Search

Daptomycin is a cyclic lipopeptide antibiotic with potent activity against gram-positive bacteria. It has a calcium-dependent mechanism of action that disrupts multiple features of the bacterial membrane function. This antibiotic is highly demanded due to its effectiveness against to microorganisms resistant to other antibiotics, including vancomycin-resistant Staphylococcus aureus (VRSA) and methicillin-resistant S. aureus (MRSA). Daptomycin is produced by fermentation of Streptomyces roseosporus, currently identified as Streptomyces filamentosus. However, low fermentation yields and high production costs are reported. This chapter describes a method of strain improvement involving random mutagenesis, rational screening by bioassay, and flask fermentation. The ultimate objective is to select mutants of S. roseosporus overproducing daptomycin in order to design a more cost-effective daptomycin production.

Background and Aims: Results from a recent study among 750 heroin users in three Mexico's northern border cities revealed an increase in white powder availability (also known as China white) and preference for this product among heroin users, as well as a general perception of increased overdose cases among this population. Here, we questioned whether those findings reflect an increased presence of heroin laced with fentanyl, which is associated with greater risks of overdose but that, until now, has not been described in Mexico., Design: We tested fentanyl using highly sensitive test strips in syringe plungers, metal cookers and drug wrappings associated with heroin use., Setting: Three injection sites in Tijuana, Baja California, México., Participants: Eighty-nine heroin users who interchanged paraphernalia for new syringes., Measurements: We tested 59 residues of 'pure' white powder. The rest were white powder with black tar (n = 5) or white powder with crystal meth (n = 9), black tar with crystal meth (n = 1), black tar only (n = 13) and crystal meth only (n = 2)., Findings: Users believed that they consumed either white powder heroin, white powder heroin with crystal meth, white powder with black tar heroin or black tar heroin only. Analyses revealed that 93% (n = 55) of the 'pure' white powder samples had fentanyl. All (n = 9) the white powder samples mixed with crystal meth and 40% (n = 2) of the white powder with black tar were also laced with fentanyl., Conclusions: In a sample of 89 heroin users in Mexico, most white powder heroin users were unknowingly exposed to fentanyl, with fentanyl detected in 93% of white powder samples., (© 2019 Society for the Study of Addiction.)

Background: Pertussis immunization during pregnancy is recommended in many countries. Data from large randomized controlled trials are needed to assess the immunogenicity, reactogenicity and safety of this approach., Methods: This phase IV, observer-blind, randomized, placebo-controlled, multicenter trial assessed immunogenicity, transplacental transfer of maternal pertussis antibodies, reactogenicity and safety of a reduced-antigen-content diphtheria-tetanus-three-component acellular pertussis vaccine (Tdap) during pregnancy. Women received Tdap or placebo at 27-36 weeks' gestation with crossover ≤ 72-hour-postpartum immunization. Immune responses were assessed before the pregnancy dose and 1 month after, and from the umbilical cord at delivery. Superiority (primary objective) was reached if the lower limits of the 95% confidence intervals (CIs) of the pertussis geometric mean concentration (GMC) ratios (Tdap/control) in cord blood were ≥ 1.5. Solicited and unsolicited adverse events (AEs) and pregnancy-/neonate-related AEs of interest were recorded., Results: 687 pregnant women were vaccinated (Tdap: N = 341 control: N = 346). Superiority of the pertussis immune response (maternally transferred pertussis antibodies in cord blood) was demonstrated by the GMC ratios (Tdap/control): 16.1 (95% CI: 13.5-19.2) for anti-filamentous hemagglutinin, 20.7 (15.9-26.9) for anti-pertactin and 8.5 (7.0-10.2) for anti-pertussis toxoid. Rates of pregnancy-/neonate-related AEs of interest, solicited general and unsolicited AEs were similar between groups. None of the serious AEs reported throughout the study were considered related to maternal Tdap vaccination., Conclusions: Tdap vaccination during pregnancy resulted in high levels of pertussis antibodies in cord blood, was well tolerated and had an acceptable safety profile. This supports the recommendation of Tdap vaccination during pregnancy to prevent early-infant pertussis disease., Clinical Trial Registration: ClinicalTrials.gov: NCT02377349., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: BC, MAC, NMes, NMey and SOK are employees of the GSK group of companies (GSK), and BC and NMes own GSK restricted shares. BT, MBEP, OGV, SAH and TN’s institutions received grants from GSK during the conduct of the study. KPP received grants from the National Health and Medical Research Council during the conduct of the study, and from MedImmune, Novavax and Pfizer outside the submitted work. FMT’s institution received financial support from GSK during the conduct of the study, as well as financial and non-financial support outside the submitted work; he also received personal fees from Pfizer, Novavax, MSD and Sanofi Pasteur; his institution also received financial support as trial fees from Ablynx, Jansen, Regeneron, Medimmune, Pfizer, MSD, Sanofi Pasteur, Novavax and Novartis, as well as non-financial support from Pfizer and MSD and grants from MSD and Astra Zeneca. LK is working as consultant for GSK. SAH is member of ad-hoc advisory committees for GSK and Sanofi Pasteur and he has a patent for novel triple adjuvant issued. AMG, CMP, GVZ, ICG, JEAF, MARZ, MCFM, MDCM, MMF, MV, PGM, PK, PM, YRE and ZS declare no conflicts of interest., (Copyright © 2019. Published by Elsevier Ltd.)

The clinical use of virtual reality (VR) has proven its efficacy, especially when used as an exposure technique. A prominent property of VR's utility is its equivalence with the reality it represents. In this study, we explored this equivalence in a clinical context using neuroimaging. A sample of 32 adults with specific phobias (i.e., to cockroaches, spiders, or lizards) was divided into two groups: One was exposed to phobic stimuli using VR and the other was exposed to real phobic images (RI). We used brain activations as a dependent measure, focusing specifically on brain areas usually associated with fear processing. Whole-brain analysis detected higher activations for RI in the hippocampus, occipital, and calcarine areas. A specific analysis of the amygdala and insula also detected higher activations and extensions in response to RI, but VR stimuli also activated those areas in a significant manner. These results suggest that even in those cases where RI stimuli activate all of the brain's fear-processing circuits, VR stimuli do so as well. This implies that VR can be useful as an exposure technique similar to RI and applied as more than a mere training mechanism.

Purpose: Sarcoidosis is a granulomatous disease of unknown etiology. Occasionally, triggering causes are identified, such as neoplasms, and they are termed sarcoid-like reactions, which may appear in any sarcoidotic target tissue. Choroidal metastases appear as part of widespread metastatic disease or as the first suggestion of neoplastic disease. They can also be a part of the differential diagnosis of a spectrum of inflammatory eye diseases. We present a case in which a lung carcinoma, pulmonary and eye sarcoid-like reactions, and choroidal metastasis take place in the same patient., Case Report: A 60-year-old male with a past history of pulmonary sarcoidosis and associated anterior uveitis was diagnosed with a lung carcinoma with no regional lymph nodes extension, so that the resection surgery was performed without additional systemic treatment. At the same time, he complained of visual acuity loss and pain in his right eye. An intense ocular inflammatory reaction and a choroidal mass compatible with metastasis were identified. A vitrectomy with an accompanied histological exam of the lesion was deemed inconclusive. Ocular symptoms progressively worsened showing mass growth, and as a result, an enucleation was performed and the histological study subsequently revealed metastasis from his lung carcinoma., Conclusion: Sarcoid-like reactions may be due to incipient malignancies. Any diagnosis of sarcoidosis requires ruling out other diseases that can produce secondary sarcoid-like reactions. In addition, any choroidal mass suggestive of metastasis requires exclusion of metastatic disease even in the absence of clinical signs indicating tumor extension.

Eastern gamagrass (Tripsacum dactyloides L.) belongs to the same tribe of the Poaceae family as maize (Zea mays L.) and grows naturally in the same region where maize is commercially produced in the USA. Although no evidence exists of gene flow from maize to eastern gamagrass in nature, experimental crosses between the two species were produced using specific techniques. As part of environmental risk assessment, the possibility of transgene flow from maize to eastern gamagrass populations in nature was evaluated with the objectives: (1) to assess the seeds of eastern gamagrass populations naturally growing near commercial maize fields for the presence of a transgenic glyphosate-tolerance gene (cp4 epsps) that would indicate cross-pollination between the two species, and (2) to evaluate the possibility of interspecific hybridization between transgenic maize used as male parent and eastern gamagrass used as female parent. A total of 46,643 seeds from 54 eastern gamagrass populations collected in proximity of maize fields in Illinois, USA were planted in a field in 2014 and 2015. Emerged seedlings were treated with glyphosate herbicide and assessed for survival. An additional 48,000 seeds from the same 54 eastern gamagrass populations were tested for the presence of the cp4 epsps transgene markers using TaqMan ® PCR method. The results from these trials showed that no seedlings survived the herbicide treatment and no seed indicated presence of the herbicide tolerant cp4 epsps transgene, even though these eastern gamagrass populations were exposed to glyphosate-tolerant maize pollen for years. Furthermore, no interspecific hybrid seeds were produced from 135 hand-pollination attempts involving 1529 eastern gamagrass spikelets exposed to maize pollen. Together, these results indicate that there is no evidence of gene flow from maize to eastern gamagrass in natural habitats. The outcome of this study should be taken in consideration when assessing for environmental risks regarding the consequence of gene flow from transgenic maize to its wild relatives.

A number of pharmaceutical steroid synthons are currently produced through the microbial side-chain cleavage of natural sterols as an alternative to multi-step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc 2 155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc 2 155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3-ketosteroid 9α-hydroxylase (KshAB) and a ketosteroid Δ 1 -dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4-androstadiene-3,17-dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039-5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_5941 (ΔkstD1)] transforms natural sterols into 4-androstene-3,17-dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc 2 155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids., (© 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.)

Introduction: This study compares the differential efficacy of three groups of treatments for agoraphobia: paroxetine combined with cognitive-behavioral therapy, paroxetine combined with cognitive-behavioral therapy and virtual reality exposure, and a group with only paroxetine., Methodology: 99 patients with agoraphobia were finally selected. Both combined treatment groups received 11 sessions of cognitive-behavioral therapy, and one of the groups was also exposed to 4 sessions of virtual reality treatment. Treatments were applied in individual sessions once a week for 3 months., Results: The three treatment groups showed statistically significant improvements. In some measures, combined treatment groups showed greater improvements. The virtual reality exposure group showed greater improvement confronting phobic stimuli., Conclusions: Treatments combining psychopharmacological and psychological therapy showed greater efficacy. Although the use of new technologies led to greater improvement, treatment adherence problems still remain.

Psychosis, dementias, anxiety, depression, suicide and suicide attempts, as well as psychiatric disorders associated to violence and poverty have increased the global burden of disease. Other related problems associated to special diets, body image, compulsive use of computers and mobile phones, and those frequently observed in migrants subjected to intense distress are reviewed as well. Information and communication technologies may have undesirable side effects affecting some individuals in their conduct and social interactions.

One source of potential harm from the cultivation of transgenic crops is their dispersal, persistence and spread in non-agricultural land. Ecological damage may result from such spread if the abundance of valued species is reduced. The ability of a plant to spread in non-agricultural habitats is called its invasiveness potential. The risks posed by the invasiveness potential of transgenic crops are assessed by comparing in agronomic field trials the phenotypes of the crops with the phenotypes of genetically similar non-transgenic crops known to have low invasiveness potential. If the transgenic and non-transgenic crops are similar in traits believed to control invasiveness potential, it may be concluded that the transgenic crop has low invasiveness potential and poses negligible ecological risk via persistence and spread in non-agricultural habitats. If the phenotype of the transgenic crop is outside the range of the non-transgenic comparators for the traits controlling invasiveness potential, or if the comparative approach is regarded as inadequate for reasons of risk perception or risk communication, experiments that simulate the dispersal of the crop into non-agricultural habitats may be necessary. We describe such an experiment for several commercial insect-resistant transgenic maize events in conditions similar to those found in maize-growing regions of Mexico. As expected from comparative risk assessments, the transgenic maize was found to behave similarly to non-transgenic maize and to be non-invasive. The value of this experiment in assessing and communicating the negligible ecological risk posed by the low invasiveness potential of insect-resistant transgenic maize in Mexico is discussed.

Carotenoids are important pigments produced by plants and many microorganisms, including fungi, microalgae, cyanobacteria, and bacteria. Marine actinomycetes are a group of bacteria that produce a variety of metabolites with economic potential. Here, we describe a general method of selecting marine actinomycetes as carotenoids' producers. The screening is carried out at two levels: the first one involves a quick selection of strains by visual color inspection, and the second consists in the analysis of the extracts by HPLC. The taxonomic analysis of the producing strains gives us an overview of the groups of actinomycetes in which carotenoids can be found.

Mucor circinelloides is a β-carotene producing zygomycete amenable to metabolic engineering using molecular tools. The crtS gene of the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous encodes the enzymatic activities β-carotene hydroxylase and ketolase, allowing this yeast to produce the xanthophyll called astaxanthin. Here we describe the fermentation of X. dendrorhous in astaxanthin producing conditions to purify mRNA for the cloning of the cDNA from the crtS gene by RT-PCR. Further construction of an expression plasmid and transformation of M. circinelloides protoplasts allow the heterologous expression of the crtS cDNA in M. circinelloides to obtain β-cryptoxanthin and zeaxanthin overproducing transformants. These two xanthophylls are hydroxylated compounds from β-carotene. These results show that the crtS gene is involved in the conversion of β-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways.

During the last 3 years the National Academy of Medicine, the School of Medicine and the Ministry of Health have analyzed several global health issues with local impact. The group of articles presented in this symposium is focused on the health systems. The Mexican health system has evolved through the years facing different challenges and showing its strengths as well as several inconsistencies, flaws and limitations. The subjects analyzed deal with several alternatives to face the health challenges. All health systems are under constant change and financial resources as well as conceptual clarity, human resources, coordination, and leadership are needed.

Amine-functionalized anion-tethered ionic liquids (ILs) trihexyl(tetradecyl)phosphonium asparaginate [P(66614)][Asn], glutaminate [P(66614)][Gln], lysinate [P(66614)][Lys], methioninate [P(66614)][Met], prolinate [P(66614)][Pro], taurinate [P(66614)][Tau], and threoninate [P(66614)][Thr] were synthesized and investigated as potential absorbents for CO(2) capture from postcombustion flue gas. Their physical properties, including density, viscosity, glass transition temperature, and thermal decomposition temperature were determined. Furthermore, the CO(2) absorption isotherms of [P(66614)][Lys], [P(66614)][Tau], [P(66614)][Pro], and [P(66614)][Met] were measured using a volumetric method, and the results were modeled with two different Langmuir-type absorption models. The most important result of this study is that the viscosity of [P(66614)][Pro] only increased by a factor of 2 when fully complexed with 1 bar of CO(2) at room temperature. This is in stark contrast to the other chemically reacted ILs investigated here and all other amino acid-based ILs reported in the literature, which dramatically increase in viscosity, typically by 2 orders of magnitude, when complexed with CO(2). The unique behavior of [P(66614)][Pro] is likely due to its ring structure, which limits the number and availability of hydrogen atoms that can participate in a hydrogen bonding network. We found that water can be used to further reduce the viscosity of the CO(2)-complexed IL, while only slightly decreasing the CO(2) capacity. Finally, from temperature-dependent isotherms, we estimate a heat of absorption of -63 kJ/mol of CO(2) for the 1:1 reaction of CO(2) with [P(66614)][Pro], when we use the two-reaction model., (© 2011 American Chemical Society)

Astaxanthin is a red xanthophyll (oxygenated carotenoid) with large importance in the aquaculture, pharmaceutical, and food industries. The green alga Haematococcus pluvialis and the heterobasidiomycetous yeast Xanthophyllomyces dendrorhous are currently known as the main microorganisms useful for astaxanthin production at the industrial scale. The improvement of astaxanthin titer by microbial fermentation is a requirement to be competitive with the synthetic manufacture by chemical procedures, which at present is the major source in the market. In this review, we show how the isolation of new strains of X. dendrorhous from the environment, the selection of mutants by the classical methods of random mutation and screening, and the rational metabolic engineering, have provided improved strains with higher astaxanthin productivity. To reduce production costs and enhance competitiveness from an industrial point of view, low-cost raw materials from industrial and agricultural origin have been adopted to get the maximal astaxanthin productivity. Finally, fermentation parameters have been studied in depth, both at flask and fermenter scales, to get maximal astaxanthin titers of 4.7 mg/g dry cell matter (420 mg/l) when X. dendrorhous was fermented under continuous white light. The industrial scale-up of this biotechnological process will provide a cost-effective method, alternative to synthetic astaxanthin, for the commercial exploitation of the expensive astaxanthin (about $2,500 per kilogram of pure astaxanthin).

An improved semi-industrial process for astaxanthin production by fermentation of Xanthophyllomyces dendrorhous has been developed. The culture medium was designed at the flask scale, reaching an astaxanthin cellular content of 3.0 mgg(-1) cell weight and a volumetric yield of 119 mgL(-1) broth. Astaxanthin production in flask was significantly improved by white light (4.0 mgg(-1) and 221 mgL(-1)), and by ultraviolet light (4.4 mgg(-1) and 235 mgL(-1)). The scale-up to 10- and 800-L fermentors was developed by feeding with glucose. Representative data for illuminated fermentation processes are presented and discussed at the 10-L scale, where 420 mgL(-1) (4.7 mgg(-1)) astaxanthin were produced, and the 800-L scale, with productivities of 350 mgL(-1) (4.1 mgg(-1)) astaxanthin. The purity of the astaxanthin in the broth was about 84%, with accumulation of the following carotenoids other than astaxanthin: 4% beta-carotene, 4% canthaxanthin, 5% HDCO, 1% zeaxanthin and 2% phoenicoxanthin. This technology can be easily scaled-up to an industrial application for the production of this xanthophyll widely demanded nowadays., (Copyright (c) 2010 Elsevier B.V. All rights reserved.)

Amino acid ionic liquid trihexyl(tetradecyl)phosphonium methioninate [P(66614)][Met] and prolinate [P(66614)][Pro] absorb CO(2) in nearly 1:1 stoichiometry, surpassing by up to a factor of 2 the CO(2) capture efficiency of previously reported ionic liquid and aqueous amine absorbants for CO(2). Room temperature isotherms are obtained by barometric measurements in an accurately calibrated stirred cell, and the product identity is confirmed using in situ IR. Density functional theory (DFT) calculations support the 1:1 reaction stoichiometry and predict reaction enthalpies in good agreement with calorimetric measurements and isotherms.

The gdhA gene encoding the NADP-dependent glutamate dehydrogenase (GDH) activity from Xanthophyllomyces dendrorhous has been cloned and characterized, and its promoter used for controlled gene expression in this red-pigmented heterobasidiomycetous yeast. We determined the nucleotide sequence of a 4701 bp DNA genomic fragment, showing an open reading frame of 1871 bp interrupted by five introns with fungal consensus splice-site junctions. The predicted protein (455 amino acids; 49 kDa) revealed high identity to GDHs, especially to those from the fungi Cryptococcus neoformans (70%), Sclerotinia sclerotiorum (66%), and several species of Aspergillus (66-67%). Gene phylogenies support the grouping of X. dendrorhous GDH close to those from the majority of the filamentous fungi. The promoter region of the gdhA gene (PgdhA) contains a TATA-like box and two large pyrimidine stretches. The use of PgdhA for gene expression was validated by electrotransformation of X. dendrorhous using an in-frame fusion with the hygromycin resistance gene (hygR) as a reporter. X. dendrorhous transformants were able to grow in YEME complex medium and in Czapek minimal medium supplemented with 50 microg/ml hygromycin, but gene expression in Czapek medium was repressed when using ammonium acetate as a nitrogen source. PgdhA is a valuable tool for controlled gene expression in Basidiomycetes.

Hydatid disease is a parasitic infection generally occurring in specific geographical areas. Exclusive involvement of the muscles is extremely uncommon, because implantation at this site would require passage through the filters of the liver and lung. We describe a patient with a mass in the popliteal fossa of the knee for 7 years who was seen for pain and was found to have hydatid disease.

Engineering halophilic bacteria to produce carotenoids is a subject of great scientific and commercial interest, as carotenoids are desirable products used as additives and colorants in the food industry, with beta-carotene the most prominent. With this target, we expressed the beta-carotene biosynthetic genes crtE, crtY, crtI, and crtB from Pantoea agglomerans and the cDNA encoding isopentenyl pyrophosphate isomerase from Haematococcus pluvialis in the halophilic bacterium Halomonas elongata obtaining a strain able to produce practically pure beta-carotene. Reverse transcription-polymerase chain reaction analysis showed crtY, crtI, and crtB heterologous expression in a selected exconjugant of H. elongata. Biosynthesis of beta-carotene was dependent on NaCl concentration in the culture medium, with the highest production (560 microg per g of dry weight) in 2% NaCl. On the contrary, no beta-carotene was detected in 15% NaCl. Successful construction of the beta-carotene biosynthetic pathway in H. elongata opens the possibility of engineering halophilic bacteria for carotenoid production.

The conversion of beta-carotene into xanthophylls is a subject of great scientific and industrial interest. We cloned the crtS gene involved in astaxanthin biosynthesis from two astaxanthin producing strains of Xanthophyllomyces dendrorhous: VKPM Y2410, an astaxanthin overproducing strain, and the wild type ATCC 24203. In both cases, the ORF has a length of 3166 bp, including 17 introns, and codes for a protein of 62.6 kDa with similarity to cytochrome-P450 hydroxylases. crtS gene sequences from strains VKPM Y2410, ATCC 24203, ATCC 96594, and ATCC 96815 show several nucleotide changes, but none of them causes any amino acid substitution, except a G2268 insertion in the 13th exon of ATCC 96815 which causes a change in the reading frame. A G1470 --> A change in the 5' splicing region of intron 8 was also found in ATCC 96815. Both point mutations explain astaxanthin idiotrophy and beta-carotene accumulation in ATCC 96815. Mutants accumulating precursors of the astaxanthin biosynthetic pathway were selected from the parental strain VKPM Y2410 (red) showing different colors depending on the compound accumulated. Two of them were blocked in the biosynthesis of astaxanthin, M6 (orange; 1% astaxanthin, 71 times more beta-carotene) and M7 (orange; 1% astaxanthin, 58 times more beta-carotene, 135% canthaxanthin), whereas the rest produced lower levels of astaxanthin (5-66%) than the parental strain. When the crtS gene was expressed in M7, canthaxanthin accumulation disappeared and astaxanthin production was partially restored. Moreover, astaxanthin biosynthesis was restored when X. dendrorhous ATCC 96815 was transformed with the crtS gene. The crtS gene was heterologously expressed in Mucor circinelloides conferring to this fungus an improved capacity to synthesize beta-cryptoxanthin and zeaxanthin, two hydroxylated compounds from beta-carotene. These results show that the crtS gene is involved in the conversion of beta-carotene into xanthophylls, being potentially useful to engineer carotenoid pathways.

Mass transfer models on supercritical carbon dioxide (SC-CO2) extraction of vegetable oils are reviewed, that may facilitate the scale-up of laboratory data for industrial design purposes. Reviewed mechanisms of oil transport within the solid matrix include the desorption from the solid, the formation of a shrinking core of condensed oil in a non-adsorbing porous matrix, and diffusion in a homogenous medium. Analyzed simplificat ions of a general mass transfer model include external control of mass transfer rates, internal control of mass transfer rates, consideration of a linear driving force, and steady state approximations, among others. More complex two-stage models, and critical comparisons of some of the proposed models are also included. Trends for the external mass transfer coefficient and effective diffusivity in the solid matrix from studies on SC-CO2 extraction of oil from vegetable substrates are thoroughly discussed and contrasted with those obtained using simpler model systems. The possible effect of the axial dispersion on the rate of extraction is also discussed. Finally, the high-pressure vegetable oil-CO2 phase equilibrium is discussed in connection with its influence on the mass transfer process. Special emphasis is given to the role of the solid matrix on high-pressure phase equilibrium.

We determined the nucleotide sequence of a 4599-bp DNA genomic fragment including the gamma-actin encoding gene from Blakeslea trispora, showing an open reading frame of 1561 bp interrupted by four introns with fungal consensus splice-site junctions. The untranslated regions of the actA gene contain a consensus TATA box, a CCAAT motif, a large pyrimidine stretch, and the polyadenylation sequence AATAAA. The predicted protein (375 amino acids) revealed high identity to gamma-actins from fungi (>90%), and gene phylogenies support the grouping of B. trispora actin close to those from the majority of the filamentous fungi. actA transcript (1.4 kb) level in beta-carotene producing conditions was faintly higher than carRA (1.9 kb) and slightly lower than carB (1.8 kb) beta-carotene biosynthetic genes. The use of the actA promoter (PactA) for heterologous gene expression was ascertained by the transformation of gene fusions with the bleomycin resistance gene (bleR) from Streptoalloteichus hindustanus and the geneticin resistance marker (aphI) from Tn903, into Escherichia coli and Acremonium chrysogenum.

We purified the beta-N-acetylglucosaminidase from the filamentous fungus Penicillium chrysogenum and its N-terminal sequence was determined, showing the presence of a mixture of two proteins (P1 and P2). A genomic DNA fragment was cloned by using degenerated oligonucleotides from the Nt sequences. The nucleotide sequence showed the presence of an ORF (nagA gene) lacking introns, with a length of 1791 bp, and coding for a protein of 66.5 kDa showing similarity to acetylglucosaminidases. The NagA deduced protein includes P1 and P2 as incomplete forms of the mature protein, and contains putative features for protein maturation: an 18-amino acid signal peptide, a KEX2 processing site, and four glycosylation motifs. The sequence just after the signal peptide corresponds to P2 and that after the KEX2 site to P1. The nagA transcript has a size of about 2.1 kb and is present until the end of the fermentation process for penicillin production. NagA is one of the most largely represented proteins in P. chrysogenum, increasing along the fermentation process. The suitability of the nagA promoter (PnagA) for gene expression in fungi was demonstrated by expressing the bleomycin resistance gene (ble(R)) from Streptoalloteichus hindustanus in P. chrysogenum.

The current situation of clinical research in Mexico is analyzed. The main findings are as follows: 10% of total number of researchers in Mexico are engaged in medical research; there is a highly centralized distribution in the Mexico City metropolitan area; there exists unequal academic development among disciplines, and there is an overwhelming number of researchers in public educational and health institutions. A substantial increase in medical publications during the last 15 years with reasonable citation impact was also found. Several urgent matters to attend were identified, such as financing problems, effect of health services descentralization completed in 1997, and the need to selectively support certain research areas such as accidents, mental health, addictions, geriatrics and chronic diseases.

An Acremonium chrysogenum strain improvement program based on the transformation with cephalosporin biosynthetic genes was carried out to enhance cephalosporin C production. Best results were obtained with cefEF and cefG genes, selecting transformants with increased cephalosporin C production and lower accumulation of biosynthetic intermediates. Phleomycin resistant transformants, designated B1 and C1, showed a single copy random integration event, higher levels of cefEF transcript and, according to immunoblotting analyses, higher amounts of deacetylcephalosporin C acetyltransferase (DAC-AT) protein than their parental strains. Moreover, DAC-AT activity was higher in the transformants. Plasmids carrying geneticin resistance markers based on the nptII gene from Tn5 and the aphI gene from Tn903 were constructed to transform again B1 and C1, showing that the cassette Pgdh-nptII-trpC was able to confer geneticin resistance to A. chrysogenum and demonstrating that geneticin is a helpful selection marker.

Aims: To estimate the prevalence, characteristics and risk factors associated with the development of benzodiazepine dependence in the users of these active ingredients., Design: A representative sample of patients currently receiving benzodiazepine treatment for 1 month or longer (mean 38.2+/-52 months, range 1-360 months) was studied., Participants: One thousand and forty eight (1048) consecutive patients attending 20 primary-care health centres of the Canary Islands Health Service participated in this study during 2002., Measurements: The severity of dependence scale (SDS) was used as a screening test of benzodiazepine dependence among patients using benzodiazepines., Findings: Of patients using benzodiazepines for more than 1 month, 47% developed dependence to these compounds. Benzodiazepine dependence was more prevalent among women who were middle aged, separated, of low educational background, unemployed or housewives. Patients using short half-life benzodiazepines registered higher rates of dependence as well as those using higher doses or the patients with longer use. However, multivariate logistic regression analysis shows that benzodiazepine dependence was closely related only to three of the variables considered: the benzodiazepine dose used, the duration of this use and to the concomitant use of antidepressants., Conclusions: The distribution of dose and length of treatment shows no evidence of pharmacological tolerance, neither in the whole sample nor in those patients with addiction (SDS+), reinforcing the idea that psychological dependence (addiction) is more relevant than pharmacological or physiological dependence in benzodiazepine chronic use.

A telepsychiatry service, using ISDN videoconferencing, was established to provide psychiatric consultations for the population of El Hierro, the most westerly of the Canary Islands. During the first year of routine operation, a total of 40 patients had 40 initial and 126 follow-up teleconsultations. The main reason for the consultation identified in the general practitioner's referral form was to establish a diagnosis (60% of patients); the second most common reason was the management of a patient previously diagnosed (20% of patients). According to the results of a questionnaire survey, patients' acceptance of and satisfaction with the technology were high. In their first teleconsultation, about a third of them said that they experienced some initial inconvenience, but this disappeared after a few minutes. Ninety per cent of the patients considered that they received the follow-up care they required. Telepsychiatry was considered by physicians and patients as an adequate vehicle for mental health-care.

Objective: To evaluate cytology laboratories and the performance of cytotechnologists for establishing efficient external quality control for Mexico's National Program for the Prevention and Control of Cervical Cancer., Material and Methods: During January and February 1998, an onsite evaluation of all cytology laboratories of the Ministry of Health found that only 70% of the microscopes were in adequate working conditions, reagents were out of date, and working conditions were sub-optimal. A program for external quality control based on proficiency testing was established for cytotechnologists. Fifty slide sets with 20 Papanicolaou slides and 10 photographic slides were prepared. The sets were given to the cytotechnologists for evaluation and again one year later by courier., Results: Twenty-one percent of microscopes were repaired and 9% replaced; reagents were distributed and laboratory facilities improved. Only 16% of cytotechnologists passed the initial proficiency test. Cytotechnologists received a refresher training course: one year later 67% of them passed the proficiency test. To ascertain that each slide was correctly diagnosed, 41 sets were rescreened by expert cytopathologists or cytologists and their diagnoses compared to the original ones. Thirty-seven sets had 86% to 96% concordance., Conclusions: This new system for external quality control of cervical cytology allowed the opportune and reliable evaluation of the performance of cytotechnologists.

Objective: To assess the antidepressant prescribing patterns of community psychiatrists, and the prescriptions issued by general practitioners and private physicians during 1999 in order to analyse and discuss the intensity and sources of variations between doctors., Methods: All reimbursed prescriptions for antidepressants written in 1999 by community psychiatrists and general practitioners working for the Canary Islands Health Service at Santa Cruz de Tenerife were collected from official sources. Prescriptions were available individualized for each psychiatrist but were collected globally for the others. Drugs were classified according to the Anatomic Therapeutic Chemical (ATC-1999 edition) System and use was quantified in terms of defined daily doses (DDDs). As an indicator of the quality of drug prescribing, the DU90% was used., Results: The total use of antidepressant in Tenerife was 21.4 DDD/1000 inhabitants/day. The most frequently prescribed substances were fluoxetine, paroxetine and sertraline, which accounted for 58% of all prescriptions. Each psychiatrist used between 10 and 20 different substances and between 15 and 26 different trade names. Prescribing by general practitioners mirrored that of psychiatrist, and private doctors (mainly psychiatrist) were found to have a different pattern of prescribing with higher use of new and uncommon antidepressants. Psychiatrists acknowledge the pressures of promotion by the pharmaceutical industry and half of them recognize a personal relationship with some 'company representatives'., Conclusion: There is a remarkable degree of variation in antidepressant prescribing by psychiatrists and general practitioners, this is due to economic and social factors as much as to morbidity differences.

In the present study, a cationic membrane conductance activated by the acetylcholine agonist carbachol was characterized in vitro in neurons of the basolateral amygdala. Extracellular perfusion of the K+ channel blockers Ba2+ and Cs+ or loading of cells with cesium acetate did not affect the carbachol-induced depolarization. Similarly, superfusion with low-Ca2+ solution plus Ba2+ and intracellular EGTA did not affect the carbachol-induced depolarization, suggesting a Ca2+-independent mechanism. On the other hand, the carbachol-induced depolarization was highly sensitive to changes in extracellular K+ or Na+. When the K+ concentration in the perfusion medium was increased from 4.7 to 10 mM, the response to carbachol increased in amplitude. In contrast, lowering the extracellular Na+ concentration from 143.2 to 29 mM abolished the response in a reversible manner. Results of coapplication of carbachol and atropine, pirenzepine or gallamine indicate that the carbachol-induced depolarization was mediated by muscarinic cholinergic receptors, but not the muscarinic receptor subtypes M1, M2 or M4, specifically. These data indicate that, in addition to the previously described reduction of a time- and voltage-independent K+ current (IKleak), a voltage- and time-dependent K+ current (IM), a slow Ca2+-activated K+ current (sIahp) and the activation of a hyperpolarization-activated inward rectifier K+ current (IQ), carbachol activated a Ca2+-independent non-selective cationic conductance that was highly sensitive to extracellular K+ and Na+ concentrations.

The electrophysiological properties, the response to cholinergic agonists and the morphological characteristics of neurons of the basolateral complex were investigated in rat amygdala slices. We have defined three types of cells according to the morphological characteristics and the response to depolarizing pulses. Sixty-six of the recorded cells (71%) responded with two to three action potentials, the second onwards having less amplitude and longer duration (burst). In a second group, consisting of 21 cells (22%), the response to depolarization was a train of spikes, all with the same amplitude (multiple spike). Finally, seven neurons (7%) showed a single action potential (single spike). Burst response and multiple-spike neurons respond to the cholinergic agonist carbachol (10-20 microM) with a depolarization that usually attained the level of firing. This effect was accompanied by decreased or unchanged input membrane resistance and was blocked by atropine (1.5 microM). The depolarizing response to superfusion with carbachol occurred even when synaptic transmission was blocked by tetrodotoxin, indicating a direct effect of carbachol. Similarly, the depolarization by carbachol was still present when the M-type conductance was blocked by 2 mM Ba2+. The carbachol-induced depolarization was prevented by superfusion with tetraethylammonium (5 mM). Injection of biocytin into some of the recorded cells and subsequent morphological reconstruction showed that "burst" cells have piriform or oval cell bodies with four or five main dendritic trunks; spines are sparse or absent on primary dendrites but abundant on secondary and tertiary dendrites. This cellular type corresponds to a pyramidal morphology. The "multiple-spike" neurons have oval or fusiform somata with four or five thick primary dendritic trunks that leave the soma in opposite directions; they have spiny secondary and tertiary dendrites. Finally, neurons which discharge with a "single spike" to depolarizing pulses are round with four or five densely spiny dendrites, affording these neurons a mossy appearance. The results indicate that most of the amygdaloid neurons respond to carbachol with a depolarization. This effect was concomitant with either decrease or no change in the membrane input resistance and was not blocked by the addition of Ba2+, an M-current blocker, indicating that a conductance pathway other than K+ is involved in the response to carbachol.