Actitud de los fisioterapeutas acerca del acceso directo y su implementación a través de un cambio en la política pública en Puerto Rico / Attitude of physiotherapists about direct access and its implementation through a change in public policy in Puerto Rico

Objetivos: Explorar las actitudes de los fisioterapeutas de Puerto Rico: 1) hacia el acceso directo (AD), 2) las implicaciones para la profesión, la práctica y los servicios de salud, y 3) la implementación del AD a través de un cambio en política pública. Métodos: El diseño fue exploratorio transversal, no experimental y con un enfoque cuantitativo. Los participantes eran fisioterapeutas con licencia vigente, mayores de 21 años de edad, con cualquier grado académico en fisioterapia y que actualmente ejercen la práctica en Puerto Rico. Fueron excluidos fisioterapeutas sin experiencia clínica, que estaban completando un grado doctoral transicional o con experiencia ejerciendo con AD. Para abordar los objetivos de investigación, se construyó un cuestionario, cuyo contenido fue validado por 4 fisioterapeutas expertos utilizando el modelo de Lawshe modificado por Tristán. Resultados: Participaron de este estudio 100 fisioterapeutas. El 96% de los participantes estuvo de acuerdo con la implementación del AD en Puerto Rico. El 83% indicó estar preparado para ejercer la profesión por AD. El 55% entienden que fisioterapeutas con grado doctoral están más preparados para ejercer por AD. El 59% indicó que el AD debe estar restringido por nivel educativo y/o experiencia. Conclusión: La actitud de los fisioterapeutas en Puerto Rico respecto al AD resultó ser favorable, independientemente del grado académico, ya que están a favor con incorporar el AD a la fisioterapia, se sienten preparados para ejercer por AD y consideran el AD beneficioso para los pacientes, la práctica y la profesión. (AU)

Objectives: To explore the attitudes of physiotherapists in Puerto Rico: (1) towards direct access (DA), (2) the implications for the profession, practice, and health services, and (3) the implementation of DA through a change in public policy. Methods: The design was cross-sectional exploratory, non-experimental, and quantitative in nature. Participants were licensed physiotherapists, over 21 years old, with any academic degree in physiotherapy, currently practicing in Puerto Rico. Physiotherapists without clinical experience, those completing a transitional doctoral degree, or with experience practicing with DA were excluded. To address the research objectives, a questionnaire was constructed, whose content was validated by 4 expert physiotherapists using the Lawshe model modified by Tristán. Results: One hundred physiotherapists participated in this study. 96% of participants agreed with the implementation of DA in Puerto Rico. However, only 83% indicated being prepared to practice the profession through DA. 55% understood that physiotherapists with doctoral degrees were better prepared to practice through DA. 59% indicated that DA should be restricted based on educational level and/or experience. Conclusion: The attitude of physiotherapists in Puerto Rico towards DA was favorable regardless of academic degree. They are in favor of incorporating DA into physiotherapy, feel prepared to practice through DA, and consider it beneficial for patients, practice, and the profession. (AU)

COVID-19 fatality in Mexico's indigenous populations.

OBJECTIVE: The aim of the study was to explore the factors that could explain the differences in fatality rates among indigenous groups with COVID-19 diagnosis compared with the rest of the population in Mexico. STUDY DESIGN: We analyzed the public data of COVID-19 surveillance, of the Mexican Ministry of Health, to estimate COVID-19 fatality rates by ethnicity. METHODS: We explored associated factors using Cox proportional hazards models stratified by outpatient and hospital management at diagnosis; analysis was conducted in three scenarios: national level, states with 89% of the indigenous population, and South Pacific region. RESULTS: A total of 412,017 COVID-19 cases were included, with 1.1% of the indigenous population. The crude fatality rate per 1000 person-weeks was 64.8% higher among indigenous than among non-indigenous people (29.97 vs. 18.18, respectively), and it increased more than twice within outpatients (5.99 vs. 2.64, respectively). Cox analysis revealed that indigenous people who received outpatient management had higher fatality rate than non-indigenous outpatients, at the national level (hazard ratio [HR] = 1.63; 95% confidence interval [CI] = 1.34-1.98), within the subgroup of 13 states (HR = 1.66; 95% CI = 1.33-2.07), and in the South Pacific region (HR = 2.35; 95% CI = 1.49-3.69). Factors associated with higher fatality rates among non-indigenous and indigenous outpatients were age, sex, and comorbidities. CONCLUSIONS: COVID-19 fatality is higher among indigenous populations, particularly within cases managed as outpatients.

Conocimiento del cardiólogo sobre el fisioterapeuta y retos al remitir pacientes con enfermedad arterial periférica en Puerto Rico / Cardiologist's knowledge of the physiotherapy and the challenges of referring patients with peripheral arterial disease in Puerto Rico

Objetivos: Esta investigación buscaba explorar el conocimiento que tiene el cardiólogo sobre el papel del fisioterapeuta en el manejo de pacientes con enfermedad arterial periférica (EAP), conocer los niveles de adherencia del médico a las guías de manejo y tratamiento de pacientes con EAP, determinar los retos que enfrenta para remitir pacientes con EAP al fisioterapeuta e identificar estrategias potenciales para promover el mismo. Métodos: La investigación fue realizada mediante un enfoque cuantitativo, utilizando un diseño transversal exploratorio, no experimental. Para abordar los objetivos planteados se construyó y validó un cuestionario, el cual fue administrado a 25 médicos con especialidad en cardiología (68%), cardiología intervencionista (14%) o cirugía vascular (18%). Los datos fueron analizados descriptivamente. Resultados: El conocimiento del médico cardiólogo sobre el papel del fisioterapeuta tanto en aspectos generales, como en la prevención y manejo de pacientes con EAP, fue de un 70%. El 62% de los médicos solo remiten hasta un 10% de sus pacientes con EAP a fisioterapia. Entre las barreras principales para remitir a estos pacientes se encuentra la poca cobertura por parte de las aseguradoras médicas y el alto costo de los servicios de rehabilitación. Conclusión: Educar a los cardiólogos sobre el alcance de la fisioterapia puede llevar a que se reconozca su efectividad. Las barreras encontradas pueden llegar a predisponer al paciente a futuras complicaciones. Aunque hay un desconocimiento sobre el fisioterapeuta, existe la disposición del médico a que se le eduque con el fin de utilizar todas las herramientas disponibles para el bienestar del paciente

Objectives: This study aims to determine the knowledge of the cardiologist of the physiotherapist (PT) role in the management of patients with peripheral arterial disease (PAD). It also set out to determine the levels of adherence of physicians to the clinical management and treatment guidelines of patients with PAD, as well as to determine the challenges the cardiologist faces when referring patients with PAD to the PT, and to identify potential strategies to promote it. Methods: This was a quantitative study, conducted using an exploratory cross-sectional, non-experimental design. To address the proposed objectives, a questionnaire was constructed and validated, which was administered to 25 physicians specialised in cardiology (68%), interventional cardiology (14%), or vascular surgery (18%). A descriptive analysis was performed on the data. Results: The level of physician's knowledge about the role of PT in general and in the prevention and management of patients with PAD was 70%, and 62% of physicians only refer up to 10% of their patients with PAD to the PT. Among the main barriers to referring these patients is the low coverage by health insurance providers, and the high cost of rehabilitation services. Conclusion: Educating cardiologists about the scope of physiotherapy can lead to recognition of its effectiveness. The barriers encountered may predispose the patient to future complications. Although there is a lack of knowledge about the PT, there is a physician's willingness to be educated in order to use all available tools for the patient's well-being

The use of postoperative prophylactic antibiotics in stented distal hypospadias repair: a systematic review and meta-analysis.

INTRODUCTION: The current literature on the use of antibiotics perioperatively for many pediatric procedures, including hypospadias, is inconsistent. There is currently no clear evidence for the use of postoperative antibiotic prophylaxis for stented distal hypospadias repair. OBJECTIVE: This study aims to synthesize and assess the available literature on the use versus non-use of postoperative antibiotic prophylaxis for stented distal hypospadias repair. METHODOLOGY: Systematic literature search was performed on March 2018 for evaluation of trials that assessed the use and non-use of postoperative prophylactic antibiotics among stented distal hypospadias repair in children. Methodological quality of the studies was assessed according to the study design as recommended by the Cochrane Collaboration. The outcome assessed includes composite overall posthypospadias repair complications of infection and wound healing complications. The event rate for each treatment group was extracted to extrapolate intervention relative risk (RR) and corresponding 95% confidence interval (CI). Mantel-Haenszel method with random effect model was used in pooling of effect estimates from the included studies. Heterogeneity was assessed with subgroup analysis performed according to the study design. Publication bias was likewise determined. The protocol of this review was registered in PROSPERO (CRD42018087301) and reported in accordance with preferred reporting items for systematic reviews and meta-analyses (PRISMA) guidelines. RESULT: A total of seven studies (four cohorts, three randomized controlled trials) with 986 stented distal hypospadias repairs (408 with no post-operative prophylactic antibiotics and 578 given postoperative prophylactic antibiotics) were included for the meta-analysis. Moderate to serious risk of bias was noted among the cohort studies, while the included randomized controlled trials (RCT) were of high risk of bias. Inconsistencies of effect estimates between subgroups and publication bias with small study effect were likely present. The overall pooled effect estimates comparing treatment groups showed no significant difference for outcomes of overall composite postoperative complication (RR 0.93, 95% CI 0.45, 1.93). Assessment of composite infection related complications and wound healing complications likewise did not show any significant between-group differences (RR 1.28, 95% CI 0.49, 3.35 and RR 1.01, 95% CI 0.48, 2.12; respectively) (Table). Asymptomatic bacteriuria was noted to be significantly higher among the intervention group with no postoperative prophylactic antibiotics (RR 4.01, 95% CI 1.11, 14.54). CONCLUSION: The available evidence to date was assessed to be of high risk. The low level of evidence generated suggests that there is limited utility in the use of postoperative prophylactic antibiotics to prevent clinically significant posthypospadias repair complications.

Evidence of Early Emergence of the Primary Dentition in a Northern Plains American Indian Population.

The purposes of this study were to describe primary tooth emergence in an American Indian (AI) population during the first 36 mo of life to compare 1) patterns of emergence between male and female children and 2) tooth emergence between these AI children and other U.S. ethnic groups. Data were derived from a birth cohort of 239 AI children from a Northern Plains tribe participating in a longitudinal study of early childhood caries, with examination data at target ages of 8, 12, 16, 22, 28, and 36 mo of age (±1 mo). Patterns of emergence in AI children were characterized and sex comparisons accomplished with interval-censored survival methodology. Numbers of erupted teeth in AI children at each age were compared via Kruskal-Wallis tests against those in children of the same age, as drawn from a cross-sectional study of dental caries patterns in Arizona; these comparisons were based on the dental examinations of 547 White non-Hispanic and 677 Hispanic children. Characterization of time to achievement of various milestones-including emergence of the anterior teeth, the first molars, and the complete primary dentition-provided no evidence of sex differences among AI children. AI children had significantly more teeth present at 8 mo (median, 3) than either White non-Hispanic (P < 0.0063) or Hispanic (P < 0.0001) children (median, 2 each). This was also true at 12 mo (P < 0.001; medians, 8 vs. 6 and 7, respectively) and 16 mo (P < 0.001; medians, 12 vs. 11 each). Less pronounced differences were seen at 22 mo (P < 0.0001). White non-Hispanic and Hispanic children did not differ at any time considered (P > 0.05). These results provide evidence of earlier tooth emergence in AI children than in the other 2 ethnicities. Although the underlying etiology of the severity of early childhood caries in AI children is likely to be multifactorial, earlier tooth emergence may be a contributing factor. Knowledge Transfer Statement: The findings of this study have practical implications for practitioners providing childhood oral health care to ethnic groups with early tooth emergence. It may be important to provide parents with information on toothbrushing, dentist visits, and other practices supportive of good oral health as early as possible to protect their children's primary dentition.

Previous Exposure to Anesthesia and Autism Spectrum Disorder (ASD): A Puerto Rican Population-Based Sibling Cohort Study.

BACKGROUND: Autism Spectrum Disorder (ASD) is characterized by impaired social interaction and communication, and by restricted and repetitive behavior, that begins usually before a child is three years old.(1) Researchers have shown that prevalence rates in the U.S. may be as high as 1 in 68.(52) A number of studies have examined the effects of early exposure to anesthesia on brain development and subsequent impairment in neurocognitive function; yet, little is known about the possible effects of anesthetic agents on social-behavioral functioning. The association between exposure to anesthesia either in uterus, during the first years of life, or later and development of Autistic Spectrum Disorder (ASD) or its severity was determined in a retrospective population based cohort study. OBJECTIVES: Identify if children who had previous exposure to anesthesia either in uterus, first years of life during their developing brain years, or later, are at risk of developing ASD and its severe form of the disease. METHODS: Data was obtained from structured interviews administered to a sample of 515 parents/guardians distributed in two groups: ASD = 262 children diagnosed with this condition and Non-ASD: 253 children (siblings of ASD group) without diagnosis (95% confidence interval) that freely decided to participate and agreed to a consent form. Variables studied include: demographics, diagnosis and severity of ASD, exposure to anesthesia, method of childbirth, and age of exposure Children less than 2 years of age were considered into have developing brain period. Data was analyzed using Chi-square or Fisher exact test. RESULTS: In contrast to non-ASD group, most of the children within ASD group were male, 76% (p=0.0001). With regards to methods of childbirth, 64% of the ASD population were vaginal delivery (VD; Non-anesthesia exposure group) and 36% cesarean delivery (CD) compared to non-autistic population with 71% VD and 29% CD, which demonstrates no statistical difference between both groups (p=0.1113). Out of the 36% of ASD population that underwent CD, 7% were performed using general anesthesia and 93% regional anesthesia, while the 29% of the CD of non-ASD, 5% were performed using general anesthesia and 95% regional anesthesia. This reveals no statistical significance (p=0.7569) with the development of ASD and the type of anesthesia used when comparing ASD with non-ASD patients. In view of severity of autism, in VD, 56% of ASD population had mild form of the disorder, 34% moderate, and 10% severe; while CD had a 54% mild form of the disorder, 33% moderate, and 13% severe. This shows no statistical association (p=0.8069) when comparing exposure to anesthesia in uterus to subsequent severe form of ASD. Of the 262 ASD patients, 99 had exposure to anesthetics before their diagnosis, while in Non-ASD population, 110 had exposure to anesthesia, demonstrating no statistically significant association between both groups (p=0.2091). Out of 99 ASD patients exposed to anesthesia prior to their diagnosis, 72 were exposed before age 2. When compared to the 110 Non-ASD patients exposed to anesthesia, 86 had exposure during this developing brain period, which indicates no statistically significant association (p=0.4207). In addition, most of the ASD children exposed to anesthesia during developing brain were diagnosed with mild degree of the disorder when compared to ASD children without any previous exposure to anesthesia (p=0.9700) during the same period. When the exposure occurred after age 2, ASD children developed mild form of the disorder as compared with ASD children without any previous exposure to anesthesia (p=0.1699) in that period. CONCLUSIONS: Children under early exposure to anesthesia in uterus, first 2 years of life, or later are not more likely to develop neither ASD nor severe form of the disorder.

Amphiphilic cationic nanogels as brain-targeted carriers for activated nucleoside reverse transcriptase inhibitors.

Progress in AIDS treatment shifted emphasis towards limiting adverse effects of antiviral drugs while improving the treatment of hard-to-reach viral reservoirs. Many therapeutic nucleoside reverse transcriptase inhibitors (NRTI) have a limited access to the central nervous system (CNS). Increased NRTI levels induced various complications during the therapy, including neurotoxicity, due to the NRTI toxicity to mitochondria. Here, we describe an innovative design of biodegradable cationic cholesterol-ε-polylysine nanogel carriers for delivery of triphosphorylated NRTIs that demonstrated high anti-HIV activity along with low neurotoxicity, warranting minimal side effects following systemic administration. Efficient CNS targeting was achieved by nanogel modification with brain-specific peptide vectors. Novel dual and triple-drug nanoformulations, analogous to therapeutic NRTI cocktails, displayed equal or higher antiviral activity in HIV-infected macrophages compared to free drugs. Our results suggest potential alternative approach to HIV-1 treatment focused on the effective nanodrug delivery to viral reservoirs in the CNS and reduced neurotoxicity.

Previous Exposure to Anesthesia and Autism Spectrum Disorder (ASD): A Puerto Rican Population-Based Sibling Cohort Study.

BACKGROUND: Autism Spectrum Disorder (ASD) is characterized by impaired social interaction and communication, and by restricted and repetitive behavior, that begins usually before a child is three years old.1 Researchers have shown that prevalence rates in the U.S. may be as high as 1 in 68.52 A number of studies have examined the effects of early exposure to anesthesia on brain development and subsequent impairment in neurocognitive function; yet, little is known about the possible effects of anesthetic agents on social-behavioral functioning. The association between exposure to anesthesia either in uterus, during the first years of life, or later and development of Autistic Spectrum Disorder (ASD) or its severity was determined in a retrospective population based cohort study. OBJECTIVES: Identify if children who had previous exposure to anesthesia either in uterus, first years of life during their developing brain years, or later, are at risk of developing ASD and its severe form of the disease. METHODS: Data was obtained from structured interviews administered to a sample of 515 parents/guardians distributed in two groups: ASD = 262 children diagnosed with this condition and Non-ASD: 253 children (siblings of ASD group) without diagnosis (p = 0.8069) when comparing exposure to anesthesia in uterus to subsequent severe form of ASD. Of the 262 ASD patients, 99 had exposure to anesthetics before their diagnosis, while in Non-ASD population, 110 had exposure to anesthesia, demonstrating no statistically significant association between both groups (p = 0.2091). Out of 99 ASD patients exposed to anesthesia prior to their diagnosis, 72 were exposed before age 2. When compared to the 110 Non-ASD patients exposed to anesthesia, 86 had exposure during this developing brain period, which indicates no statistically significant association (p = 0.4207). In addition, most of the ASD children exposed to anesthesia during developing brain were diagnosed with mild degree of the disorder when compared to ASD children without any previous exposure to anesthesia (p = 0.9700) during the same period. When the exposure occurred after age 2, ASD children developed mild form of the disorder as compared with ASD children without any previous exposure to anesthesia (p = 0.1699) in that period. CONCLUSIONS: Children under early exposure to anesthesia in uterus, first 2 years of life, or later are not more likely to develop neither ASD nor severe form of the disorder. INDEX WORDS: Anesthesia, Autism Spectrum Disorder, Puerto Rico. (95% confidence interval) that freely decided to participate and agreed to a consent form. Variables studied, include: demographics, diagnosis and severity of ASD, exposure to anesthesia, method of childbirth, and age of exposure. Children less than 2 years of age were considered into have developing brain period. Data was analyzed using Chi-square or Fisher exact test. RESULTS: In contrast to non-ASD group, most of the children within ASD group were male, 76% (p = 0.0001). With regards to methods of childbirth, 64% of the ASD population were vaginal delivery (VD; Non-anesthesia exposure group) and 36% cesarean delivery (CD) compared to non-autistic population with 71% VD and 29% CD, which demonstrates no statistical difference between both groups (p = 0.1113). Out of the 36% of ASD population that underwent CD, 7% were performed using general anesthesia and 93% regional anesthesia, while the 29% of the CD of non-ASD, 5% were performed using general anesthesia and 95% regional anesthesia. This reveals no statistical significance (p = 0.7569) with the development of ASD and the type of anesthesia used when comparing ASD with non-ASD patients. In view of severity of autism, in VD, 56% of ASD population had mild form of the disorder, 34% moderate, and 10% severe; while CD had a 54% mild form of the disorder, 33% moderate, and 13% severe. This shows no statistical association.

A taxonomic and phylogenetic revision of the Penicillium sclerotiorum complex.

The morphological concept of Penicillium sclerotiorum (subgenus Aspergilloides) includes strains with monoverticillate, vesiculate conidiophores, and vivid orange to red colony colours, with colourful sclerotia sometimes produced. Multigene phylogenetic analyses with the nuclear ribosomal internal transcribed spacer (ITS) region, cytochrome c oxidase subunit 1 (cox1), ß-tubulin (benA), translation elongation factor 1-α (tef1-α), and calmodulin (cmd), reveal that the P. sclerotiorum morphospecies is a complex of seven phylogenetically distinct species, three of which were recently described, namely P. guanacastense, P. mallochii, and P. viticola. Three previously unidentified species are described here as P. cainii, P. jacksonii, and P. johnkrugii. The phylogenetic species are morphologically similar, but differ in combinations of colony characters, sclerotium production, conidiophore stipe roughening and branching, and conidial shape. Ecological characters and differences in geographical distribution further characterise some of the species, but increased sampling is necessary to confirm these differences. The fungal DNA barcode, the ITS, and the animal DNA barcode, cox1, have lower species resolving ability in our phylogenetic analyses, but still allow identification of all the species. Tef1-α and cmd were superior in providing fully resolved, statistically well-supported phylogenetic trees for this species complex, whereas benA resolved all species but had some issues with paraphyly. Penicilliumadametzioides and P. multicolor, considered synonyms of P. sclerotiorum by some previous authors, do not belong to the P. sclerotiorum complex. TAXONOMIC NOVELTIES: New species:Penicillium cainii K.G. Rivera, Malloch & Seifert, P. jacksonii K.G. Rivera, Houbraken & Seifert, P. johnkrugii K.G. Rivera, Houbraken & Seifert.

[Undergraduate teaching of internal medicine in a private hospital]. / Docencia de pregrado en medicina interna en un hospital privado: diez años de experiencia.

There is a 10 years teaching experience for fourth year medical students and interns in a Chilean private hospital. The students attend an eight weeks practical course. The interns rotate during 16 weeks by specialties and make shifts. The hospital structure with Clinical Services and Medical-Surgical departments facilitates the teaching process. There are approximately 30,000 admissions per year with a mean stay of 3.7 days, that allow the students to be in touch with patients with different diseases that are managed with updated technology. We emphasize the ethical and clinical management of concrete problems of patients, learning and communication skills. The students evaluate their stay answering surveys and with semi structured interviews. Teaching is assessed by tutors and heads of departments, in clinical rounds, sometimes prepared by the students, by a thorough revision of problem oriented medical records and with practical and theoretical tests. The results of the program have been quite satisfactory for participants.

Docencia de pregrado en medicina interna en un hospital privado: Diez años de experiencia / Undergraduate teaching of internal medicine in a private hospital

There is a 10 years teaching experience for fourth year medical students and interns in a Chilean private hospital. The students attend an eight weeks practical course. The interns rotate during 16 weeks by specialties and make shifts. The hospital structure with Clinical Services and Medical-Surgical departments facilitates the teaching process. There are approximately 30,000 admissions per year with a mean stay of 3.7 days, that allow the students to be in touch with patients with different diseases that are managed with updated technology. We emphasize the ethical and clinical management of concrete problems of patients, self ¡earning and communication skills. The students evaluate their stay answering surveys and with semi structured interviews. Teaching is assessed by tutors and heads of departments, in clinical rounds, sometimes prepared by the students, by a thorough revision of problem oriented medical records and with practical and theoretical tests. The results of the program have been quite satisfactory for participants.

An antibiotic complex from Lysobacter enzymogenes strain C3: antimicrobial activity and role in plant disease control.

Lysobacter enzymogenes C3 is a bacterial biological control agent that exhibits antagonism against multiple fungal pathogens. Its antifungal activity was attributed in part to lytic enzymes. In this study, a heat-stable antifungal factor (HSAF), an antibiotic complex consisting of dihydromaltophilin and structurally related macrocyclic lactams, was found to be responsible for antagonism by C3 against fungi and oomycetes in culture. HSAF in purified form exhibited inhibitory activity against a wide range of fungal and oomycetes species in vitro, inhibiting spore germination, and disrupting hyphal polarity in sensitive fungi. When applied to tall fescue leaves as a partially-purified extract, HSAF at 25 mug/ml and higher inhibited germination of conidia of Bipolaris sorokiniana compared with the control. Although application of HSAF at 12.5 mug/ml did not reduce the incidence of conidial germination, it inhibited appressorium formation and suppressed Bipolaris leaf spot development. Two mutant strains of C3 (K19 and DeltaNRPS) that were disrupted in different domains in the hybrid polyketide synthase-nonribosomal peptide synthetase gene for HSAF biosynthesis and had lost the ability to produce HSAF were compared with the wild-type strain for biological control efficacy against Bipolaris leaf spot on tall fescue and Fusarium head blight, caused by Fusarium graminearum, on wheat. Both mutant strains exhibited decreased capacity to reduce the incidence and severity of Bipolaris leaf spot compared with C3. In contrast, the mutant strains were as efficacious as the wild-type strain in reducing the severity of Fusarium head blight. Thus, HSAF appears to be a mechanism for biological control by strain C3 against some, but not all, plant pathogenic fungi.

Biosynthesis of sphinganine-analog mycotoxins.

Sphinganine-analog mycotoxins (SAMT) are polyketide-derived natural products produced by a number of plant pathogenic fungi and are among the most economically important mycotoxins. The toxins are structurally similar to sphinganine, a key intermediate in the biosynthesis of ceramides and sphingolipids, and competitive inhibitors for ceramide synthase. The inhibition of ceramide and sphingolipid biosynthesis is associated with several fatal diseases in domestic animals and esophageal cancer and neural tube defects in humans. SAMT contains a highly reduced, acyclic polyketide carbon backbone, which is assembled by a single module polyketide synthase. The biosynthesis of SAMT involves a unique polyketide chain-releasing mechanism, in which a pyridoxal 5'-phosphate-dependent enzyme catalyzes the termination, offloading and elongation of the polyketide chain. This leads to the introduction of a new carbon-carbon bond and an amino group to the polyketide chain. The mechanism is fundamentally different from the thioesterase/cyclase-catalyzed polyketide chain releasing found in bacterial and other fungal polyketide biosynthesis. Genetic data suggest that the ketosynthase domain of the polyketide synthase and the chain-releasing enzyme are important for controlling the final product structure. In addition, several post-polyketide modifications have to take place before SAMT become mature toxins.

Functional replacement of the ketosynthase domain of FUM1 for the biosynthesis of fumonisins, a group of fungal reduced polyketides.

The genetic manipulation of the biosynthesis of fungal reduced polyketides has been challenging due to the lack of knowledge on the biosynthetic mechanism, the difficulties in the detection of the acyclic, non-aromatic metabolites, and the complexity in genetically manipulating filamentous fungi. Fumonisins are a group of economically important mycotoxins that contaminate maize-based food and feed products worldwide. Fumonisins contain a linear dimethylated C18 chain that is synthesized by Fum1p, which is a single module polyketide synthase (PKS). Using a genetic system that allows the specific manipulation of PKS domains in filamentous fungus Fusarium verticillioides, we replaced the KS domain of fumonisin FUM1 with the KS domain of T-toxin PKS1 from Cochliobolus heterostrophus. Although PKS1 synthesizes different polyketides, the F. verticillioides strain carrying the chimeric PKS produced fumonisins. This represents the first successful domain swapping in PKSs for fungal reduced polyketides and suggests that KS domain alone may not be sufficient to control the product's structure. To further test if the whole fumonisin PKS could be functionally replaced by a PKS that has a similar domain architecture, we replaced entire FUM1 with PKS1. This strain did not produce any fumonisin or new metabolites, suggesting that the intrinsic interactions between the intact PKS and downstream enzymes in the biosynthetic pathway may play a role in the control of fungal reduced polyketides.

Facile purification of fibrinogen fragments using a computer-based model with general applicability to the generation of salt gradients.

We and others have recently shown that specific fragments of cross-linked fibrin affect cell behavior. In order to develop a facile method for the preparative scale purification of fibrin fragment D dimer, a simple gradient generating system for conventional chromatography was developed and validated, and methods of fibrin fragment D dimer purification were compared. The experimentally measured salt concentration/time relationship fell directly on the model-predicted line. Model-predicted changes in the reservoir volume and/or salt concentration in the limit buffer affected both the initial slope and the shape of the concentration/time relationship. This gradient generation method was used to separate the D domains of fibrin(ogen) from the amino terminal region E domain using anion-exchange chromatography. While the predicted salt gradient was achieved, a salt-dependent separation was found to be less optimal than that of a pH-dependent separation, as validated by Coomassie-stained SDS-PAGE and by immunoblotting. In conclusion, a facile, user-friendly, computer-based method to predict and generate salt gradients was written and validated by direct experimentation. While fibrinogen fragment purification was acceptable using this system, both separation and yields of fibrinogen and fibrin fragments were superior using a pH-based separation technique.

Adenovirally mediated gene transfer of functional human tissue-type plasminogen activator to murine lungs.

As several forms of lung injury are associated with alveolar fibrin deposition, and fibrin has been pathogenically implicated in the lung fibrotic response, we sought to develop an in vivo gene transfer model of fibrinolytic protease overexpression. To this end, human tissue-type plasminogen activator (t-PA) possesses a high degree of specificity for proteolytic activation of fibrin-bound plasminogen to its active form, plasmin. To construct an effective vector, the cDNA for human t-PA was inserted downstream of a cytomegalovirus early enhancer-promoter into the E1 position of a replication-deficient adenovirus. The adenovirally expressed t-PA was found to be of the expected size and appropriate functional activity both in vitro and in vivo. A single intratracheal instillation of the adenoviral-t-PA construct resulted in a dose- dependent, tissue-specific expression of increased levels of t-PA antigen (100-fold) and t-PA protease activity (4-fold) for at least 2 wk in whole lung lysates. The expressed protein localized to the bronchiolar epithelium and peribronchiolar alveolar cells and did not result in increases in total lung protein or alveolar cell counts at 3 d after instillation. In conclusion, a single intratracheal instillation of adenoviral-cytomegalovirus-t-PA construct will generate dramatic bronchoalveolar compartment overexpression of functional recombinant human t-PA for at least 2 wk. This vector can now be utilized for the determination of the therapeutic potential of t-PA in a number of in vivo model systems.

Polymerization of fibrinogen in murine bleomycin-induced lung injury.

Bleomycin lung injury in mice leads to an acute alveolitis followed by a fibroproliferative response characterized by the accumulation of extracellular matrix. Because distinct regions of the fibrin(ogen) molecule have unique in vitro biological effects on cells, we quantified, localized, and biochemically characterized the molecular form of extravascular fibrin(ogen) in methoxyflurane anesthetized, bleomycin-injured mice. Bleomycin or saline (controls) was administered intratracheally, and lung tissue was harvested and analyzed at several times thereafter. Immunoreactive fibrin tissue content increased to a maximal 50-fold over controls in a temporal and spatial pattern paralleling that of alveolitis and maximal fibroproliferation. The generation of gamma-gamma-chain dimers and alpha-chain polymers, together with the loss of free alpha- and gamma-chains, indicates that the fibrin is predominantly covalently cross-linked. In fibroproliferative-phase lungs, the fibrin fibrils are branched and colocalize with those of collagen at the electron microscopic level. These observations strongly suggest that fibrin is a significant molecular effector of the in vivo fibroproliferative response after lung injury.

Regulation of type I plasminogen activator inhibitor by fibrin degradation products in rat lung fibroblasts.

Persistent fibrin deposition in tissues characterizes the early pathology of many types of injury. In an animal model of bleomycin-induced lung fibrosis, increased expression of type 1 plasminogen activator inhibitor (PAI-1) is associated with accumulation of fibrin in fibroproliferative lesions. Plasmin proteolysis of cross-linked fibrin generates fibrin degradation products (FDPs) with multiple biological activities in several cell types. We reasoned that fibrin fragments may also regulate fibroblast-mediated fibrinolysis. In this study, we describe induction of PAI-1 mRNA, protein, and activity by soluble FDPs and fibrinogen in rat lung fibroblast monolayers. FDPs are more potent than fibrinogen, inducing a concentration-dependent, maximal 3.7 (+/- 0.9)-fold increase in PAI-1 mRNA as measured by northern blotting and a 9.0 (+/- 1.3)-fold induction of PAI-1 antigen levels. Active PAI-1 is demonstrated in fibrinogen- and FDP-stimulated conditioned media. Further characterization of this response shows that PAI-1 expression is induced by the DD/D fragments, but not by immunopurified fragment E. Experiments using Actinomycin D and puromycin indicate that the induction appears to be transcriptionally regulated and is not dependent on new protein synthesis. FDP induction of PAI-1 suggests a matrix-cell feedback process in which a fibrin fragment modulates expression of an important regulator of fibrinolysis.

Learning deficits in congenitally hydrocephalic rats and prevention by early shunt treatment.

Shunt surgery is the usual treatment for infantile hydrocephalus; however, the extent to which it avoids subsequent neurological deficits is uncertain. The effect of early-onset hydrocephalus was tested in H-Tx rats using the Morris water maze. Spatial learning was assessed at 21 days after birth in control (n = 18), hydrocephalic (n = 18) and hydrocephalic rats shunt-treated at 4-5 (n = 7) or at 10-12 days of life (n = 13). The time taken to find a hidden platform was measured in five trials on 2 consecutive days and the data analyzed by one- and two-way ANOVA and t-tests. The latencies of the control rats decreased significantly between the first and second trial on the 1st day, and learning was retained until the 2nd day. The hydrocephalic group had longer latencies than controls on both days, with no significant decrease between any trials. Performance was not significantly different between the two shunt groups. Overall, the shunted rats had latencies which were not significantly different from controls but were significantly lower than hydrocephalics. Despite this, the shunted rats did not perform as well as the controls. It is concluded that, although shunt treatment improved learning, some effects of early-onset hydrocephalus may not be reversible and/or a longer recovery time is required.