02.044 -
Plant Biotechnology (Tokyo, Japan) Dec 2023In early developing tomato ( L.) fruit, starch accumulates at high levels and is used by various primary metabolites in ripening fruits. ADP-glucose pyrophosphorylase is...
In early developing tomato ( L.) fruit, starch accumulates at high levels and is used by various primary metabolites in ripening fruits. ADP-glucose pyrophosphorylase is responsible for the first key step of starch biosynthesis. Although it has been reported that and isoforms are mainly expressed in early developing fruit, their regulatory mechanism has not been elucidated. The present study investigated the transcriptional response of and to various metabolizable sugars, nonmetabolizable sugar analogues, hexokinase inhibitors and proline by an experimental system using half-cut fruits. was upregulated in response to sucrose and constituted hexoses such glucose, whereas the gene almost did not exhibit a prominent sugar response. Further analyses revealed that other disaccharides such maltose and trehalose did not show a remarkable effect on both and expressions. These results indicate that there are two distinct regulatory mechanisms, namely, sugar metabolism-dependent and -independent, for the regulation of AGPase gene expression. Interestingly, the ADP treatment, a hexokinase inhibitors, cancelled the sugar response of , indicating that hexokinase-mediated sugar signaling should be involved in the sugar response of These results suggest that sugar-dependent () and sugar-independent () pathways coordinatively regulate starch biosynthesis in immature tomato fruit.
PubMed: 38434118
DOI: 10.5511/plantbiotechnology.23.1004a