Disaccharide Trehalose Research Articles

Disaccharide Type Affected Phenolic and Volatile Compounds of Citrus Fiber-Blackberry Cream Fillings.

The food industry is continuously developing ingredients, processing methods and packaging materials to improve the quality of fruit products. The aim of this work was to study the possibility of using citrus fiber in the preparation of blackberry cream fillings in combination with disaccharides (sucrose, maltose and trehalose). Evaluations of the phenolics, proanthocyanidins, antioxidant activity, color and volatiles of blackberry cream fillings were conducted after preparation and after three months of storage. Blackberry cream fillings were prepared from citrus fiber (5%), blackberry juice and disaccharides (50%). Disaccharide type had an effect on all investigated parameters. The highest phenol content was in fillings with trehalose (4.977 g/100 g) and the lowest was in fillings prepared with sucrose (4.249 g/100 g). The same tendency was observed after storage. Fillings with maltose had the highest proanthocyanidins content (473.05 mg/100 g) while fillings with sucrose had the lowest amount (299.03 mg/100 g) of these compounds. Regarding volatile compounds, terpenes and aldehydes and ketones were evaluated in the highest concentration. Terpenes were determined in the highest concentration in fillings with trehalose (358.05 µg/kg), while aldehydes and ketones were highest in fillings with sucrose (250.87 µg/kg). After storage, concentration of volatiles decreased. These results indicate that the selection of adequate disaccharides is very important since it can influence the final quality of the product.

Repression of the TreR transcriptional regulator in Streptococcus mutans by the global regulator, CcpA

Streptococcus mutans, the etiologic agent of dental caries in humans, is considered a dominating force in the oral microbiome due to its highly-evolved propensity for survival. The oral pathogen encodes an elaborate array of regulatory elements, including the carbon catabolite-responsive regulator, CcpA, a global regulator key in the control of sugar metabolism and in stress tolerance response mechanisms. The recently characterized trehalose utilization operon, integral for the catabolism of the disaccharide trehalose, is controlled by a local regulator, TreR, which has been implicated in a number of cellular functions outside of trehalose catabolism. Electrophoretic mobility shift assays demonstrated that CcpA bound a putative cre site in the treR promoter. Loss of ccpA resulted in elevated expression of treR in cultures of the organism grown in glucose or trehalose, indicating that CcpA not only acts as a repressor of trehalose catabolism genes, but also the local regulator. The loss of both CcpA and TreR in S. mutans resulted in an impaired growth rate and fitness response, supporting the hypothesis that these regulators are involved in carbon catabolism control and in induction of components of the organism's stress response.

Enhancing the preservation of liposomes: The role of cryoprotectants, lipid formulations and freezing approaches

In the last decades, liposomes acquired a striking success in the biomedical field thanks to their biocompatibility and drug delivery ability. Many liposomal drug formulations have been already approved by the Food and Drug Administration (FDA) and used for the treatment of a wide range of pathologies with or without further engineering. Their clinical application requires strict compliance with high standard quality rules, and it is crucial to employ storage methods that do not affect the integrity of the vesicles and preventing the leakage of their cargo.In this work, the design of a suitable formulation for freeze-drying had been investigated for two different liposomes, DOPC-DOTAP and the PEGylated counterpart, DOPC-DOTAP-DSPE-PEG. The role of various cryoprotectants was evaluated paying attention to their ability to preserve the structural integrity of liposomes. At first, the study was focused on freezing and two methodologies were investigated, quenching in liquid nitrogen and shelf-ramped freezing. This analysis showed that the disaccharides (cellobiose, glucose, lactose, sucrose, and trehalose) and the polyol (mannitol) protected successfully the integrity of liposomes, while during the process, in the presence of a surfactant, liposomes were strongly damaged and fragmented by the ice crystals. Furthermore, the choice of the rate of freezing depended on the different compositions of the lipid bilayer. Finally, the effects of lyophilization on liposomes with and without additives were studied; cellobiose, lactose and trehalose showed encouraging results for the maintenance of the morpho-functional parameters of liposomes during the entire freeze-drying process.

CryptoCEST: A promising tool for spatially resolved identification of fungal brain lesions and their differentiation from brain tumors with MRI

Infectious brain lesions caused by the pathogenic fungi Cryptococcus neoformans and C. gattii, also referred to as cryptococcomas, could be diagnosed incorrectly as cystic brain tumors if only based on conventional magnetic resonance (MR) images. Previous MR spectroscopy (MRS) studies showed high local concentrations of the fungal disaccharide trehalose in cryptococcomas. The aim of this study was to detect and localize fungal brain lesions caused by Cryptococcus species based on Chemical Exchange Saturation Transfer (CEST) MR imaging of endogenous trehalose, and hereby to distinguish cryptococcomas from gliomas. In phantoms, trehalose and cryptococcal cells generated a concentration-dependent CEST contrast in the 0.2 – 2 ppm chemical shift range, similar to glucose, but approximately twice as strong. In vivo single voxel MRS of a murine cryptococcoma model confirmed the presence of trehalose in cryptococcomas, but mainly for lesions that were large enough compared to the size of the MRS voxel. With CEST MRI, combining the more specific CEST signal at 0.7 ppm with the higher signal-to-noise ratio signal at 4 ppm in the CryptoCEST contrast enabled localization and distinction of cryptococcomas from the normal brain and from gliomas, even for lesions smaller than 1 mm3. Thanks to the high endogenous concentration of the fungal biomarker trehalose in cryptococcal cells, the CryptoCEST contrast allowed identification of cryptococcomas with high spatial resolution and differentiation from gliomas in mice. Furthermore, the CryptoCEST contrast was tested to follow up antifungal treatment of cryptococcomas. Translation of this non-invasive method to the clinic holds potential for improving the differential diagnosis and follow-up of cryptococcal infections in the brain.

Kinetic stability of the water-forming NADH oxidase from Giardia lamblia: implications for biotechnological processes

NAD(P)H oxidases (NOXs) catalyze the oxidation of NAD(P)H by reducing molecular O2 to H2O2 or H2O. Water-forming NOXs have been highlighted as promising biocatalyst systems for regeneration of NAD+ or NADP+ in industrial applications. The NADH oxidase of Giardia lamblia (GlNOX) uses naturally both nicotinamide cofactors producing only water as byproduct; therefore, it can be considered a multipurpose and innocuous regeneration system for redox reactions using either NAD+ or NADP+. From a biotechnological perspective, kinetic stability (and not thermodynamic stability) must be the most important aspect to consider for enzymes involved in industrial applications; therefore, understanding the kinetic stability of a technologically interesting enzyme is an indispensable step toward its application. In this work, we analyze the thermal inactivation kinetics of GlNOX and the influence of diverse additives on it. A combination of 1,4-Dithiothreitol as reducing agent and the disaccharide trehalose protects remarkably GlNOX from inactivation. Structural studies indicate that inactivation is not related to denaturation, whereas overoxidation of the active site cysteine seems to be the main factor related to loss of the enzyme activity. It is concluded that understanding the factors that influence the protein inactivation of GlNOX allowed to improve its kinetic stability, making it biotechnologically valuable.

Zika virus is transmitted in neural progenitor cells via cell-to-cell spread and infection is inhibited by the autophagy inducer trehalose.

Zika virus (ZIKV) is a mosquito-borne human pathogen that causes congenital Zika syndrome and neurological symptoms in some adults. There are currently no approved treatments or vaccines for ZIKV, and exploration of therapies targeting host processes could avoid viral development of drug resistance. The purpose of our study was to determine if the non-toxic and widely used disaccharide trehalose, which showed antiviral activity against Human Cytomegalovirus (HCMV) in our previous work, could restrict ZIKV infection in clinically relevant neural progenitor cells (NPCs). Trehalose is known to induce autophagy, the degradation and recycling of cellular components. Whether autophagy is proviral or antiviral for ZIKV is controversial and depends on cell type and specific conditions used to activate or inhibit autophagy. We show here that trehalose treatment of NPCs infected with recent ZIKV isolates from Panama and Puerto Rico significantly reduces viral replication and spread. In addition, we demonstrate that ZIKV infection in NPCs spreads primarily cell-to-cell as an expanding infectious center, and NPCs are infected via contact with infected cells far more efficiently than by cell-free virus. Importantly, ZIKV was able to spread in NPCs in the presence of neutralizing antibody.Importance Zika virus causes birth defects and can lead to neurological disease in adults. While infection rates are currently low, ZIKV remains a public health concern with no treatment or vaccine available. Targeting a cellular pathway to inhibit viral replication is a potential treatment strategy that avoids development of antiviral resistance. We demonstrate in this study that the non-toxic autophagy-inducing disaccharide trehalose reduces spread and output of ZIKV in infected neural progenitor cells (NPCs), the major cells infected in the fetus. We show that ZIKV spreads cell-to-cell in NPCs as an infectious center and that NPCs are more permissive to infection by contact with infected cells than by cell-free virus. We find that neutralizing antibody does not prevent the spread of the infection in NPCs. These results are significant in demonstrating anti-ZIKV activity of trehalose and in clarifying the primary means of Zika virus spread in clinically relevant target cells.

Photovoltage generation by photosystem II core complexes immobilized onto a Millipore filter on an indium tin oxide electrode.

The light-induced functioning of photosynthetic pigment-protein complex of photosystem II (PSII) is linked to the vectorial translocation of charges across the membrane, which results in the formation of voltage. Direct measurement of the light-induced voltage (∆V) generated by spinach oxygen-evolving PSII core complexes adsorbed onto a Millipore membrane filter (MF) on an indium tin oxide (ITO) electrode under continuous illumination has been performed. PSII was shown to participate in electron transfer from water to the ITO electrode, resulting in ∆V generation. No photovoltage was detected in PSII deprived of the water-oxidizing complex. The maximal and stable photoelectric signal was observed in the presence of disaccharide trehalose and 2,6-dichloro-1,4-benzoquinone, acting as a redox mediator between the primary quinone acceptor QA of PSII and electrode surface. Long time preservation of the steady-state photoactivity at room temperature in a simple in design ITO|PSII-MF|ITO system may be related to the retention of water molecules attached to the PSII surface in the presence of trehalose.

Social Chemical Communication Determines Recovery From L1 Arrest via DAF-16 Activation.

In a population, chemical communication determines the response of animals to changing environmental conditions, what leads to an enhanced resistance against stressors. In response to starvation, the nematode Caenorhabditis elegans arrest post-embryonic development at the first larval stage (L1) right after hatching. As arrested L1 larvae, C. elegans become more resistant to diverse stresses, allowing them to survive for several weeks expecting to encounter more favorable conditions. L1 arrested at high densities display an enhanced resistance to starvation, dependent on soluble compounds released beyond hatching and the first day of arrest. Here, we show that this chemical communication also influences recovery after prolonged periods in L1 arrest. Animals at high density recovered faster than animals at low density. We found that the density effect on survival depends on the final effector of the insulin signaling pathway, the transcription factor DAF-16. Moreover, DAF-16 activation was higher at high density, consistent with a lower expression of the insulin-like peptide DAF-28 in the neurons. The improved recovery of animals after arrest at high density depended on soluble compounds present in the media of arrested L1s. In an effort to find the nature of these compounds, we investigated the disaccharide trehalose as putative signaling molecule, since its production is enhanced during L1 arrest and it is able to activate DAF-16. We detected the presence of trehalose in the medium of arrested L1 larvae at a low concentration. The addition of this concentration of trehalose to animals arrested at low density was enough to rescue DAF-28 production and DAF-16 activation to the levels of animals arrested at high density. However, despite activating DAF-16, trehalose was not capable of reversing survival and recovery phenotypes, suggesting the participation of additional signaling molecules. With all, here we describe a molecular mechanism underlying social communication that allows C. elegans to maintain arrested L1 larvae ready to quickly recover as soon as they encounter nutrient sources.

Application of isomelezitose as an osmoprotectant for biological control agent preservation during drying and storage

ABSTRACT Isomelezitose (IMZ) is of interest as a potential osmoprotectant, but it has not been previously characterised even though the structurally similar trisaccharide melezitose has been previously shown to have excellent protective qualities. The recent discovery that sucrose can be converted to IMZ during glucansucrase reactions suggests that IMZ might become an economical choice as an osmoprotectant for microbial inoculants, such as biological control agents (BCAs). Because of this opportunity, its osmoprotective function was explored in this work using two strains of Pseudomonas fluorescens that are BCAs used to control maladies of potatoes stored postharvest. BCA cell viability and growth recovery rate after drying and storage at 25 oC were assessed. The data showed that IMZ and impure IMZ reaction products can function similarly to melezitose and trehalose to significantly reduce viable cell and activity losses during air drying and storage. Biocontrol treatments formulated with IMZ retained the ability to suppress Fusarium dry rot disease by up to 50% under severe disease challenge, even after storage 28 d at 25 oC in vacuum-sealed bags. Biocontrol treatments with IMZ suppressed disease by 15–35% more thancontrols without any added osmoprotectant. Neither IMZ nor two impure enzyme reaction products impacted germination of dry rot causative Fusarium sambucinum conidia or subsequent hyphal growth under gnotobiotic conditions. For the first time, the osmoprotective capability of IMZ has been demonstrated and shown to be potentially superior to others previously reported, including the structurally related compound melezitose and the currently marketed disaccharide trehalose.

Exogenous trehalose ameliorates methyl viologen induced oxidative stress through regulation of stomatal pore opening and glutathione metabolism in tomato seedlings

Oxidative burst is the common thread linking all abiotic and biotic stressors. So in this study non-reducing disaccharide trehalose (Tre) has been examined for attenuation of oxidative stress, imposed through methyl viologen (MV) foliar spray in tomato seedlings. Initially the ameliorating property was ascertained by leaf disc senescence and stomatal pore opening assay, before carrying out the foliar spray on seedlings. Both these assays showed Tre mediated mitigation of MV induced oxidative stress. It was also observed that trehalose dose determines the extent of stomatal pore opening. MV treated seedlings showed more reactive oxygen species (ROS) accumulation compared to control, Tre and MV + Tre treated plants both histochemically and quantitatively. Damage due to ROS accumulation was documented by quantifying malondialdehye and lipoxygenase activity. Finally, the response of the tomato seedlings to oxidative stress was monitored by quantifying the antioxidant enzyme activities and non-enzymatic antioxidant content under stressed and unstressed conditions. Also transcript levels of glutathione synthetase (GS) and gamma-glutamylcysteine synthetase (γ-ECS), enzymes responsible for GSH synthesis; and transcription factors no apical meristem ATAF and cup-shaped cotyledon (NAC2) and dehydration-responsive element-binding (DREB) were found to increase on application of Tre. All of these experimentations showed better adaptability and survivability of the seedlings treated exogenously with both MV and Tre in comparison to those treated only with MV.

Measurement of the diffusion coefficient and hydrogen bonds of water in a dry-protective solution by microscopic near-infrared spectroscopy

The water diffusion coefficients in highly concentrated ε-poly-l-lysine (PLL) and the disaccharides trehalose and sucrose were obtained by measuring the water distribution in sample solutions during the unsteady one-dimensional drying. The combination peak area of water in the near-infrared absorbance spectrum was measured to evaluate the water retained in the sample. The water diffusion coefficients were calculated by solving the differential equation of the diffusion coefficient that is derived from Fick’s law. Additionally, the measured combination peak areas of water allow for evaluating the hydrogen bond states of water molecules in sample solution. The evaluated hydrogen bond states of PLL and the disaccharide solutions showed drastic changes at approximately 50wt% water mass concentration, below which the water diffusion coefficients with respect to the water mass concentration are not identical. From these results, the conformation changes in PLL and the strong interactions of water and sample solutes are suggested.

Abstract 227: Maltose enhanced the growth of bladder and colon cancer cells unlike some other disaccharides: Cellobiose, isomaltose, lactose, and sucrose

Abstract The addiction of most cancer cells to the metabolism of glucose is well known. The metabolism by cancer cells of other saccharides is less well characterized. We have studied the impact on growth of human bladder and colon cancer cells of mono- and disaccharides. The standard RPMI 1640 medium contains 2 mg glucose per ml. Substitution for glucose by other monosaccharides (fructose, galactose and mannose) resulted in similar growth in some cell lines but greatly diminished growth in others. For the Caco-2 and HT29 colon cancer cells, co-incubation with 2-deoxyglucose was more inhibitory for growth with fructose than with glucose as substrate. There was a similar situation with some bladder cancer cell lines (5637, HT1197 and RT4) whereas with other bladder cancer cells (HT1376, T24 and UM-UC-3) 2-deoxyglucose caused greater inhibition with glucose. In those cell lines in which alkaline phosphatase activity could be induced after incubation with butyrate (Caco-2, HT29, 5637, HT1197 and HT1376), induction by butyrate was observed with any of the monosaccharides. Similarly with the disaccharides lactose, maltose and trehalose there was induction of alkaline phosphatase by butyrate. In the course of this work it was apparent that maltose could enhance growth to an extent that was sometimes not significantly different to that seen with glucose and was not seen with cellobiose, isomaltose, lactose or sucrose. HT29 cells were the only cell line to have a substantial increased growth with trehalose. The increased growth with maltose was inhibited by 2-deoxyglucose. In conclusion, maltose increased growth of bladder and colon cancer cells without a period of adaptation. Citation Format: Michael A. Lea, Charles desBordes. Maltose enhanced the growth of bladder and colon cancer cells unlike some other disaccharides: Cellobiose, isomaltose, lactose, and sucrose [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 227.

Structural Analysis of Binding Determinants of Salmonella typhimurium Trehalose-6-phosphate Phosphatase Using Ground-State Complexes.

Trehalose-6-phosphate phosphatase (T6PP) catalyzes the dephosphorylation of trehalose 6-phosphate (T6P) to the disaccharide trehalose. The enzyme is not present in mammals but is essential to the viability of multiple lower organisms as trehalose is a critical metabolite, and T6P accumulation is toxic. Hence, T6PP is a target for therapeutics of human pathologies caused by bacteria, fungi, and parasitic nematodes. Here, we report the X-ray crystal structures of Salmonella typhimurium T6PP (StT6PP) in its apo form and in complex with the cofactor Mg2+ and the substrate analogue trehalose 6-sulfate (T6S), the product trehalose, or the competitive inhibitor 4-n-octylphenyl α-d-glucopyranoside 6-sulfate (OGS). OGS replaces the substrate phosphoryl group with a sulfate group and the glucosyl ring distal to the sulfate group with an octylphenyl moiety. The structures of these substrate-analogue and product complexes with T6PP show that specificity is conferred via hydrogen bonds to the glucosyl group proximal to the phosphoryl moiety through Glu123, Lys125, and Glu167, conserved in T6PPs from multiple species. The structure of the first-generation inhibitor OGS shows that it retains the substrate-binding interactions observed for the sulfate group and the proximal glucosyl ring. The OGS octylphenyl moiety binds in a unique manner, indicating that this subsite can tolerate various chemotypes. Together, these findings show that these conserved interactions at the proximal glucosyl ring binding site could provide the basis for the development of broad-spectrum therapeutics, whereas variable interactions at the divergent distal subsite could present an opportunity for the design of potent organism-specific therapeutics.

Lactulose and Melibiose Attenuate MPTP-Induced Parkinson's Disease in Mice by Inhibition of Oxidative Stress, Reduction of Neuroinflammation and Up-Regulation of Autophagy.

Parkinson’s disease (PD) is a common neurodegenerative disease characterized by the progressive loss of dopaminergic (DAergic) neurons in the ventral brain. A disaccharide trehalose has demonstrated the potential to mitigate the DAergic loss in disease models for PD. However, trehalose is rapidly hydrolyzed into glucose by trehalase in the intestine, limiting its potential for clinical practice. Here, we investigated the neuroprotective potential of two trehalase-indigestible analogs, lactulose and melibiose, in sub-chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD. Treatment with MPTP generated significant motor deficits, inhibited dopamine levels, and down-regulated dopamine transporter (DAT) in the striatum. Expression levels of genes involved in anti-oxidative stress pathways, including superoxide dismutase 2 (SOD2), nuclear factor erythroid 2-related factor 2 (NRF2), and NAD(P)H dehydrogenase (NQO1) were also down-regulated. Meanwhile, expression of the oxidative stress marker 4-hydroxynonenal (4-HNE) was up-regulated along with increased microglia and astrocyte reactivity in the ventral midbrain following MPTP treatment. MPTP also reduced the activity of autophagy, evaluated by the autophagosomal marker microtubule-associated protein 1 light chain 3 (LC3)-II. Lactulose and melibiose significantly rescued motor deficits, increased dopamine in the striatum, reduced microglia and astrocyte reactivity as well as decreased levels of 4-HNE. Furthermore, lactulose and melibiose up-regulated SOD2, NRF2, and NQO1 levels, as well as enhanced the LC3-II/LC3-I ratio in the ventral midbrain with MPTP treatment. Our findings indicate the potential of lactulose and melibiose to protect DAergic neurons in PD.

Trehalose alleviates oxidative stress-mediated liver injury and Mallor-Denk body formation via activating autophagy in mice.

Autophagy is a degradation pathway for long-lived cytoplasmic proteins or damaged organelles and also for many aggregate-prone and disease-causing proteins. Endoplasmic reticulum (ER) stress and oxidative stress are associated with the pathophysiology of various liver diseases. These stresses induce the accumulation of abnormal proteins, Mallory-Denk body (MDB) formation and apoptosis in hepatocytes. A disaccharide trehalose had been reported to induce autophagy and decrease aggregate-prone proteins and cytotoxicity in neurodegenerative disease models. But the effects of trehalose in hepatocytes have not been fully understood. We examined the effect of trehalose on autophagy, ER stress and oxidative stress-mediated cytotoxicity and MDB formation in hepatocytes using mice model with 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) treatment for 3months. We administered trehalose by intraperitoneal injection of water containing 10% trehalose (0.02mg/g body weight) every other day for 3months. Our results demonstrated that trehalose induced autophagy and reduced ER stress, oxidative stress, MDB formation and apoptosis in hepatocytes of DDC-fed mice by Western blotting and immunostaining analyses. Electron microscopy revealed that trehalose induced autolysosome formation, which located is close to the MDBs. Thus, our findings suggest that trehalose can become a therapeutic agent for oxidative stress-related liver diseases via activating autophagy.

Kramers’ Theory and the Dependence of Enzyme Dynamics on Trehalose-Mediated Viscosity

The disaccharide trehalose is accumulated in the cytoplasm of some organisms in response to harsh environmental conditions. Trehalose biosynthesis and accumulation are important for the survival of such organisms by protecting the structure and function of proteins and membranes. Trehalose affects the dynamics of proteins and water molecules in the bulk and the protein hydration shell. Enzyme catalysis and other processes dependent on protein dynamics are affected by the viscosity generated by trehalose, as described by the Kramers’ theory of rate reactions. Enzyme/protein stabilization by trehalose against thermal inactivation/unfolding is also explained by the viscosity mediated hindering of the thermally generated structural dynamics, as described by Kramers’ theory. The analysis of the relationship of viscosity–protein dynamics, and its effects on enzyme/protein function and other processes (thermal inactivation and unfolding/folding), is the focus of the present work regarding the disaccharide trehalose as the viscosity generating solute. Finally, trehalose is widely used (alone or in combination with other compounds) in the stabilization of enzymes in the laboratory and in biotechnological applications; hence, considering the effect of viscosity on catalysis and stability of enzymes may help to improve the results of trehalose in its diverse uses/applications.

TFEB is a master regulator of tumor-associated macrophages in breast cancer

BackgroundTumor-associated macrophages (TAMs) play key roles in the development of many malignant solid tumors including breast cancer. They are educated in the tumor microenvironment (TME) to promote tumor growth, metastasis, and therapy resistance. However, the phenotype of TAMs is elusive and how to regulate them for therapeutic purpose remains unclear; therefore, TAM-targeting therapies have not yet achieved clinical success. The purposes of this study were to examine the role of transcription factor EB (TFEB) in regulating TAM gene expression and function and to determine if TFEB activation can halt breast tumor development.MethodsMicroarrays were used to analyze the gene expression profile of macrophages (MΦs) in the context of breast cancer and to examine the impact of TFEB overexpression. Cell culture studies were performed to define the mechanisms by which TFEB affects MΦ gene expression and function. Mouse studies were carried out to investigate the impact of MΦ TFEB deficiency or activation on breast tumor growth. Human cancer genome data were analyzed to reveal the prognostic value of TFEB and its regulated genes.ResultsTAM-mimic MΦs display a unique gene expression profile, including significant reduction in TFEB expression. TFEB overexpression favorably modulates TAM gene expression through multiple signaling pathways. Specifically, TFEB upregulates suppressor of cytokine signaling 3 (SOCS3) and peroxisome proliferator-activated receptor γ (PPARγ) expression and autophagy/lysosome activities, inhibits NLRP3 (NLR Family Pyrin Domain Containing 3) inflammasome and hypoxia-inducible factor (HIF)-1α mediated hypoxia response, and thereby suppresses an array of effector molecules in TAMs including arginase 1, interleukin (IL)-10, IL-1β, IL-6 and prostaglandin E2. MΦ-specific TFEB deficiency promotes, while activation of TFEB using the natural disaccharide trehalose halts, breast tumor development by modulating TAMs. Analysis of human patient genome database reveals that expression levels of TFEB, SOCS3 and PPARγ are positive prognostic markers, while HIF-1α is a negative prognostic marker of breast cancer.ConclusionsOur study identifies TFEB as a master regulator of TAMs in breast cancer. TFEB controls TAM gene expression and function through multiple autophagy/lysosome-dependent and independent pathways. Therefore, pharmacological activation of TFEB would be a promising therapeutic approach to improve the efficacy of existing treatment including immune therapies for breast cancer by favorably modulating TAM function and the TME.

Lactulose and Melibiose Inhibit α-Synuclein Aggregation and Up-Regulate Autophagy to Reduce Neuronal Vulnerability.

Parkinson’s disease (PD) is a neurodegenerative disease characterized by selective dopaminergic (DAergic) neuronal degeneration in the substantia nigra (SN) and proteinaceous α-synuclein-positive Lewy bodies and Lewy neuritis. As a chemical chaperone to promote protein stability and an autophagy inducer to clear aggregate-prone proteins, a disaccharide trehalose has been reported to alleviate neurodegeneration in PD cells and mouse models. Its trehalase-indigestible analogs, lactulose and melibiose, also demonstrated potentials to reduce abnormal protein aggregation in spinocerebellar ataxia cell models. In this study, we showed the potential of lactulose and melibiose to inhibit α-synuclein aggregation using biochemical thioflavin T fluorescence, cryogenic transmission electron microscopy (cryo-TEM) and prokaryotic split Venus complementation assays. Lactulose and melibiose further reduced α-synuclein aggregation and associated oxidative stress, as well as protected cells against α-synuclein-induced neurotoxicity by up-regulating autophagy and nuclear factor, erythroid 2 like 2 (NRF2) pathway in DAergic neurons derived from SH-SY5Y cells over-expressing α-synuclein. Our findings strongly indicate the potential of lactulose and melibiose for mitigating PD neurodegeneration, offering new drug candidates for PD treatment.

MtrP, a putative methyltransferase in Corynebacteria, is required for optimal membrane transport of trehalose mycolates

Pathogenic bacteria of the genera Mycobacterium and Corynebacterium cause severe human diseases such as tuberculosis (Mycobacterium tuberculosis) and diphtheria (Corynebacterium diphtheriae). The cells of these species are surrounded by protective cell walls rich in long-chain mycolic acids. These fatty acids are conjugated to the disaccharide trehalose on the cytoplasmic side of the bacterial cell membrane. They are then transported across the membrane to the periplasm where they act as donors for other reactions. We have previously shown that transient acetylation of the glycolipid trehalose monohydroxycorynomycolate (hTMCM) enables its efficient transport to the periplasm in Corynebacterium glutamicum and that acetylation is mediated by the membrane protein TmaT. Here, we show that a putative methyltransferase, encoded at the same genetic locus as TmaT, is also required for optimal hTMCM transport. Deletion of the C. glutamicum gene NCgl2764 (Rv0224c in M. tuberculosis) abolished acetyltrehalose monocorynomycolate (AcTMCM) synthesis, leading to accumulation of hTMCM in the inner membrane and delaying its conversion to trehalose dihydroxycorynomycolate (h2TDCM). Complementation with NCgl2764 normalized turnover of hTMCM to h2TDCM. In contrast, complementation with NCgl2764 derivatives mutated at residues essential for methyltransferase activity failed to rectify the defect, suggesting that NCgl2764/Rv0224c encodes a methyltransferase, designated here as MtrP. Comprehensive analyses of the individual mtrP and tmaT mutants and of a double mutant revealed strikingly similar changes across several lipid classes compared with WT bacteria. These findings indicate that both MtrP and TmaT have nonredundant roles in regulating AcTMCM synthesis, revealing additional complexity in the regulation of trehalose mycolate transport in the Corynebacterineae.

Bacterial Natural Disaccharide (Trehalose Tetraester): Molecular Modeling and in Vitro Study of Anticancer Activity on Breast Cancer Cells.

Isolation and characterization of new biologically active substances affecting cancer cells is an important issue of fundamental research in biomedicine. Trehalose lipid was isolated from Rhodococcus wratislaviensis strain and purified by liquid chromatography. The effect of trehalose lipid on cell viability and migration, together with colony forming assays, were performed on two breast cancer (MCF7—low metastatic; MDA-MB231—high metastatic) and one “normal” (MCF10A) cell lines. Molecular modeling that details the structure of the neutral and anionic form (more stable at physiological pH) of the tetraester was carried out. The tentative sizes of the hydrophilic (7.5 Å) and hydrophobic (12.5 Å) portions of the molecule were also determined. Thus, the used trehalose lipid is supposed to interact as a single molecule. The changes in morphology, adhesion, viability, migration, and the possibility of forming colonies in cancer cell lines induced after treatment with trehalose lipid were found to be dose and time dependent. Based on the theoretical calculations, a possible mechanism of action and membrane asymmetry between outer and inner monolayers of the bilayer resulting in endosome formation were suggested. Initial data suggest a mechanism of antitumor activity of the purified trehalose lipid and its potential for biomedical application.