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Fragment-based drug discovery (FBDD) is a crucial strategy for developing new drugs that have been applied to diverse targets, from neglected infectious diseases to cancer. With at least seven drugs already launched to the market, this approach has gained interest in both academics and industry in the last 20 years. FBDD relies on screening small libraries with about 1000-2000 compounds of low molecular weight (about 300 Da) using several biophysical methods. Because of the reduced size of the compounds, the chemical space and diversity can be better explored than large libraries used in high throughput screenings. This review summarises the most common biophysical techniques used in fragment screening and orthogonal validation. We also explore the advantages and drawbacks of the different biophysical techniques and examples of applications and strategies., (© 2024 Wiley-VCH GmbH.)

Sexually Transmitted Infections (STIs) are a public health burden rising in developed and developing nations. The World Health Organization estimates nearly 374 million new cases of curable STIs yearly. Global efforts to control their spread have been insufficient in fulfilling their objective. As there is no vaccine for many of these infections, these efforts are focused on education and condom distribution. The development of vaccines for STIs is vital for successfully halting their spread. The field of immunoinformatics is a powerful new tool for vaccine development, allowing for the identification of vaccine candidates within a bacterium's genome and allowing for the design of new genome-based vaccine peptides. The goal of this review was to evaluate the usage of immunoinformatics in research focused on non-viral STIs, identifying fields where research efforts are concentrated. Here we describe gaps in applying these techniques, as in the case of Treponema pallidum and Trichomonas vaginalis.

Bacteria of the Leuconostoc genus are Gram-positive bacteria that are commonly found in raw milk and persist in fermented dairy products and plant food. Studies have already explored the probiotic potential of L. mesenteroides, but not from a probiogenomic perspective, which aims to explore the molecular features responsible for their phenotypes. In the present work, probiogenomic approaches were applied in strains F-21 and F-22 of L. mesenteroides isolated from human milk to assess their biosafety at the molecular level and to correlate molecular features with their potential probiotic characteristics. The complete genome of strain F-22 is 1.99 Mb and presents one plasmid, while the draft genome of strain F-21 is 1.89 Mb and presents four plasmids. A high percentage of average nucleotide identity among other genomes of L. mesenteroides (≥ 96%) corroborated the previous taxonomic classification of these isolates. Genomic regions that influence the probiotic properties were identified and annotated. Both strains exhibited wide genome plasticity, cell adhesion ability, proteolytic activity, proinflammatory and immunomodulation capacity through interaction with TLR-NF-κB and TLR-MAPK pathway components, and no antimicrobial resistance, denoting their potential to be candidate probiotics. Further, the strains showed bacteriocin production potential and the presence of acid, thermal, osmotic, and bile salt resistance genes, indicating their ability to survive under gastrointestinal stress. Taken together, our results suggest that L. mesenteroides F-21 and F-22 are promising candidates for probiotics in the food and pharmaceutical industries., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The quality of green coffee beans (GCBs) is possibly affected by storage conditions. Edible polymer coatings for GCBs can help preserve flavors and improve shelf life of GCBs. This study aimed to incorporate α-tocopherol, a powerful antioxidant, in thermoplastic starch [TPS] and chitosan [TPC] and determined the best cavitation energy (960-3840 J·mL -1 ) using an ultrasonic probe. Then, we evaluated the incorporation of bentonite (0% and 2% m/m) and α-tocopherol (0% and 10% m/m) in the best energy cavitation/biopolymer combination. The TPS and TPC coatings demonstrated good adherence to the GCBs, measured by surface energy. The dispersion of α-tocopherol in TPC, with cavitation energy 960 J·mL -1 , promoted greater stability (greater zeta potential), thereby increasing antioxidant activity by 28% compared to TPS, therefore, was selected for a second stage. Incorporation of 2% bentonite into the TPC, with 10% α-tocopherol, resulted in a 3.7 × 10 -10 g·m -1 ·s -1 ·Pa -1 water vapor permeability, which is satisfactory for prevented of moisture gain during storage. The compressive load showed values of 375 N to the non-coated GCB and around 475 N with the insertion of coatings to the GCB. Thus, a TPC/α-tocopherol/bentonite combination, dispersed with 960 J·mL -1 energy, was highly effective in the development of biopolymeric coatings for the GCBs., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

We investigated the effect in vitro and in vivo of doxycycline hyclate (Dx), a broad-spectrum antibiotic inhibitor of matrix metaloproteinases (MMPs), on adult Schistosoma mansoni worms and granulomatous liver inflammation in infected mice. Adult S. mansoni worms in culture treated with different concentrations of Dx (50-180 μg/mL) were studied for eight days to assess its morphology, eggs production, and mortality. Uninfected mice and those infected with S. mansoni, untreated and treated with praziquantel (Pz; 200 mg/kg) or Dx (50 mg/kg), were evaluated for 60 days. Our results indicated that Dx induced dose-dependent integumentary lesions (bubbles, tubercle collapse, spicule disappearance, peeling, and erosion), reduced mating rate and eggs-laying in adult S. mansoni worms. The effective lethal dose required to kill 50% of worms was 112.0 μg/mL Dx (DL 50 ). In mice, S. mansoni infection induced hepatomegaly, intense IL-4, IL-10, TNF-α and TGF-β production, granulomatous inflammation and hepatic glycogen depletion. The number and size of the granulomas was similar in untreated and Dx-treated animals. Untreated animals showed a predominance of productive granulomas, and intense MMP-2 and MMP-9 activities. Dx-treated mice exhibited a significant increase in IL-4 levels, tissue inflammation, proportion of involutive granulomas, and hepatic collagenogenesis, as well as attenuated MMP-2 and MMP-9 activities. Our findings indicated that Dx is toxic to adult S. mansoni worms in vitro. However, in vitro beneficial effects were not reproduced in vivo, since Dx treatment increased liver granulomatous inflammation and collagenogenesis in S. mansoni-infected mice by a process potentially associated with Dx-mediated hepatic MMP-2 and MMP-9 inhibition., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The objective of this paper was to evaluate the effect of bioactive whey protein isolate/montmorillonite films containing citric acid on the inhibition of enzymatic browning and physicochemical properties in minimally processed apples. Whey protein isolate films incorporated with montmorillonite (3 g/100 g) and citric acid (5 and 10 g/100 g) were applied to the apples slices. All samples were packaged in polypropylene trays (14.6 cm × 11.4 cm × 6.5 cm) and stored at 5 ± 2 °C and 85 ± 3% RH for eight days. Every two days, the apples samples were evaluated for color, acidity, pH, soluble solids, water activity and polyphenol oxidase and peroxidase enzyme activity. The enzymatic browning of the apples slices was reduced for all films during storage. However, the films containing citric acid maintained the color characteristics, reducing the loss of quality associated the maintenance of acidity, soluble solids, water activity, reduction of polyphenol oxidase and peroxidase activity, thus prolonging the shelf life of the apples., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Folate pathway is a key target for the development of new drugs against infectious diseases since the discovery of sulfa drugs and trimethoprim. The knowledge about this pathway has increased in the last years and the catalytic mechanism and structures of all enzymes of the pathway are fairly understood. In addition, differences among enzymes from prokaryotes and eukaryotes could be used for the design of specific inhibitors. In this review, we show a panorama of progress that has been achieved within the folate pathway obtained in the last years. We explored the structure and mechanism of enzymes, several genetic features, strategies, and approaches used in the design of new inhibitors that have been used as targets in pathogen chemotherapy.

Prion protein modulates many cellular functions including the secretion of trophic factors by astrocytes. Some of these factors are found in exosomes, which are formed within multivesicular bodies (MVBs) and secreted into the extracellular space to modulate cell-cell communication. The mechanisms underlying exosome biogenesis were not completely deciphered. Here, we demonstrate that primary cultures of astrocytes and fibroblasts from prnp-null mice secreted lower levels of exosomes than wild-type cells. Furthermore, prnp-null astrocytes exhibited reduced MVB formation and increased autophagosome formation. The reconstitution of PRNP expression at the cell membrane restored exosome secretion in PRNP-deficient astrocytes, whereas macroautophagy/autophagy inhibition via BECN1 depletion reestablished exosome release in these cells. Moreover, the PRNP octapeptide repeat domain was necessary to promote exosome secretion and to impair the formation of the CAV1-dependent ATG12-ATG5 cytoplasmic complex that drives autophagosome formation. Accordingly, higher levels of CAV1 were found in lipid raft domains instead of in the cytoplasm in prnp-null cells. Collectively, these findings demonstrate that PRNP supports CAV1-suppressed autophagy to protect MVBs from sequestration into phagophores, thus facilitating exosome secretion.

Objective: To evaluate whether patient age has a significant impact on mefloquine concentrations in the plasma and erythrocytes over the course of treatment for uncomplicated falciparum malaria., Methods: A total of 20 children aged between 8 and 11 years and 20 adult males aged between 22 and 41 years with uncomplicated falciparum malaria were enrolled in the study. Mefloquine was administered to patients in both age groups at a dose of 20mgkg(-1). The steady-state drug concentrations were measured by reversed-phase high performance liquid chromatography., Results: All patients had an undetectable mefloquine concentration on day 0. In adults, the plasma mefloquine concentrations ranged from 770 to 2930ngmL(-1) and the erythrocyte concentrations ranged from 2000 to 6030ngmL(-1). In children, plasma mefloquine concentrations ranged from 881 to 3300ngmL(-1) and erythrocyte concentrations ranged from 3000 to 4920ngmL(-1). There was no significant correlation between mefloquine concentrations in the plasma and erythrocytes in either adults or children., Conclusion: In the present study, we observed no effect of patient age on the steady-state concentrations of mefloquine in the plasma and erythrocytes. We found that the mefloquine concentration in the erythrocytes was approximately 2.8-times higher than in the plasma. There were no significant correlations between mefloquine concentrations in the erythrocytes and plasma for either age group., (Copyright © 2016 Sociedade Brasileira de Infectologia. Published by Elsevier Editora Ltda. All rights reserved.)

The A33 protein, expressed in colorectal tumors, is a target for improving treatment of patients with colorectal cancer. Over the last decade, studies have tested anti-A33 antibody as a therapeutic agent for these patients. Preclinical results were promising, but clinical trials did not confirm positive results. Here, immunohistochemistry in colorectal cancer tissue showed that samples from well-differentiated tumors presented a strong A33 membrane staining, whereas poorly differentiated tumors and mucinous adenocarcinomas showed weak cytoplasmic and nuclear staining. Moderately differentiated tumors presented variable staining. We suggest that in future clinical trials, patients should be selected on the basis of membrane expression of A33.

Colorectal cancer (CRC) is one of the most frequently diagnosed malignancies. The generation of conventional treatments has improved, but approximately 50 % of patients with CRC who undergo potentially curative surgery ultimately relapse and die, usually as a consequence of metastatic disease. Our previous findings showed that engagement of the cellular prion protein (PrP(C)) to its ligand HSP70/90 heat shock organizing protein (HOP) induces proliferation of glioblastomas. In addition, PrP(C) has been described as an important modulator of colorectal tumor growth. Here, we investigated the biological relevance of the PrP(C)-HOP interaction in CRC cells. We demonstrate that HOP induced the migration and invasion of CRC cell lines in a PrP(C)-dependent manner and that phosphorylation of the ERK1/2 pathway is a downstream mediator of these effects. Additionally, we show that a HOP peptide with the ability to bind PrP(C) and abolish the PrP(C)-HOP interaction inhibited the migration and invasion of CRC cells. Together, these data indicate that the disruption of the PrP(C)-HOP complex could be a potential therapeutic target for modulating the migratory and invasive cellular properties that lead to metastatic CRC.

This chapter is derived from our experience in the study of stress-Inducible Protein 1 (STI1) in extracellular vesicles. We used different techniques to isolate, explore, and characterize the extracellular vesicles that contained this protein. Ultracentrifugation and gel chromatography were used to isolate extracellular vesicles of different sizes, nanotracking particle analysis (NTA) determined number and size of vesicles, while flow cytometry and ELISA were used to determine the specific protein content of vesicles.

Objectives: The purpose of this study was to review the epidemiological aspects of displacement fractures of the acetabulum that had been treated surgically at the National Institute of Traumatology and Orthopedics (INTO)., Methods: We retrospectively analyzed 126 acetabulum fractures that had been treated surgically at INTO between March 2006 and November 2008. The following factors were taken into account: age, sex, trauma mechanism, injury classification, time elapsed between trauma and surgery, affected side and associated bone injuries., Results: 76.8% were male; the mean age was 39.6 years. The trauma mechanism was traffic accidents in 59%; the time that elapsed between injury and surgery was on average 16.4 days; 55% of the cases were on the right side; 30% of the patients presented associated fractures., Conclusion: Most of the patients were male, in an economically active age group, and were victims of traffic accidents. Edge and/or posterior column fractures were the most frequent types. Associated injuries were common and most of the fractures operated in our service came to us late.

Tetrahydropyran rings are a common feature of complex polyketide natural products, but much remains to be learned about the enzymology of their formation. The enzyme SalBIII from the salinomycin biosynthetic pathway resembles other polyether epoxide hydrolases/cyclases of the MonB family, but SalBIII plays no role in the conventional cascade of ring opening/closing. Mutation in the salBIII gene gave a metabolite in which ring A is not formed. Using this metabolite in vitro as a substrate analogue, SalBIII has been shown to form pyran ring A. We have determined the X-ray crystal structure of SalBIII, and structure-guided mutagenesis of putative active-site residues has identified Asp38 and Asp104 as an essential catalytic dyad. The demonstrated pyran synthase activity of SalBIII further extends the impressive catalytic versatility of α+β barrel fold proteins., (© 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.)

Riparian vegetation is one of the most important abiotic components determining the water flow pattern in lotic ecosystems, influencing the composition, richness, and diversity of invertebrates. We have identified whether differences in the structure of the assemblages of invertebrates between riffles and pools may influence the responses of fauna to the effects of land use. In addition, we investigated which fauna metrics are responsible for the differentiation between riffles and pools in streams subject to different land uses. During the dry season of 2012, the main substrates of riffles and pools were sampled (Surber collector) from nine streams within forest, pasture, and urban areas. Principal component analysis (PCA) and Permanova showed differences in the set of environmental variables between streams and mesohabitats. The first PCA axis distinguished the forest and pasture streams from the urban area streams and was related to variables indicative of nutrient enrichment and land use, while the second axis was formed by velocity flow and by the quantities of ultrafine and coarse sand, which distinguished the riffles and pools of the streams. The faunal composition distinguished the streams in pasture and forest areas from the urban streams. Riffles and pools were not concordant in the representation of the invertebrate fauna, indicating the importance of sampling both mesohabitats in the types of streams investigated. The richness, taxonomic composition, and relative abundance of families of Ephemeroptera, Plecoptera, and Trichoptera showed robust responses in riffles to the effects of environmental changes, while in pools, only the richness showed a significant response. It was possibly concluded that riffles were more sensitive in detecting the effects of land use. The information from this study help to understand how the community of invertebrates and the types of habitats in streams may be affected by anthropogenic impacts.

Conclusion: We demonstrated an early increase in aquaporin 2 (AQP2) expression in a motor nerve (extratemporal facial nerve, FN) following acute peripheral compression (crush), concomitant to effective development of motor dysfunction (facial palsy). The early increase in AQP2 expression that occurred concomitantly with the appearance of a deficit in a peripheral motor nerve suggests that this protein is involved in the physiological events associated with post-injury edema, similar to the already demonstrated behavior of AQP4 in the central nervous system (CNS)., Objective: The aim of this study was to assess the expression of AQP2 in the FN of rats up to 7 days after crush., Methods: The extratemporal trunk of the right FN of rats was subjected to mechanical crush, and the expression of AQP2 in the affected (right) and non-affected (left) FN was measured by means of western blotting at days 1, 3, and 7 after injury. Behavioral analysis of the development of facial palsy was also performed over the same time period., Results: Increased expression of AQP2 was shown in the affected FN compared with its corresponding control at day 1 after compression, simultaneously with the appearance of facial palsy.

Objective: The purposes of this study were to describe the CT features of isolated posterior acetabular wall fractures with associated marginal impaction and to discuss the potential therapeutic implications of recognizing this type of fracture., Conclusion: Marginal impaction is an important cause of articular incongruity that adversely affects prognosis. Radiologists should be capable of identifying posterior acetabular wall fracture patterns because they may be the first to suggest diagnoses.

3-Dehydroquinase, the third enzyme in the shikimate pathway, is a potential target for drugs against tuberculosis. Whilst a number of potent inhibitors of the Mycobacterium tuberculosis enzyme based on a 3-dehydroquinate core have been identified, they generally show little or no in vivo activity, and were synthetically complex to prepare. This report describes studies to develop tractable and drug-like aromatic analogues of the most potent inhibitors. A range of carbon-carbon linked biaryl analogues were prepared to investigate the effect of hydrogen bond acceptor and donor patterns on inhibition. These exhibited inhibitory activity in the high-micromolar range. The addition of flexible linkers in the compounds led to the identification of more potent 3-nitrobenzylgallate- and 5-aminoisophthalate-based analogues., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Nanocomposites of chitosan (CS) were developed and characterized in a full factorial design with varying levels of montmorillonite (MMTNa) and encapsulated tocopherol (toc-encap). The structural properties (XRD, FTIR), morphology (TEM), hygroscopic properties (water vapour permeability, hydrophobicity, sorption isotherms) and optical properties (haze, CIELab parameters) of the resulting materials were evaluated. Toc-encap contents up to 10% influenced the intercalation of MMTNa in the CS matrix, resulting in films with reduced water vapour permeability (3.48×10(-11)(g/msPa)), increased hydrophobicity (ΔGHydroph |7.93-59.54|mJm(-2)) and lower equilibrium moisture content (EMC), thus showing potential for active food packaging materials. At levels above 10%, toc-encap agglomerates occurred, which deteriorated the properties of the resulting films, as shown with the TEM. As the toc-encap content increased, the films became slightly more yellow, more irregular and less transparent, with a higher haze index., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The objective of this study was to perceive the death-dying process from the perspective of nursing students. This is an exploratory, descriptive and qualitative research study. Data were collected between June and July 2013, from three focus groups with six nursing students at a University Center located in the central region of Rio Grande do Sul, Brazil. The meetings were organized with an approach to increase discussions about the death-dying process from the perspective of the complex thinking. Data were analyzed by means of the Strategic Focal Analysis, and three categories were created: Death: a process of rupture or continuity?; Recognizing weaknesses in the undergraduate educational process; and Outlining strategies to broaden academic discussions. It is possible to conclude that the death/dying process is minimally discussed in undergraduate courses, and when it is discussed, it happens in a fragmented and disjunctive manner, without integrating it into the human living process. Descriptors: Death. Education, nursing. Attitude to death.

Inhibition of the biosynthesis of tetrahydrofolate (THF) has long been a focus in the treatment of both cancer and infectious diseases. Dihydrofolate reductase (DHFR), which catalyzes the last step, is one of the most thoroughly explored targets of this pathway, but there are no DHFR inhibitors used for tuberculosis treatment. Here, we report a structural, site-directed mutagenesis and calorimetric analysis of Mycobacterium tuberculosis DHFR (MtDHFR) in complex with classical DHFR inhibitors. Our study provides insights into the weak inhibition of MtDHFR by trimethoprim and other antifolate drugs, such as pyrimethamine and cycloguanil. The construction of the mutant Y100F, together with calorimetric studies, gives insights into low affinity of MtDHFR for classical DHFR inhibitors. Finally, the structures of MtDHFR in complex with pyrimethamine and cycloguanil define important interactions in the active site and provide clues to the more effective design of antibiotics targeted against MtDHFR., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

We developed antimicrobial packaging incorporated with allyl isothiocyanate (AIT) and carbon nanotube (CNT), and this packaging was used for shredded cooked chicken meat inoculated with Salmonella Choleraesuis. The following parameters were analysed during the 40 days of storage: microbial counts, colour characteristics and changes in the oxidation of the meat as well as changes in the mechanical properties of the film, the structure of the antimicrobial film and the diffusion of the antimicrobial agent into the food. The incorporation of AIT into the films increased the elongation at the break (E) value of the films and decreased the tensile strength (TS) value of the films. The CNT was important to retain the AIT which is a volatile substance in the film. The diffusion of the AIT from the film into the chicken reduced the microbial contamination, controlled oxidation and reduced the colour changes. Thus, these packages were effective for the 40 days of storage., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The co-chaperone stress-inducible protein 1 (STI1) is released by astrocytes, and has important neurotrophic properties upon binding to prion protein (PrP(C)). However, STI1 lacks a signal peptide and pharmacological approaches pointed that it does not follow a classical secretion mechanism. Ultracentrifugation, size exclusion chromatography, electron microscopy, vesicle labeling, and particle tracking analysis were used to identify three major types of extracellular vesicles (EVs) released from astrocytes with sizes ranging from 20-50, 100-200, and 300-400 nm. These EVs carry STI1 and present many exosomal markers, even though only a subpopulation had the typical exosomal morphology. The only protein, from those evaluated here, present exclusively in vesicles that have exosomal morphology was PrP(C). STI1 partially co-localized with Rab5 and Rab7 in endosomal compartments, and a dominant-negative for vacuolar protein sorting 4A (VPS4A), required for formation of multivesicular bodies (MVBs), impaired EV and STI1 release. Flow cytometry and PK digestion demonstrated that STI1 localized to the outer leaflet of EVs, and its association with EVs greatly increased STI1 activity upon PrP(C)-dependent neuronal signaling. These results indicate that astrocytes secrete a diverse population of EVs derived from MVBs that contain STI1 and suggest that the interaction between EVs and neuronal surface components enhances STI1-PrP(C) signaling.

Selenophosphate synthetase (SPS) plays an indispensable role in selenium metabolism, being responsible for catalyzing the activation of selenide with adenosine 5'-triphosphate (ATP) to generate selenophosphate, the essential selenium donor for selenocysteine synthesis. Recombinant full-length Leishmania major SPS (LmSPS2) was recalcitrant to crystallization. Therefore, a limited proteolysis technique was used and a stable N-terminal truncated construct (ΔN-LmSPS2) yielded suitable crystals. The Trypanosoma brucei SPS orthologue (TbSPS2) was crystallized by the microbatch method using paraffin oil. X-ray diffraction data were collected to resolutions of 1.9 Å for ΔN-LmSPS2 and 3.4 Å for TbSPS2.

Objective: To evaluate the remodeling of the extracellular matrix in intervertebral disc degeneration through the experimental model of intervertebral disc degeneration., Methods: The model of disc degeneration induction, using needle 20G and 360° rotation, was applied for 30 seconds between the 6(th)/7(th), and 8(th)/9(th) coccygeal vertebrae of Wistar rats. The intermediary level, between the 7(th) and 8(th) vertebrae, was taken as control, not being subjected puncture. The distribution of the extracellular matrix components involved in the remodeling and inflammation process, such as proteoglycans (aggrecan, decorin, biglycan), growth factors (TGFβ), heparanase isoforms (HPSE1, HPSE2), metaloprotesasis-9 (MMP9) and interleukins (IL-6, IL-10) was analyzed during the post-injury period (15 to 30 days) and in the control group (discs collected immediately after the puncture, day zero). On the 15(th) day, acute phase of the disease, a reduced expression of extracellular matrix components had been observed, whilst there were no differences in the interleukins expression. At 30 days, the molecules followed a very similar pattern of expression in the control group (not affected by disc degeneration)., Results: The results show that during the acute phase significant alterations in the extracellular matrix components occur and in the late phase intervertebral disc returns to a profile similar to noninvolved tissue, probably due to extensive remodeling process of the extracellular matrix that is capable of regenerating the damaged tissue., Conclusion: : The experimental model used demonstrated the occurrence of significant changes in the extracellular matrix during the period analyzed after induction of intervertebral disc degeneration. Laboratory investigation.

Rational ligand design: Schaeffer's acid analogues were identified as novel inhibitors of M. tuberculosis type II dehydroquinase, a key enzyme of the shikimate pathway. Their likely binding mode was predicted using a combination of ensemble docking and flexible active site residues. Potentially, this scaffold could provide a good starting point for the design of antitubercular agents., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The shikimate pathway is essential in Mycobacterium tuberculosis and its absence from humans makes the enzymes of this pathway potential drug targets. In the present paper, we provide structural insights into ligand and inhibitor binding to 3-dehydroquinate dehydratase (dehydroquinase) from M. tuberculosis (MtDHQase), the third enzyme of the shikimate pathway. The enzyme has been crystallized in complex with its reaction product, 3-dehydroshikimate, and with six different competitive inhibitors. The inhibitor 2,3-anhydroquinate mimics the flattened enol/enolate reaction intermediate and serves as an anchor molecule for four of the inhibitors investigated. MtDHQase also forms a complex with citrazinic acid, a planar analogue of the reaction product. The structure of MtDHQase in complex with a 2,3-anhydroquinate moiety attached to a biaryl group shows that this group extends to an active-site subpocket inducing significant structural rearrangement. The flexible extensions of inhibitors designed to form π-stacking interactions with the catalytic Tyr24 have been investigated. The high-resolution crystal structures of the MtDHQase complexes provide structural evidence for the role of the loop residues 19-24 in MtDHQase ligand binding and catalytic mechanism and provide a rationale for the design and efficacy of inhibitors.

Due to the great volume of banana skin resulting from the industrialization of banana and to their high pectin content, the objectives of the present study were to evaluate the effect of the following factors: extract/sugar, pectin and citric acid on the chemical, physical and sensory qualities of the jellies obtained. A complete factorial experimental design was used (2(3)) with 3 central points to evaluate the influence of the factors on the dependent variables, testing the linear models. The chemical properties underwent few alterations and the instrumental and sensory texture attributes were mainly affected by the extract/sugar ratio and the pectin level. The brittleness, elasticity and gumminess increased with increases in the extract/ sugar ratio and pectin level. According to the sensory analysis and the purchasing intention, the best formulations were those obtained using a higher extract/sugar ratio (60/40) and lower pectin level (0.5 g/ 100), combined with the highest (20 mL) or lowest volumes of citric acid (15 mL), with scores for all the attributes in the range from 'I liked slightly' to 'I liked moderately'.

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr(42), His(76), and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg(120) located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity.

Glycodiversification, an invaluable tool for generating biochemical diversity, can be catalyzed by glycosyltransferases, which attach activated sugar "donors" onto "acceptor" molecules. However, many glycosyltransferases can tolerate only minor modifications to their native substrates, thus making them unsuitable tools for current glycodiversification strategies. Here we report the production of functional chimeric glycosyltransferases by mixing and matching the N- and C-terminal domains of glycopeptide glycosyltransferases. Using this method we have generated hybrid glycopeptides and have demonstrated that domain swapping can result in a predictable switch of substrate specificity, illustrating that N- and C-terminal domains predominantly dictate acceptor and donor specificity, respectively. The determination of the structure of a chimera in complex with a sugar donor analog shows that almost all sugar-glycosyltransferase binding interactions occur in the C-terminal domain.

Transposon elements are important tools for gene function analysis, for example they can be used to easily create genome-wide collections of insertion mutants. Transposons may also carry sequences coding for an epitope or fluorescent marker useful for protein expression and localization analysis. We have developed three new Tn5-based transposons that incorporate a GFP (green fluorescent protein) coding sequence to generate fusion proteins in the important fungal pathogen Candida albicans. Each transposon also contains the URA3 and Kan(R) genes for yeast and bacterial selection, respectively. After in vitro transposition, the insertional allele is transferred to the chromosomal locus by homologous recombination. Transposons Tn5-CaGFP and Tn5-CaGFP-URA3::FLIP can generate C-terminal truncated GFP fusions. A URA3 flipper recycling cassette was incorporated into the transposon Tn5-CaGFP-URA3::FLIP. After the induction of Flip recombinase to excise the marker, the heterozygous strain is transformed again in order to obtain a GFP-tagged homozygous strains. In the Tn5-CaGFP-FL transposon the markers are flanked by a rare-cutting enzyme. After in vitro transposition into a plasmid-borne target gene, the markers are eliminated by restriction digestion and religation, resulting in a construct coding for full-length GFP-fusion proteins. This transposon can generate plasmid libraries of GFP insertions in proteins where N- or C-terminal tagging may alter localization. We tested our transposon system by mutagenizing the essential septin CDC3 gene. The results indicate that the Cdc3 C-terminal extension is important for correct septin filament assembly. The transposons described here provide a new system to obtain global gene expression and protein localization data in C. albicans.

The resumption of tuberculosis led to an increased need to understand the molecular mechanisms of drug action and drug resistance, which should provide significant insight into the development of newer compounds. Isoniazid (INH), the most prescribed drug to treat TB, inhibits an NADH-dependent enoyl-acyl carrier protein reductase (InhA) that provides precursors of mycolic acids, which are components of the mycobacterial cell wall. InhA is the major target of the mode of action of isoniazid. INH is a pro-drug that needs activation to form the inhibitory INH-NAD adduct. Missense mutations in the inhA structural gene have been identified in clinical isolates of Mycobacterium tuberculosis resistant to INH. To understand the mechanism of resistance to INH, we have solved the structure of two InhA mutants (I21V and S94A), identified in INH-resistant clinical isolates, and compare them to INH-sensitive WT InhA structure in complex with the INH-NAD adduct. We also solved the structure of unliganded INH-resistant S94A protein, which is the first report on apo form of InhA. The salient features of these structures are discussed and should provide structural information to improve our understanding of the mechanism of action of, and resistance to, INH in M. tuberculosis. The unliganded structure of InhA allows identification of conformational changes upon ligand binding and should help structure-based drug design of more potent antimycobacterial agents.

The increase in incidence of infectious diseases worldwide, particularly in developing countries, is worrying. Each year, 14 million people are killed by infectious diseases, mainly HIV/AIDS, respiratory infections, malaria and tuberculosis.. Despite the great burden in the poor countries, drug discovery to treat tropical diseases has come to a standstill. There is no interest by the pharmaceutical industry in drug development against the major diseases of the poor countries, since the financial return cannot be guaranteed. This has created an urgent need for new therapeutics to neglected diseases. A possible approach has been the exploitation of the inhibition of unique targets, vital to the pathogen such as the shikimate pathway enzymes, which are present in bacteria, fungi and apicomplexan parasites but are absent in mammals. The chorismate synthase (CS) catalyses the seventh step in this pathway, the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. The strict requirement for a reduced flavin mononucleotide and the anti 1,4 elimination are both unusual aspects which make CS reaction unique among flavin-dependent enzymes, representing an important target for the chemotherapeutic agents development. In this review we present the main biochemical features of CS from bacterial and fungal sources and their difference from the apicomplexan CS. The CS mechanisms proposed are discussed and compared with structural data. The CS structures of some organisms are compared and their distinct features analyzed. Some known CS inhibitors are presented and the main characteristics are discussed. The structural and kinetics data reviewed here can be useful for the design of inhibitors.

Bacteria, fungi and plants can convert carbohydrate and phosphoenolpyruvate into chorismate, which is the precursor of various aromatic compounds. The seven enzymes of the shikimate pathway are responsible for this conversion. Shikimate kinase (SK) is the fifth enzyme in this pathway and converts shikimate to shikimate-3-phosphate. In this work, the conformational changes that occur on binding of shikimate, magnesium and chloride ions to SK from Mycobacterium tuberculosis (MtSK) are described. It was observed that both ions and shikimate influence the conformation of residues of the active site of MtSK. Magnesium influences the conformation of the shikimate hydroxyl groups and the position of the side chains of some of the residues of the active site. Chloride seems to influence the affinity of ADP and its position in the active site and the opening length of the LID domain. Shikimate binding causes a closing of the LID domain and also seems to influence the crystallographic packing of SK. The results shown here could be useful for understanding the catalytic mechanism of SK and the role of ions in the activity of this protein.

In bacteria, fungi, plants, and apicomplexan parasites, the aromatics compounds, such as aromatics amino acids, are synthesized through seven enzymes from the shikimate pathway, which are absent in mammals. The absence of this pathway in mammals make them potential targets for development of new therapy against infectious diseases, such as tuberculosis, which is the world's second commonest cause of death from infectious disease. The last enzyme of shikimate pathway is the chorismate synthase (CS), which is responsible for conversion of the 5-enolpyruvylshikimate-3-phosphate to chorismate. Here, we report the crystallographic structure of CS from Mycobacterium tuberculosis (MtCS) at 2.65 A resolution. The MtCS structure is similar to other CS structures, presenting beta-alpha-beta sandwich structural topology, in which each monomer of MtCS consists of a central helical core. The MtCS can be described as a tetramer formed by a dimer of dimers. However, analytical ultracentrifugation studies suggest the MtCS is a dimer with a more asymmetric shape than observed on the crystallographic dimer and the existence of a low equilibrium between dimer and tetramer. Our results suggest that the MtCS oligomerization is concentration dependent and some conformational changes must be involved on that event.

Tuberculosis remains the leading cause of mortality arising from a bacterial pathogen (Mycobacterium tuberculosis). There is an urgent need for the development of new antimycobacterial agents. The aromatic amino-acid pathway is essential for the survival of this pathogen and represents a target for structure-based drug design. Accordingly, the M. tuberculosis prephenate dehydratase has been cloned, expressed, purified and crystallized by the hanging-drop vapour-diffusion method using PEG 400 as a precipitant. The crystal belongs to the orthorhombic space group I222 or I2(1)2(1)2(1), with unit-cell parameters a = 98.26, b = 133.22, c = 225.01 angstroms, and contains four molecules in the asymmetric unit. A complete data set was collected to 3.2 angstroms resolution using a synchrotron-radiation source.

The development of new therapies against infectious diseases is vital in developing countries. Among infectious diseases, tuberculosis is considered the leading cause of death. A target for development of new drugs is the tryptophan pathway. The last enzyme of this pathway, tryptophan synthase (TRPS), is responsible for conversion of the indole 3-glycerol phosphate into indol and the condensation of this molecule with serine-producing tryptophan. The present work describes the molecular models of TRPS from Mycobacterium tuberculosis (MtTRPS) complexed with six inhibitors, the indole 3-propanol phosphate and five arylthioalkyl-phosphonated analogs of substrate of the alpha-subunit. The molecular models of MtTRPS present good stereochemistry, and the binding of the inhibitors is favorable. Thus, the generated models can be used in the design of more specific drugs against tuberculosis and other infectious diseases.

Purine nucleoside phosphorylase (PNP) is a ubiquitous enzyme, which plays a key role in the purine salvage pathway, and PNP deficiency in humans leads to an impairment of T-cell function, usually with no apparent effects on B-cell function. Human PNP has been submitted to intensive structure-based design of inhibitors, most of them using low-resolution structures of human PNP. Here we report the crystal structure of human PNP in complex with hypoxanthine, refined to 2.6A resolution. The intermolecular interaction between ligand and PNP is discussed.

The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3A resolution, clearly revealing the amino acid residues involved in shikimate binding. In MtSK, the Glu61 strictly conserved in SK forms a hydrogen bond and salt-bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81, and Arg136, and hydroxyl groups with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for elucidation of the mechanism of SK-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.

Tuberculosis made a resurgence in the mid-1980s and now kills approximately 3 million people a year. The re-emergence of tuberculosis as a public health threat, the high susceptibility of HIV-infected persons and the proliferation of multi-drug-resistant strains have created a need to develop new drugs. Shikimate kinase and other enzymes in the shikimate pathway are attractive targets for development of non-toxic antimicrobial agents, herbicides and anti-parasitic drugs, because the pathway is essential in these species whereas it is absent from mammals. The crystal structure of shikimate kinase from Mycobacterium tuberculosis (MtSK) complexed with MgADP and shikimic acid (shikimate) has been determined at 2.3 A resolution, clearly revealing the amino-acid residues involved in shikimate binding. This is the first three-dimensional structure of shikimate kinase complexed with shikimate. In MtSK, the Glu61 residue that is strictly conserved in shikimate kinases forms a hydrogen bond and salt bridge with Arg58 and assists in positioning the guanidinium group of Arg58 for shikimate binding. The carboxyl group of shikimate interacts with Arg58, Gly81 and Arg136 and the hydroxyl groups interact with Asp34 and Gly80. The crystal structure of MtSK-MgADP-shikimate will provide crucial information for the elucidation of the mechanism of the shikimate kinase-catalyzed reaction and for the development of a new generation of drugs against tuberculosis.

The enzymes of the shikimate pathway are potential targets for the development of new therapies because they are essential for bacteria but absent from mammals. The last step in this pathway is performed by chorismate synthase (CS), which catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate to chorismate. Optimization of crystallization trials allowed the crystallization of homogeneous recombinant CS from Mycobacterium tuberculosis (MtCS). The crystals of MtCS belong to space group P6(4)22 (or P6(2)22) and diffract to 2.8 A resolution, with unit-cell parameters a = b = 129.7, c = 156.8 A. There are two molecules in the asymmetric unit. Molecular-replacement trials were not successful. Heavy-atom derivative screening is in progress.

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. More recently, the 3-D structure of human PNP has been refined to 2.3A resolution, which allowed a redefinition of the residues involved in the substrate-binding sites and provided a more reliable model for structure-based design of inhibitors. This work reports crystallographic study of the complex of Human PNP:guanine (HsPNP:Gua) solved at 2.7A resolution using synchrotron radiation. Analysis of the structural differences among the HsPNP:Gua complex, PNP apoenzyme, and HsPNP:immucillin-H provides explanation for inhibitor binding, refines the purine-binding site, and can be used for future inhibitor design.

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. PNP is a target for inhibitor development aiming at T-cell immune response modulation. This work reports on the crystallographic study of the complex of human PNP-immucillin-H (HsPNP-ImmH) solved at 2.6A resolution using synchrotron radiation. Immucillin-H (ImmH) inhibits the growth of malignant T-cell lines in the presence of deoxyguanosine without affecting non-T-cell tumor lines. ImmH inhibits activated normal human T cells after antigenic stimulation in vitro. These biological effects of ImmH suggest that this agent may have utility in the treatment of certain human diseases characterized by abnormal T-cell growth or activation. This is the first structural report of human PNP complexed with immucillin-H. The comparison of the complex HsPNP-ImmH with recent crystallographic structures of human PNP explains the high specificity of immucillin-H for human PNP.