Cholesterol and 27-hydroxycholesterol promote thyroid carcinoma aggressiveness.

Cholesterol mediates its proliferative and metastatic effects via the metabolite 27-hydroxycholesterol (27-HC), at least in breast and endometrial cancer. We determined the serum lipoprotein profile, intratumoral cholesterol and 27-HC levels in a cohort of patients with well-differentiated papillary thyroid carcinoma (PTC; low/intermediate and high risk), advanced thyroid cancers (poorly differentiated, PDTC and anaplastic thyroid carcinoma, ATC) and benign thyroid tumors, as well as the expression of genes involved in cholesterol metabolism. We investigated the gene expression profile, cellular proliferation, and migration in Nthy-ori 3.1 and CAL-62 cell lines loaded with human low-density lipoprotein (LDL). Patients with more aggressive tumors (high-risk PTC and PDTC/ATC) showed a decrease in blood LDL cholesterol and apolipoprotein B. These changes were associated with an increase in the expression of the thyroid's LDL receptor, whereas 3-hydroxy-3-methylglutaryl-CoA reductase and 25-hydroxycholesterol 7-alpha-hydroxylase were downregulated, with an intratumoral increase of the 27-HC metabolite. Furthermore, LDL promoted proliferation in both the Nthy-ori 3.1 and CAL-62 thyroid cellular models, but only in ATC cells was its cellular migration increased significantly. We conclude that cholesterol and intratumoral accumulation of 27-HC promote the aggressive behavior process of PTC. Targeting cholesterol metabolism could be a new therapeutic strategy in thyroid tumors with poor prognosis.

Evaluation of inhibitors for development of a selective medium for isolation of Leptospira spp. from clinical samples.

This study was conducted to develop a selective medium for the detection of Leptospira spp. in clinical samples. Serovars of Leptospira spp., environmental bacteria and the fungus from contaminated cultures of patients with suspected leptospirosis were inoculated into EMJH medium containing amphotericin B, 5-fluorouracil (5-FU), furazolidone and neomycin used singly or combined. Medium with 5-FU at the concentration of 200 µg ml-1 did not show any inhibitory effect against the fungus, Gram-negative bacilli and any of the leptospira strains except serovar Pyrogenes. The highest concentration of neomycin and furazolidone that did not inhibit the growth of leptospires was 4 µg ml-1 . All strains of Leptospira spp. grew on 5-FU (100 µg ml-1 ) in combination with neomycin (4 µg ml-1 ) and on 5-FU (100 µg ml-1 ) in combination with furazolidone (4 µg ml-1 ). The highest concentration of amphotericin B (500 µg ml-1 ) that inhibited the growth of the fungus also inhibited the bacteria and most of serovars of Leptospira spp. The most effective antibiotic combinations that inhibited the majority of environmental bacteria growth without affecting leptospiral growth were EMJH with 5-FU (100 µg ml-1 ) in combination with neomycin (4 µg ml-1 ). In conclusion, these findings will help the development of new selective media to isolate leptospires. SIGNIFICANCE AND IMPACT OF THE STUDY: Leptospirosis is one of the most widespread zoonotic diseases in the world. Since certain serovars are often associated with the symptoms and severity of the disease, the isolation and identification of the leptospires usually permits the prediction of sources of infection. Attempts to isolate Leptospira spp. from clinical specimens are often frustrated by overgrowth of the slow-growing bacteria by more rapidly growing contaminants. In this study, we evaluated selective agents to develop a new selective medium to isolate leptospires. The results demonstrated that the association of drugs in concentrations that allowed the growth of leptospires is to be more effective in inhibiting bacterial contaminants.

The effects of smokeless cookstoves on peak expiratory flow rates in rural Honduras.

BACKGROUND: The use of biomass fuel for cooking in traditional cookstove designs negatively affects respiratory health of communities in developing countries. Indoor pollution affects particularly women and children, who are participating in food preparation. The effects of smokeless cookstove designs on indoor pollution are well documented, but few studies exist to assess the effects of improved stove designs on the respiratory health of community members. METHODS: This study uses peak expiratory flow rate (PEFR) measurements in a before-and-after format to assess respiratory function of inhabitants of all 30 houses of Buenas Noches in central Honduras. PEFRs are measured before and 6 months after the installation of Justa stoves in people's homes. Health behaviors, respiratory symptoms and fire wood use are evaluated in a door-to-door survey format. RESULTS: A total of 137 eligible women and children between 6 and 14 years participated in the study. PEFR improved by 9.9-18.5% (P < 0.001) depending on the participants' exposure to indoor pollution. Health complaints like cough and behaviors like clinic visits did not change with the introduction of smokeless cookstove technology. CONCLUSIONS: Smokeless stoves improve respiratory health in an environment of high levels of indoor pollution.

Efficacy of serum samples stored on filter paper for the detection of antibody to Leptospira spp. by microagglutination test (MAT).

The aim of this study was to investigate the microagglutination test (MAT) results in serum samples dried on filter paper and stored at different temperatures during 1day, 7days, 30days and 1year to determine the stability of sera antibody against leptospires. Serum samples collected onto filter paper for the detection of leptospires antibody was compared with MAT in a study of 300 serum samples from patients with suspected leptospirosis. Among 300 fresh serum samples analyzed by MAT 156 (52%) were positive and 144 (48%) negative. All the negative fresh serum samples were negative when dried on filter paper (specificity 100%). The sensitivity of MAT performed on dried serum samples was 100%. Storage on filter paper at room temperature and at 4°C for 1 and 7days did not affect the MAT titers. For up to 7days, 98.72% of dried serum samples had titers identical to those of the corresponding serum samples, and 1.18% of dried serum samples showed 1 dilution of difference. After a storage period of one month a prozone phenomenon was observed. After a storage period of one year all serum samples were negative. Serum samples collected onto filter paper are a convenient source of antibodies for serological diagnosis and epidemiological surveys.

Analysis of multilocus sequence typing for identification of Leptospira isolates in Brazil.

A collection of 101 Leptospira isolates was tested by multilocus sequence typing (MLST) and by traditional serotyping. MLST divided the isolates into 4 sequence types (STs), while serotyping classified them into 6 serogroups. Two isolates failed to generate products for some genes by MLST. MLST was less discriminatory than serotyping for uncommonly occurring isolates from humans in Brazil.

Leptospiral glycolipoprotein as a candidate antigen for serodiagnosis of human leptospirosis.

AIMS: Development of a simple, specific, rapid and inexpensive Dot-ELISA test for early diagnosis of human leptospirosis. METHODS AND RESULTS: Serum samples from 90 patients diagnosed with leptospirosis were analysed by Dot-ELISA test incorporating Glycolipoprotein (GLP) antigen from serovars Copenhageni and Patoc. Results were compared with those obtained with microscopic agglutination test, currently, the gold standard reference serological method. Serum samples from healthy blood bank donors and patients diagnosed with diseases other than leptospirosis were used as negative controls. The specificities of both GLP-based assays were 97.1% and 100% with serum samples from patients with other diseases and with serum samples from healthy control group, respectively. With serum samples from patients with acute leptospirosis, sensitivity was 76.6% with Dot-ELISA Copenhageni and 90.0% with Dot-ELISA Patoc. With serum samples from patients in convalescence, sensitivity was 100% with both GLP-based assays. CONCLUSIONS: This Dot-ELISA provides a candidate antigen for serodiagnosis of leptospirosis during all phases of illness and could be a good alternative method for the early diagnosis of leptospirosis. SIGNIFICANCE AND IMPACT OF THE STUDY: The Dot-ELISA test is simple, specific, rapid and inexpensive. It is suitable for identifying a large number of samples and, hence, reducing the death rate of patients with leptospirosis.

Application of pulsed-field gel electrophoresis for the discrimination of leptospiral isolates in Brazil.

AIMS: Leptospirosis is a public health problem worldwide. Traditionally, microscopic agglutination test (MAT) and cross-agglutinin absorption test (CAAT) are used to identify leptospires. However, these techniques are laborious and time-consuming, requiring the maintenance of a collection of more than 200 reference strains and correspondent rabbit antisera. The purpose of this study was to evaluate the pulsed-field gel electrophoresis (PFGE) method for discrimination of Leptospira serovars. METHODS AND RESULTS: Fourteen clinical isolates of Leptospira spp. were analysed by MAT before being characterized by PFGE. The isolates were compared with a library of 206 different reference Leptospira serovars. All the isolates gave clear profiles with high resolution. PFGE and MAT results were in agreement for all clinical isolates evaluated. Twelve isolates were classified as serovar Icterohaemorrhagiae/Copenhageni by PFGE. By MAT, these isolates were classified as serogroup Icterohaemorrhagiae with titres ranging from 3200 to 25 600. Two isolates were classified as serovar Canicola by PFGE, and as serogroup Canicola by MAT with titres higher than 3200. CONCLUSIONS: PFGE offers the advantages of simple, reliable and reproducible results. SIGNIFICANCE AND IMPACT OF THE STUDY: PFGE provides a convenient tool for the identification of clinical isolates.

Reactivation strategies by unfolding/refolding of chymotrypsin derivatives after inactivation by organic solvents.

Immobilized enzyme derivatives, in organic media at neutral pH and moderate temperatures, should be mainly and perhaps uniquely inactivated by promotion of conformational changes on their 3D structure. Subsequent irreversible inactivation mechanisms (intermolecular aggregations, chemical modifications, thiol-disulfide exchanges) are thus impossible. However, simple reincubation in aqueous medium of enzymes previously inactivated by solvents usually yields significant but slow and incomplete reactivations. Disruption of incorrect protein structures by denaturing agents (urea, guanidine) is proposed as a new strategy to get rapid, complete and technologically feasible reactivations. By using multipoint immobilized chymotrypsin derivatives, we have evaluated the possibility of unfolding and further refolding of native (non-inactivated) derivatives by different denaturing conditions. After unfolding in 8 M guanidine, derivatives were quickly and completely refolded up to 100% of catalytic activity in 10 minutes. Besides, successive cycles of unfolding and refolding could be exactly reproduced. Finally we checked the possibility to reactivate chymotrypsin derivatives inactivated by dioxane. Simple reincubations in aqueous media yielded a poor reactivation even after 24 hours. However, unfolding in 8 M guanidine enabled complete reactivation in less than 2 hours. From this point of view, by working under 'chemically inert conditions' (moderate pH and temperatures), fully dispersed covalently immobilized enzyme derivatives seem to behave as almost everlasting catalysts despite the very deleterious effect of organic media.

Effect of thermodynamic water activity on amino-acid ester synthesis catalyzed by agarose-chymotrypsin in 3-pentanone.

Chymotrypsin linked to agarose beads by multi-point covalent attachment catalyzes synthesis of Ac-Trp-OEt in 3-pentanone even when the thermodynamic water activity (aw) of the system is reduced to as low as 0.4. If fully hydrated catalyst is added to the reaction mixture before removal of water, product is formed linearly once aw has stabilized. The initial rate is reduced from that if aw is kept close to 1 (0.47 mmol s-1 (kg enzyme)-1), to 50% (aw 0.9), 25% (aw 0.4) and < 1% (aw 0.25). The large drop between aw of 1 and 0.9 probably reflects the effects of water removal on the agarose gel structure. Catalyst partly dried (even only to aw 0.86) before adding to the organic phase is inactive. At reduced aw, the equilibrium (when reached) is shifted in favor of the ester, as expected.

The equilibrium and kinetics of N-acetyl-tryptophan phenylethyl ester synthesis by agarose-chymotrypsin in organic media.

The synthesis of N-acetyl tryptophan phenylethyl ester in organic media is catalyzed by suspended agarose beads with multipoint covalently attached chymotrypsin. A dilute aqueous phase is trapped within the gel beads and may be manipulated separately from the organic phase. The equilibrium position becomes more favorable as the solvent polarity decreases, with K(eq) increasing 38 times between 2-butanone and 1,1,1-trichloroethane. The more apolar solvents also give faster kinetics. Addition of cosolvents (up to 10% dimethylformamide or 20% acetonitrile) does not affect the rate but does substantially reduce the equilibrium yield, presumably also by making the organic phase more polar. With trichloroethane as solvent the enzyme appears to be kinetically saturated with 1M phenylethanol. Doubling this concentration also does not cause the expected increase in equilibrium conversion, probably again because K(eq) is reduced in the more polar organic phase. Increased temperature raises the reaction rate as expected but has little effect on the equilibrium.

Effect of immiscible organic solvents on activity/stability of native chymotrypsin and immobilized-stabilized derivatives.

We have developed different activity/stability tests to evaluate the possibilities of fully dispersed chymotrypsin derivatives as industrial catalysts in biphasic systems. We have tested different immiscible organic solvents (log P ranged from 0.65 to 2.8) and used different enzyme derivatives (soluble chymotrypsin and one-point and multipoint covalent attached derivatives). Special emphasis has been given to the role of the "exact composition of the aqueous phase."High phosphate concentrations largely protect every hymotrypsin derivative from the distorting effects of dissolved solvent molecules. The effects on the activity and stability of soluble chymotrypsin due to saturating solvent concentrations in an aqueous solution, and the much more severe effects of contact with the phase interface in a stirred biphasic system, all show the opposite trend for the influence of solvent polarity to that generally observed for biocatalysts. For example, deleterious effects decline in the order chloroform, dichloromethane, ethyl acetate. On the contrary, with or without stirring, our stabilized chymotrypsin-agarose derivatives are much more stable against these water-immiscible solvents, and their relative effects follow the normal trend. From these integrated activity and stability tests we can conclude that fully dispersed immobilized-stabilized derivatives seem to be an interesting alternative to develop industrial biphasic processes catalyzed by chymotrypsin.

Enzyme reaction engineering: design of peptide synthesis by stabilized trypsin.

By using very active and very stable trypsin agarose derivatives, we have optimized the design of the synthesis of a model dipeptide, benzoylarginine leucinamide, by two different strategies: (i) kinetically controlled synthesis (KCS), by using benzoyl arginine ethyl ester and leucinamide as substrates, and (ii) thermodynamically controlled synthesis (TCS), by using benzoyl arginine and leucinamide as substrates. In each strategy, we have studied the integrated effect of a number of variables that define the reaction medium on different parameters of industrial interest, e.g. time course of peptide synthesis, higher synthetic yields, and stability of the catalyst, as well as aminolysis/hydrolysis ratios and rate of peptide hydrolysis in the case of KCS. Both synthetic approaches were carried out in monophasic water or water-organic cosolvent systems. We have mainly tested a number of variables, e.g. temperature, polarity of the reaction medium (presence of cosolvents, presence of ammonium sulfate), and exact structure of the trypsin derivatives. Optimal experimental conditions for these synthetic approaches were established in order to simultaneously obtain good values for all industrial parameters. The use of previously stabilized trypsin derivatives greatly improves the design of these synthetic approaches (e.g. by using drastic experimental conditions: 1 M ammonium sulfate (KCS) or 90% organic cosolvents (TCS]. In these conditions, our derivatives preserve more than 95% of activity after 2 months and we have been able to reach synthetic productivities of 180 (KCS) and 1 (TCS) tons of dipeptide per year per liter of catalyst.

Stabilizing effect of penicillin G sulfoxide, a competitive inhibitor of penicillin G acylase: its practical applications.

We have found that penicillin G sulfoxide (pen G SO) behaves as a general stabilizing agent of two bacterial penicillin G acylases (PGAs) from E. coli and from K. citrophila), and this role is related to a strong inhibitory effect on the enzymes. The stabilizing effect has been observed during two different inactivation processes: (i) thermal inactivation of soluble enzymes at alkaline pH, and (ii) inactivation of immobilized enzymes as a consequence of covalent multiinteraction with highly activated agarose aldehyde gels. At the same time, pen G SO behaves as a strong competitive inhibitor of these two enzymes. The inhibition constant is more than 10-fold lower than the one corresponding to another smaller competitive inhibitor, phenylacetic acid (PAA), the structure of which is exactly the acyl donor moiety corresponding to pen G SO. In turn, PAA hardly exerts any stabilizing effect on PGAs. The stabilizing effect of pen G SO allowed the preparation of derivatives of these PGAs preserving full catalytic activity in spite of being 1,400- and 650-fold more stable than the corresponding soluble or one-point attached immobilized enzymes.

Immobilization-stabilization of proteases as a tool to improve the industrial design of peptide synthesis.

Synthesis of dipeptides benzoyl Arginine leucinamide and kyotorphin catalyzed by highly stabilized derivatives of trypsin and chymotrypsin have been performed. Extreme experimental conditions could be tested and parameters of industrial interest could be improved provided the high activity and stability of the derivatives in these unfavourable environments. Thermodynamically controlled synthesis catalyzed by trypsin could be optimized and 97% conversion was obtained in 90% organic cosolvents. 100% yields were achieved in kinetically controlled synthesis catalyzed by trypsin in aqueous medium in the presence of IM Ammonium Sulphate. Higher starting concentrations of poorly soluble substrates of chymotrypsin could be used in a reaction medium containing 50% DMF and 95% yield were obtained.

Immobilization-stabilization of penicillin G acylase from Escherichia coli.

We have developed a strategy for immobilization-stabilization of penicillin G acylase from E. coli, PGA, by multipoint covalent attachment to agarose (aldehyde) gels. We hve studied the role of three main variables that control the intensity of these enzyme-support multiinteraction processes: 1. surface density of aldehyde groups in the activated support; 2. temperature; and 3. contact-time between the immobilized enzyme and the activated support prior to borohydride reduction of the derivatives. Different combinations of these three variables have been tested to prepare a number of PGA-agarose derivatives. All these derivatives preserve 100% of catalytic activity corresponding to the soluble enzyme that has been immobilized but they show very different stability. The less stable derivative has exactly the same thermal stability of soluble penicillin G acylase and the most stable one is approximately 1,400 fold more stable. A similar increase in the stability of the enzyme against the deleterious effect of organic solvents was also observed. On the other hand, the agarose aldehyde gels present a very great capacity to immobilize enzymes through multipoint covalent attachment. In this way, we have been able to prepare very active and very stable PGA derivatives containing up to 200 International Units of catalytic activity per mL. of derivative with 100% yields in the overall immobilization procedure.