Circadian rhythm and disease: Relationship, new insights, and future perspectives.

The circadian system is responsible for internal functions and regulation of the organism according to environmental cues (zeitgebers). Circadian rhythm dysregulation or chronodisruption has been associated with several diseases, from mental to autoimmune diseases, and with life quality change. Following this, some therapies have been developed to correct circadian misalignments, such as light therapy and chronobiotics. In this manuscript, we describe the circadian-related diseases so far investigated, and studies reporting relevant data on this topic, evidencing this relationship, are included. Despite the actual limitations in published work, there is clear evidence of the correlation between circadian rhythm dysregulation and disease origin/development, and, in this way, clock-related therapies emerge as great progress in the clinical field. Future improvements in such interventions can lead to the development of successful chronotherapy strategies, deeply contributing to enhanced therapeutic outcomes.

Antifouling Marine Coatings with a Potentially Safer and Sustainable Synthetic Polyphenolic Derivative.

The development of harmless substances to replace biocide-based coatings used to prevent or manage marine biofouling and its unwanted consequences is urgent. The formation of biofilms on submerged marine surfaces is one of the first steps in the marine biofouling process, which facilitates the further settlement of macrofoulers. Anti-biofilm properties of a synthetic polyphenolic compound, with previously described anti-settlement activity against macrofoulers, were explored in this work. In solution this new compound was able to prevent biofilm formation and reduce a pre-formed biofilm produced by the marine bacterium, Pseudoalteromonas tunicata. Then, this compound was applied to a marine coating and the formation of P. tunicata biofilms was assessed under hydrodynamic conditions to mimic the marine environment. For this purpose, polyurethane (PU)-based coating formulations containing 1 and 2 wt.% of the compound were prepared based on a prior developed methodology. The most effective formulation in reducing the biofilm cell number, biovolume, and thickness was the PU-based coating containing an aziridine-based crosslinker and 2 wt.% of the compound. To assess the marine ecotoxicity impact of this compound, its potential to disrupt endocrine processes was evaluated through the modulation of two nuclear receptors (NRs), peroxisome proliferator-activated receptor γ (PPARγ), and pregnane X receptor (PXR). Transcriptional activation of the selected NRs upon exposure to the polyphenolic compound (10 µM) was not observed, thus highlighting the eco-friendliness towards the addressed NRs of this new dual-acting anti-macro- and anti-microfouling agent towards the addressed NRs.

Overcoming environmental problems of biocides: Synthetic bile acid derivatives as a sustainable alternative.

Marine biofouling represents a global economic and ecological challenge. Some marine organisms produce bioactive metabolites, such as steroids, that inhibit the settlement and growth of fouling organisms. The aim of this work was to explore bile acids as a new scaffold with antifouling (AF) activity by using chemical synthesis to produce a series of bile acid derivatives with optimized AF performance and understand their structure-activity relationships. Seven bile acid derivatives were successfully synthesized in moderate to high yields, and their structures were elucidated through spectroscopic methods. Their AF activities were tested against both macro- and microfouling communities. The most potent bile acid against the settlement of Mytilus galloprovincialis larvae was the methyl ester derivative of cholic acid (10), which showed an EC50 of 3.7 µM and an LC50/EC50 > 50 (LC50 > 200 µM) in AF effectiveness vs toxicity studies. Two derivatives of deoxycholic acid (5 and 7) potently inhibited the growth of biofilm-forming marine bacteria with EC50 values < 10 µM, and five bile acids (1, 5, and 7-9) potently inhibited the growth of diatoms, showing EC50 values between 3 and 10 µM. Promising AF profiles were achieved with some of the synthesized bile acids by combining antimacrofouling and antimicrofouling activities. Initial studies on the incorporation of one of these promising bile acid derivatives in polymeric coatings, such as a marine paint, demonstrated the ability of these compounds to generate coatings with antimacrofouling activity.

Synthesis of New Glycosylated Flavonoids with Inhibitory Activity on Cell Growth.

Natural flavonoids and xanthone glycosides display several biological activities, with the glycoside moiety playing an important role in the mechanism of action of these metabolites. Herein, to give further insights into the inhibitory activity on cell growth of these classes of compounds, the synthesis of four flavonoids (5, 6, 9, and 10) and one xanthone (7) containing one or more acetoglycoside moieties was carried out. Acetyl groups were introduced using acetic anhydride and microwave irradiation. The introduction of one or two acetoglycoside moieties in the framework of 3,7-dihydroxyflavone (4) was performed using two synthetic methods: the Michael reaction and the Koenigs-Knorr reaction. The in vitro cell growth inhibitory activity of compounds 5, 6, 7, 9, and 10 was investigated in six human tumor cell lines: A375-C5 (malignant melanoma IL-1 insensitive), MCF-7 (breast adenocarcinoma), NCI-H460 (non-small cell lung cancer), U251 (glioblastoma astrocytoma), U373 (glioblastoma astrocytoma), and U87MG (glioblastoma astrocytoma). The new flavonoid 3-hydroxy-7-(2,3,4,6-tetra-O-acetyl-ß-glucopyranosyl) flavone (10) was the most potent compound in all tumor cell lines tested, with GI50 values < 8 µM and a notable degree of selectivity for cancer cells.

Therapeutic Potential of Genistein: Preclinical Studies, Clinical Evidence, and Nanotechnology Application.

Genistein (4',5,7-trihydroxyisoflavone) is a natural plant-derived phytoestrogen that can be found, for example, in soybean seeds. Genistein is present mainly in the human diet and is a common precursor in the antimicrobial phytoalexins biosynthesis and phytoanticipins in vegetables. The interest in genistein has increased due to its pharmacological effects, including anti-cancer activity, neuroprotective effects, cardiovascular protection, anti-inflammatory effects, antioxidant activity, and prevention of obesity. The most challenging issue for improving genistein is its low oral bioavailability, which has led to many animal and human pharmacokinetic studies and numerous clinical trials. Several drug delivery systems have been developed to protect and stabilize genistein to overcome the challenge of low bioavailability. This work concerns a revision of the literature reporting nano and microformulations for genistein encapsulation, including lipid nanoparticles, liposomes, tocotrienol-rich nanoemulsions, polymeric nanoparticles, dextran complexes, chitosan complexes, and Fe3O4 nanoparticles with carboxymethylated chitosan. Regarding the enormous potential of genistein, several clinical trials and marketed formulations can be found in the market.

Production of rumen- and gastrointestinal-resistant nanoparticles to deliver lysine to dairy cows.

Supplementing diets with rumen-protected lysine is a common strategy to meet the nutritional needs of high-producing dairy cows. This work addressed two separate but crucial issues: the lysine protection degree across the entire digestive tract as well as the production scalability of the proposed delivery systems. This was achieved by evaluating, in vitro or ex vivo, previously developed rumen-resistant lipid nanoparticles regarding their stability in the digestive tract and in the bloodstream of the dairy cow as well as how their production could be scaled-up. Results showed that the developed nanoparticles were able to resist digestion along the digestive tract but were degraded in the blood over 24 h. Thus, releasing their content to be used by the animal. In vitro viability assays were also performed, with the nanoparticles being found not to be inherently toxic when using nanoparticle concentrations up to 1 mg/mL. Results showed that neither the purity of the used lipids nor the production method significantly altered the nanoparticles' properties or their ruminal resistance. Furthermore, the shelf-life of these nanoparticles was assessed, and they were found to retain their properties and remain usable after at least 1 month of storage. Moreover, a pilot-scale production allowed the production of nanoparticles with similar properties to the previous ones made using standard methods. To summarize, the proposed rumen-resistant nanoparticles presented potential as orally ingested lysine delivery systems for dairy cattle supplementation, being capable of a large-scale production using cheaper components while maintaining their properties and without any efficiency loss. It should however be noted that these results were obtained mainly in vitro and further in vivo bioavailability and production experiments are needed before this technology can be confirmed as a viable way of delivering lysine to dairy cows.

The Influence of Circadian Rhythm on Cancer Cells Targeting and Transfection Efficiency of a Polycation-Drug/Gene Delivery Vector.

The conception of novel anticancer delivery systems and the combination of chronobiology with nanotechnology may provide a powerful tool to optimize cancer therapy. In this work, polyethylenimine (PEI) has been used to complex p53 encoded plasmid DNA (pDNA), and the anticancer drug methotrexate (MTX) has also been loaded into the vectors. To investigate the influence of circadian clock on drug/gene delivery efficiency, HeLa, C33A and fibroblast cells have been transfected with developed PEI/pDNA/MTX delivery vectors at six different time points. Phenomena as the cellular uptake/internalization, drug/gene delivery and p53 protein production have been evaluated. The cell-associated MTX fluorescence have been monitored, and p53 protein levels quantified. In HeLa and C33A cancer cells, significant levels of MTX were found for T8 and T12. For these time points, a high amount of p53 protein was quantified. Confocal microscopy images showed successful HeLa cell's uptake of PEI/pDNA/MTX particles, at T8. In comparison, poor levels of MTX and p53 protein were found in fibroblasts; nevertheless, results indicated rhythmicity. Data demonstrate the influence of circadian rhythm on both cancer-cells targeting ability and transfection performance of PEI/pDNA/MTX carriers and seemed to provide the optimum time for drug/gene delivery. This report adds a great contribution to the field of cancer chronobiology, highlighting the relationship between circadian rhythm and nanodelivery systems, and charting the path for further research on a, yet, poorly explored but promising topic.

Exploring the link between chronobiology and drug delivery: effects on cancer therapy.

Circadian clock is an impressive timing system responsible for the control of several metabolic, physiological and behavioural processes. Nowadays, the connection between the circadian clock and cancer occurrence and development is consensual. Therefore, the inclusion of circadian timing into cancer therapy may potentially offer a more effective and less toxic approach. This way, chronotherapy has been shown to improve cancer treatment efficacy. Despite this relevant finding, its clinical application is poorly exploited. The conception of novel anticancer drug delivery systems and the combination of chronobiology with nanotechnology may provide a powerful tool to optimize cancer therapy, instigating the incorporation of the circadian timing into clinical practice towards a more personalized drug delivery. This review focuses on the recent advances in the field of cancer chronobiology, on the link between cancer and the disruption of circadian rhythms and on the promising targeted drug nanodelivery approaches aiming the clinical application of cancer chronotherapy.

Methods to improve the immunogenicity of plasmid DNA vaccines.

DNA vaccines have emerged as innovative approaches that have great potential to overcome the limitations of current conventional vaccines. Plasmid DNA vaccines are often safer than other vaccines because they carry only antigen genetic information, are more stable and easier to produce, and can stimulate both humoral and cellular immune responses. Although the results of ongoing clinical trials are very promising, some limitations compromise the immunogenicity of these vaccines. Thus, this review describes different strategies that can be explored to improve the immunogenicity of plasmid DNA vaccines, including the optimization of the plasmid vector backbone, the use of different methods for vaccine delivery, the use of alternative administration routes and the inclusion of adjuvants. In combination, these improvements could lead to the successful clinical use of plasmid DNA vaccines.

Cold Atmospheric Plasma, a Novel Approach against Bladder Cancer, with Higher Sensitivity for the High-Grade Cell Line.

Antitumor therapies based on Cold Atmospheric Plasma (CAP) are an emerging medical field. In this work, we evaluated CAP effects on bladder cancer. Two bladder cancer cell lines were used, HT-1376 (stage III) and TCCSUP (stage IV). Cell proliferation assays were performed evaluating metabolic activity (MTT assay) and protein content (SRB assay). Cell viability, cell cycle, and mitochondrial membrane potential (Δψm) were assessed using flow cytometry. Reactive oxygen and nitrogen species (RONS) and reduced glutathione (GSH) were evaluated by fluorescence. The assays were carried out with different CAP exposure times. For both cell lines, we obtained a significant reduction in metabolic activity and protein content. There was a decrease in cell viability, as well as a cell cycle arrest in S phase. The Δψm was significantly reduced. There was an increase in superoxide and nitric oxide and a decrease in peroxide contents, while GSH content did not change. These results were dependent on the exposure time, with small differences for both cell lines, but overall, they were more pronounced in the TCCSUP cell line. CAP showed to have a promising antitumor effect on bladder cancer, with higher sensitivity for the high-grade cell line.

Kinetics of radium-223 and its effects on survival, proliferation and DNA damage in lymph-node and bone metastatic prostate cancer cell lines.

BACKGROUND: Metastatic castration-resistant prostate cancer (mCRPC) is associated with a very unfavorable prognosis. At this advanced stage of the disease, there are several therapeutic strategies approved in recent times, being one of them Radium-223 dichloride (Radium-223). However, its mechanisms of action and the process that conducts to cell death are not fully understood. Given this, our main goal is to characterize the radiobiological effects induced by Radium-223 and to evaluate its kinetics on metastatic Prostate Cancer (mPCa) cells. MATERIALS AND METHODS: In vitro studies were conducted using two mPCa cell lines, the LNCaP and PC3, the first being derived from lymph node metastasis and the second from bone metastasis. Kinetic studies were conducted to access the capacity of these cell lines to uptake, retain and internalize the Radium-223. For the assessment of radiobiological effects, cells were first exposed to different doses of Radium-223 and the clonogenic assay was done to evaluate cell survival and to determine lethal doses (LD50). Then, the effects were also evaluated in terms of proliferation, oxidative stress, morphological changes and cell damage. RESULTS: Radium-223 is uptaken by mPCa cells and reaches the nucleus, where it is retained over time. Irradiation decreases cell survival and proliferation, with LNCaP cells (LD50 = 1.73mGy) being more radiosensitive than PC3 cells (LD50 = 4.20mGy). Irradiated cells showed morphological changes usually associated with apoptosis and a dose-dependent increase in DNA damage. Moreover, activation of cell cycle checkpoints occurs through ATM/CHK2 pathway, which is involved in cell cycle arrest and cell death. CONCLUSIONS: The cytotoxic and anti-proliferative effects on both cell lines showed that Radium-223 can decrease the aggressiveness of tumor cells by decreasing the cell survival and proliferation and, also, by increasing the DNA damage. The similar results observed in both cell lines indicated that Radium-223 may have the potential to be used as a therapeutic option also for mCRPC patients with lymph node metastasis. The activation of DNA Damage Response pathways allows the possibility to understand the importance of these checkpoints as targets for new combined therapies.

Characterization of the individual glucose uptake systems of Lactococcus lactis: mannose-PTS, cellobiose-PTS and the novel GlcU permease.

According to previous reports, Lactococcus lactis imports glucose via two distinct phosphoenolpyruvate:phosphotransferase systems (mannose-PTS and cellobiose-PTS) and one or more unknown non-PTS permease(s). GlcU was identified as the sole non-PTS permease involved in the transport of glucose. Additionally, the biochemical properties of PTS(Man), PTS(Cel) and GlcU were characterized in double knockout mutants with glucose uptake restricted to a single system. Transport susceptibility to protonophores indicated that glucose uptake via GlcU is proton-motive force dependent. Competition assays revealed a high specificity of GlcU for glucose. Furthermore, the permease has low affinity for glucose and displays strong preference for the beta-anomer as shown by the profiles of consumption of the two glucose anomers studied by (13)C-NMR. Similar kinetic properties were found for PTS(Cel), while PTS(Man) is a high-affinity system recognizing equally well the two anomeric forms of glucose. Transcripts of the genes encoding the three transporters are present simultaneously in the parent strain NZ9000 as shown by reverse transcription-PCR. Investigation of the distribution of GlcU homologues among bacteria showed that these proteins are restricted to the low-GC Gram-positive Firmicutes. This work completes the identification of the glucose transport systems in L. lactis MG1363.

Towards enhanced galactose utilization by Lactococcus lactis.

Accumulation of galactose in dairy products due to partial lactose fermentation by lactic acid bacteria yields poor-quality products and precludes their consumption by individuals suffering from galactosemia. This study aimed at extending our knowledge of galactose metabolism in Lactococcus lactis, with the final goal of tailoring strains for enhanced galactose consumption. We used directed genetically engineered strains to examine galactose utilization in strain NZ9000 via the chromosomal Leloir pathway (gal genes) or the plasmid-encoded tagatose 6-phosphate (Tag6P) pathway (lac genes). Galactokinase (GalK), but not galactose permease (GalP), is essential for growth on galactose. This finding led to the discovery of an alternative route, comprising a galactose phosphotransferase system (PTS) and a phosphatase, for galactose dissimilation in NZ9000. Introduction of the Tag6P pathway in a galPMK mutant restored the ability to metabolize galactose but did not sustain growth on this sugar. The latter strain was used to prove that lacFE, encoding the lactose PTS, is necessary for galactose metabolism, thus implicating this transporter in galactose uptake. Both PTS transporters have a low affinity for galactose, while GalP displays a high affinity for the sugar. Furthermore, the GalP/Leloir route supported the highest galactose consumption rate. To further increase this rate, we overexpressed galPMKT, but this led to a substantial accumulation of α-galactose 1-phosphate and α-glucose 1-phosphate, pointing to a bottleneck at the level of α-phosphoglucomutase. Overexpression of a gene encoding α-phosphoglucomutase alone or in combination with gal genes yielded strains with galactose consumption rates enhanced up to 50% relative to that of NZ9000. Approaches to further improve galactose metabolism are discussed.

Pyruvate formate lyase is required for pneumococcal fermentative metabolism and virulence.

Knowledge of the in vivo physiology and metabolism of Streptococcus pneumoniae is limited, even though pneumococci rely on efficient acquisition and metabolism of the host nutrients for growth and survival. Because the nutrient-limited, hypoxic host tissues favor mixed-acid fermentation, we studied the role of the pneumococcal pyruvate formate lyase (PFL), a key enzyme in mixed-acid fermentation, which is activated posttranslationally by PFL-activating enzyme (PFL-AE). Mutations were introduced to two putative pfl genes, SPD0235 and SPD0420, and two putative pflA genes, SPD0229 and SPD1774. End-product analysis showed that there was no formate, the main end product of the reaction catalyzed by PFL, produced by mutants defective in SPD0420 and SPD1774, indicating that SPD0420 codes for PFL and SPD1774 for putative PFL-AE. Expression of SPD0420 was elevated in galactose-containing medium in anaerobiosis compared to growth in glucose, and the mutation of SPD0420 resulted in the upregulation of fba and pyk, encoding, respectively, fructose 1,6-bisphosphate aldolase and pyruvate kinase, under the same conditions. In addition, an altered fatty acid composition was detected in SPD0420 and SPD1774 mutants. Mice infected intranasally with the SPD0420 and SPD1774 mutants survived significantly longer than the wild type-infected cohort, and bacteremia developed later in the mutant cohort than in the wild type-infected group. Furthermore, the numbers of CFU of the SPD0420 mutant were lower in the nasopharynx and the lungs after intranasal infection, and fewer numbers of mutant CFU than of wild-type CFU were recovered from blood specimens after intravenous infection. The results demonstrate that there is a direct link between pneumococcal fermentative metabolism and virulence.

Interplay Between Capsule Expression and Uracil Metabolism in Streptococcus pneumoniae D39.

Pyrimidine nucleotides play an important role in the biosynthesis of activated nucleotide sugars (NDP-sugars). NDP-sugars are the precursors of structural polysaccharides in bacteria, including capsule, which is a major virulence factor of the human pathogen S. pneumoniae. In this work, we identified a spontaneous non-reversible mutant of strain D39 that displayed a non-producing capsule phenotype. Whole-genome sequencing analysis of this mutant revealed several non-synonymous single base modifications, including in genes of the de novo synthesis of pyrimidines and in the -10 box of capsule operon promoter (Pcps). By directed mutagenesis we showed that the point mutation in Pcps was solely responsible for the drastic decrease in capsule expression. We also demonstrated that D39 subjected to uracil deprivation shows increased biomass and decreased Pcps activity and capsule amounts. Importantly, Pcps expression is further decreased by mutating the first gene of the de novo synthesis of pyrimidines, carA. In contrast, the absence of uracil from the culture medium showed no effect on the spontaneous mutant strain. Co-cultivation of the wild-type and the mutant strain indicated a competitive advantage of the spontaneous mutant (non-producing capsule) in medium devoid of uracil. We propose a model in that uracil may act as a signal for the production of different capsule amounts in S. pneumoniae.

The lactate dehydrogenases encoded by the ldh and ldhB genes in Lactococcus lactis exhibit distinct regulation and catalytic properties - comparative modeling to probe the molecular basis.

Lactococcus lactis FI9078, a construct carrying a disruption of the ldh gene, converted approximately 90% of glucose into lactic acid, like the parental strain MG1363. This unexpected lactate dehydrogenase activity was purified, and ldhB was identified as the gene encoding this protein. The activation of ldhB was explained by the insertion of an IS905-like element that created a hybrid promoter in the intergenic region upstream of ldhB. The biochemical and kinetic properties of this alternative lactate dehydrogenase (LDHB) were compared to those of the ldh-encoded enzyme (LDH), purified from the parental strain. In contrast to LDH, the affinity of LDHB for NADH and the activation constant for fructose 1,6-bisphosphate were strongly dependent on pH. The activation constant increased 700-fold, whereas the K(m) for NADH increased more than 10-fold, in the pH range 5.5-7.2. The two enzymes also exhibited different pH profiles for maximal activity. Moreover, inorganic phosphate acted as a strong activator of LDHB. The impact of replacing LDH by LDHB on the physiology of L. lactis was assessed by monitoring the evolution of the pools of glycolytic intermediates and cofactors during the metabolism of glucose by in vivo NMR. Structural analysis by comparative modeling of the two proteins showed that LDH has a slightly larger negative charge than LDHB and a greater concentration of positive charges at the interface between monomers. The calculated pH titration curves of the catalytic histidine residues explain why LDH maintains its activity at low pH as compared to LDHB, the histidines in LDH showing larger pH titration ranges.

Structure-activity relationship studies for multitarget antithrombotic drugs.

Thrombosis is a complex process involving multiple pathways. Currently, therapy relies on the combination of two or more antithrombotic drugs, showing that inhibiting more than one target provides benefits in the prevention and treatment of thrombosis. This review focuses on structure-activity relationship studies of molecules possessing multiple actions against thrombosis, namely, dual inhibitors of coagulation, dual inhibitors of coagulation and platelet aggregation, and also dual inhibitors of platelet aggregation. EP217609 has just entered clinical trials, which raise the expectations on the multitarget strategy to prevent or treat thrombosis.

Nanostructured lipid carriers loaded with resveratrol modulate human dendritic cells.

Dendritic cells (DCs) are promising targets for drug delivery, as they can induce immunity or tolerance. The current study aims to examine the potential of using nanostructured lipid carriers (NLC) as delivery systems for human DC by evaluating nanoparticle internalization, cell labeling, and drug activity. NLC were formulated incorporating the fluorochrome fluorescein isothiocyanate (FITC-NLC) or the natural anti-inflammatory molecule resveratrol (rsv-NLC). Primary human DCs were differentiated from peripheral blood monocytes, and the innovative imaging flow cytometry technique was used to examine FITC-NLC internalization. The capacity of rsv-NLC to inhibit DC activation in response to proinflammatory cytokine tumor necrosis factor-α (TNF- α) was investigated by conventional flow cytometry. A combination of imaging and conventional flow cytometry was used to assess NLC cytotoxicity. The results obtained indicate that both NLC formulations were stable over time, with mean diameter <200 nm and highly negative zeta potential (about -30 mV). When DCs were placed in contact with NLC, imaging flow cytometry clearly showed that DCs efficiently internalized FITC-NLC, with nearly 100% of cells internalizing nanoparticles upon 1 hour of incubation. Both immature and mature DCs internalized NLC to high and comparable levels, and without cytotoxicity. Stimulating DC with TNF-α in the presence of rsv-NLC revealed that, using these nanoparticles, very small concentrations of rsv were sufficient to significantly decrease surface expression of activation marker CD83 (5 µM) and major histocompatibility complex-class II molecule human leukocyte antigen - antigen D related (10 µM), both upregulated in response to TNF-α stimulation. Rsv-NLC were compared with free rsv; at 5 µM, rsv-NLC were able to inhibit nuclear factor κ beta phosphorylation and significantly decrease the level of interleukin-12/23, both upregulated in response to TNF-α, while 10 µM free rsv were needed to promote a similar effect. Taken together, the results presented show that NLC are suitable carriers of fluorescent labels or bioactive molecules for human DCs, leading to inflammation modulation.

Transcriptional and metabolic effects of glucose on Streptococcus pneumoniae sugar metabolism.

Streptococcus pneumoniae is a strictly fermentative human pathogen that relies on carbohydrate metabolism to generate energy for growth. The nasopharynx colonized by the bacterium is poor in free sugars, but mucosa lining glycans can provide a source of sugar. In blood and inflamed tissues glucose is the prevailing sugar. As a result during progression from colonization to disease S. pneumoniae has to cope with a pronounced shift in carbohydrate nature and availability. Thus, we set out to assess the pneumococcal response to sugars found in glycans and the influence of glucose (Glc) on this response at the transcriptional, physiological, and metabolic levels. Galactose (Gal), N-acetylglucosamine (GlcNAc), and mannose (Man) affected the expression of 8 to 14% of the genes covering cellular functions including central carbon metabolism and virulence. The pattern of end-products as monitored by in vivo (13)C-NMR is in good agreement with the fermentation profiles during growth, while the pools of phosphorylated metabolites are consistent with the type of fermentation observed (homolactic vs. mixed) and regulation at the metabolic level. Furthermore, the accumulation of α-Gal6P and Man6P indicate metabolic bottlenecks in the metabolism of Gal and Man, respectively. Glc added to cells actively metabolizing other sugar(s) was readily consumed and elicited a metabolic shift toward a homolactic profile. The transcriptional response to Glc was large (over 5% of the genome). In central carbon metabolism (most represented category), Glc exerted mostly negative regulation. The smallest response to Glc was observed on a sugar mix, suggesting that exposure to varied sugars improves the fitness of S. pneumoniae. The expression of virulence factors was negatively controlled by Glc in a sugar-dependent manner. Overall, our results shed new light on the link between carbohydrate metabolism, adaptation to host niches and virulence.