Caves are stable environments with characteristics favoring the development of microorganisms. The allocthonous input of organic matter and microbes into the warm Neotropical caves may favor the ...development of filamentous fungi, including pathogenic species. Histoplasma capsulatum is a pathogenic species commonly found in caves and associated with bat and bird guano. Many Brazilian caves have been historically visited due to scenic and religious tourism. The objective of this study was to perform a microbiology study for a management plan of a show cave in Brazil, focusing on the presence and distribution of pathogenic and opportunistic fungi in the cave. Statistic analysis was used to verify the influence of touristic activity on airborne fungi spore load. Fungi were isolated from air and guano in Lapa Nova Cave. Samples were obtained through serial dilution, direct and settle plate techniques. For H. capsulatum, samples were incubated in specific media and conditions. Airborne fungal spore load was compared prior and during visitation and statistically analyzed. A total of 2,575 isolates from the genera Aspergillus, Calcarisporium, Chaetomium, Cladosporium, Curvularia, Emericella, Eurotium, Fusarium, Geotrichum, Gliocladium, Mucor, Purpureocillium, Paeciiomyces, Penicillium, Rhizopus and Trichoderma were identified. Histoplasma capsulatum was not isolated from the cave. Eleven opportunistic species were identified. Significant (p<0.05) variations on fungal richness in the air occurred due to cave visitation. Areas of potential microbiologie risks were indicated and management actions suggested. The results suggest a diverse community inhabiting the cave. Possible opportunistic species should be monitored in show caves and microbiota should always be included in the elaboration of cave management plans. This is the first detailed microbiologie study for a management plan of a show cave in the country. It provides relevant information for future management plans.
Cryptococcus gattii is the main pathogen of cryptococcosis in healthy patients and is treated mainly with fluconazole and amphotericin B. The combination of these drugs has been questioned because ...the mechanisms of action could lead to a theoretical antagonistic interaction. We evaluated distinct parameters involved in the in vitro combination of fluconazole and amphotericin B against Cryptococcus gattii. Fourteen strains of C. gattii were used for the determination of MIC, fractional inhibitory concentration, time-kill curve, and postantifungal effect (PAFE). Ergosterol quantification was performed to evaluate the influence of ergosterol content on the interaction between these antifungals. Interaction between the drugs varied from synergistic to antagonistic depending on the strain and concentration tested. Increasing fluconazole levels were correlated with an antagonistic interaction. A total of 48 h was necessary for reducing the fungal viability in the presence of fluconazole, while 12 h were required for amphotericin B. When these antifungals were tested in combination, fluconazole impaired the amphotericin B activity. The ergosterol content decreased with the increase of fluconazole levels and it was correlated with the lower activity of amphotericin B. The PAFE found varied from 1 to 4 h for fluconazole and from 1 to 3 h for amphotericin B. The interaction of fluconazole and amphotericin B was concentration-dependent and special attention should be directed when these drugs are used in combination against C. gattii.
In a broad variety of bilaterian species the trunk central nervous system (CNS) derives from three primary rows of neuroblasts. The fates of these neural progenitor cells are determined in part by ...three conserved transcription factors: vnd/nkx2.2, ind/gsh and msh/msx in Drosophila melanogaster/vertebrates, which are expressed in corresponding non-overlapping patterns along the dorsal-ventral axis. While this conserved suite of "neural identity" gene expression strongly suggests a common ancestral origin for the patterning systems, it is unclear whether the original regulatory mechanisms establishing these patterns have been similarly conserved during evolution. In Drosophila, genetic evidence suggests that Bone Morphogenetic Proteins (BMPs) act in a dosage-dependent fashion to repress expression of neural identity genes. BMPs also play a dose-dependent role in patterning the dorsal and lateral regions of the vertebrate CNS, however, the mechanism by which they achieve such patterning has not yet been clearly established. In this report, we examine the mechanisms by which BMPs act on cis-regulatory modules (CRMs) that control localized expression of the Drosophila msh and zebrafish (Danio rerio) msxB in the dorsal central nervous system (CNS). Our analysis suggests that BMPs act differently in these organisms to regulate similar patterns of gene expression in the neuroectoderm: repressing msh expression in Drosophila, while activating msxB expression in the zebrafish. These findings suggest that the mechanisms by which the BMP gradient patterns the dorsal neuroectoderm have reversed since the divergence of these two ancient lineages.
Heptosyltransferase I (HepI) is responsible for the transfer of l-glycero-d-manno-heptose to a 3-deoxy-α-d-oct-2-ulopyranosonic acid (Kdo) of the growing core region of lipopolysaccharide (LPS). The ...catalytic efficiency of HepI with the fully deacylated analogue of Escherichia coli HepI LipidA is 12-fold greater than with the fully acylated substrate, with a k cat/K m of 2.7 × 106 M–1 s–1, compared to a value of 2.2 × 105 M–1 s–1 for the Kdo2-LipidA substrate. Not only is this is the first demonstration that an LPS biosynthetic enzyme is catalytically enhanced by the absence of lipids, this result has significant implications for downstream enzymes that are now thought to utilize deacylated substrates.
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•Alkylated monosaccharides inhibit Heptosyltransferase I.•Compounds are uncompetitive with sugar acceptor substrate.•Compounds have mixed inhibition with sugar donor ...substrate.•Docking revealed putative allosteric inhibition pocket.
Gram-negative bacteria comprise the majority of microbes that cause infections that are resistant to pre-existing antibiotics. The complex cell wall architecture contributes to their ability to form biofilms, which are often implicated in hospital-acquired infections. Biofilms promote antibiotic resistance by enabling the bacteria to survive hostile environments such as UV radiation, pH shifts, and antibiotics. The outer membrane of Gram-negative bacteria contains lipopolysaccharide (LPS), which plays a role in adhesion to surfaces and formation of biofilms. The main focus of this work was the synthesis of a library of glycolipids designed to be simplified analogues of the Lipid A, the membrane embedded portion component of LPS, to be tested as substrates or inhibitors of Heptosyltransferase I (HepI or WaaC, a glycosyltransferase enzyme involved in the biosynthesis of LPS). Fourteen analogues were synthesized successfully and characterized. While these compounds were designed to function as nucleophilic substrates of HepI, they all demonstrated mild inhibition of HepI. Kinetic characterization of inhibition mechanism identified that the compounds exhibited uncompetitive and mixed inhibition of HepI. Since both uncompetitive and mixed inhibition result in the formation of an Enzyme-Substrate-inhibitor complex, molecular docking studies (using AutoDock Vina) were performed, to identify potential allosteric binding site for these compounds. The inhibitors were shown to bind to a pocket formed after undergoing a conformational change from an open to a closed active site state. Inhibition of HepI via an allosteric site suggest that disruption of protein dynamics might be a viable mechanism for the inhibition of HepI and potentially other enzymes of the GT-B structural class.
Heptosyltransferase I (HepI) catalyzes the addition of l-glycero-β-d-manno-heptose to Kdo2-Lipid A, as part of the biosynthesis of the core region of lipopolysaccharide (LPS). Gram-negative bacteria ...with gene knockouts of HepI have reduced virulence and enhanced susceptibility to hydrophobic antibiotics, making the design of inhibitors of HepI of interest. Because HepI protein dynamics are partially rate-limiting, disruption of protein dynamics might provide a new strategy for inhibiting HepI. Discerning the global mechanism of HepI is anticipated to aid development of inhibitors of LPS biosynthesis. Herein, dynamic protein rearrangements involved in the HepI catalytic cycle were probed by combining mutagenesis with intrinsic tryptophan fluorescence and circular dichroism analyses. Using wild-type and mutant forms of HepI, multiple dynamic regions were identified via changes in Trp fluorescence. Interestingly, Trp residues (Trp199 and Trp217) in the C-terminal domain (which binds ADP-heptose) are in a more hydrophobic environment upon binding of ODLA to the N-terminal domain. These residues are adjacent to the ADP-heptose binding site (with Trp217 in van der Waals contact with the adenine ring of ADP-heptose), suggesting that the two binding sites interact to report on the occupancy state of the enzyme. ODLA binding was also accompanied by a significant stabilization of HepI (heating to 95 °C fails to denature the protein when it is in the presence of ODLA). These results suggest that conformational rearrangements, from an induced fit model of substrate binding to HepI, are important for catalysis, and the disruption of these conformational dynamics may serve as a novel mechanism for inhibiting this and other glycosyltransferase enzymes.
The earliest publications in the field of marriage and family therapy introduced interventions conducted with families experiencing complex health conditions. This strategic review captures an ...evaluation of efficacy for 87 couple and family interventions published between 2010 and 2019 with a focus on the leading causes of mortality in the United States. These health conditions include chromosomal anomalies and accidents with infants aged 0–4 years; accidents and cancer among children aged 5–14; accidents among adolescents aged 15–24; and heart disease, cancer, accidents, chronic lower respiratory diseases, stroke, Alzheimer's disease, diabetes, influenza/pneumonia, and nephritis/nephrosis among adults 25 and older. Results support the need for greater inclusion of couples and families in assessments and interventions. The greatest chasm in efficacy research was with minoritized couples and families. Implications include ways to initiate couple and family interventions in the context of health conditions with attention given to accessibility, recruitment, retention, and evaluation.
Plasmodium falciparum is unable to synthesize purine bases and relies upon purine salvage and purine recycling to meet its purine needs. We report that purines formed as products of polyamine ...synthesis are recycled in a novel pathway in which 5′-methylthioinosine is generated by adenosine deaminase. The action of P. falciparum purine nucleoside phosphorylase is a convergent step of purine salvage, converting both 5′-methylthioinosine and inosine to hypoxanthine. We used accelerator mass spectrometry to verify that 5′-methylthioinosine is an active nucleic acid precursor in P. falciparum. Prior studies have shown that inhibitors of purine salvage enzymes kill malaria, but potent malaria-specific inhibitors of these enzymes have not been described previously. 5′-Methylthio-immucillin-H, a transition state analogue inhibitor that is selective for malarial relative to human purine nucleoside phosphorylase, kills P. falciparum in culture. Immucillins are currently in clinical trials for other indications and may also have application as anti-malarials.
A total of 233 specimens obtained from suspected cases of dermatomycosis from 189 patients were examined for causative fungi from December 2009 to May 2010 in a tertiary care hospital in the city of ...Belo Horizonte, state of Minas Gerais, southeastern Brazil. Yeast and fungal isolates obtained from specimens were regarded as conclusive diagnosis of mycoses in 82 cases (35.19 %), with the exception of two patients with pityriasis versicolor (2.4 %), in which the diagnosis was made only by direct examination plus clinical diagnostics of individuals. Forty-four subjects (23.28 %) were infected in more than one anatomical site. There was a higher occurrence on female patients (146, 77.2 %) than male (43, 22.8 %). Most of the infected patients were aged between 41 and 70 years (68.29 %). There were no statistically significant differences between occurrence of fungal infection and gender, presence of secondary disease and contact with animals. The largest number of examined material occurred in samples from toenails, which resulted in 50 % of positive cultures.
Candida
species were the most frequent group causing dermatomycosis in many anatomical sites, mainly in toenails and fingernails.
Candida parapsilosis
was the most representative (40.24 %) among all agents causing dermatomycosis of toenails and fingernails, followed by
Candida tropicalis
(20.73 %) and
Trichophyton rubrum
(10.98 %). Among the dermatophytes,
Trichophyton
genus represented over 80 % of the isolates, with
T. rubrum
representing 64.29 %,
T. interdigitale
(
T. mentagrophytes
) (21.43 %) and
Microsporum gypseum
(14.29 %).
Oxygen-sensitive proteins, including those enzymes which utilize oxygen as a substrate, can have reduced stability when purified using traditional aerobic purification methods. This manuscript ...illustrates the technical details involved in the anaerobic purification process, including the preparation of buffers and reagents, the methods for column chromatography in a glove box, and the desalting of the protein prior to kinetics. Also described are the methods for preparing and using an oxygen electrode to perform kinetic characterization of an oxygen-utilizing enzyme. These methods are illustrated using the dioxygenase enzyme DesB, a gallate dioxygenase from the bacterium Sphingobium sp. strain SYK-6.
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