ABSTRACT
BACKGROUND: Medical curricula have historically been designed in a top-down approach, usually excluding students. While Delphi panels have been used as a tool for medical education curricula design, none have been conducted in Ecuador. In addition, no such approach has ever included students both as panelists and researchers. MATERIAL AND METHODS: Four Delphi panels were developed and conducted using a participatory approach that allowed medical students to take part both as expert panelists and researchers: specifically, students developed the questionnaire and conducted a qualitative synthesis. Questionnaire responses were anonymized and dispatched online to panelists. The information was organized and collected to develop the qualitative syntheses and prepare the final statements. RESULTS: Thirty-two medical students participated between February and May 2018. A total of 32 questions were developed, corresponding to five different categories. For some questions, consensus was reached; for other questions, general statements were obtained.Discussion and conclusion: Developing the questionnaire, responding to it and analyzing the answers allowed students to raise significant concerns regarding medical education topics proposing relevant policy and curricula change. Participatory Delphi panels can be an efficient tool to obtain organized feedback, improve student class involvement, and promote research skills.
Subject(s)
Education, Medical, Undergraduate , Education, Medical , Students, Medical , Curriculum , Delphi Technique , Ecuador , HumansABSTRACT
The cooperative breeding hypothesis (CBH) states that cooperative breeding, a social system in which group members help to rear offspring that are not their own, has important socio-cognitive consequences. Thornton & McAuliffe (2015; henceforth T&M) critiqued this idea on both conceptual and empirical grounds, arguing that there is no reason to predict that cooperative breeding should favour the evolution of enhanced social cognition or larger brains, nor any clear evidence that it does. In response to this critique, Burkart & van Schaik (2016 henceforth B&vS) attempt to clarify the causal logic of the CBH, revisit the data and raise the possibility that the hypothesis may only apply to primates. They concede that cooperative breeding is unlikely to generate selection pressures for enhanced socio-cognitive abilities, but argue instead that the CBH operates purely through cooperative breeding reducing social or energetic constraints. Here, we argue that this revised hypothesis is also untenable because: (1) it cannot explain why resources so released would be allocated to cognitive traits per se rather than any other fitness-related traits, (2) key assumptions are inconsistent with available evidence and (3) ambiguity regarding the predictions leaves it unclear what evidence would be required to falsify it. Ultimately, the absence of any compelling evidence that cooperative breeding is associated with elevated cognitive ability or large brains (indeed data suggest the opposite is true in non-human primates) also casts doubt on the capacity of the CBH to explain variation in cognitive traits.
ABSTRACT
BACKGROUND: Do evolutionary specializations lead to evolutionary constraint? This appears plausible, particularly when specialization leads to loss of complex adaptations. In the owl monkey lineage, nocturnality clearly arose from a diurnal ancestor. This behavioural shift was accompanied by morphological changes in the eye and orbit and complete loss of colour vision via missense mutations in the gene encoding the short-wave sensitive visual pigment (SWS opsin). Interestingly, at least one subspecies of owl monkey, Azara's owl monkey (Aotus azarae azarae), has regained activity in daylight. Given that all primate species that are active in daylight, including primarily diurnal species and species that are active during both day and night, have at least dichromatic colour vision, it seems reasonable to propose that dichromacy would be adaptive in A. a. azarae. With a disabled SWS opsin, the main avenue available for Azara's owl monkeys to re-evolve colour vision is via a polymorphism in the intact X-linked opsin locus, which commonly occurs in other New World monkeys. To examine this possibility we assayed variation in the X-linked opsin of A. a. azarae, focusing on the three exons (3, 4 and 5) that control spectral sensitivity. RESULTS: We found low opsin genetic variation on a population level, and no differences at the three main sites that lead to variation in spectral sensitivity in the opsins of other New World monkeys. Two rare alleles with single amino acid variants are segregating in the population, but previous functional studies indicate that these are unlikely to affect spectral sensitivity. CONCLUSIONS: Genetic constraint on the re-evolution of colour vision is likely operating in Azara's owl monkey, which may affect the niche that this subspecies is able to occupy.
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INTRODUCTION: Negative pressure therapy (NPT) is a widely recognised procedure for the temporary closure of open abdominal wounds. In this study, we compare two NPT products, the V.A.C.® abdominal dressing (AD) system and the new ABThera™ (ABT) system, in terms of the primary closure rates achieved, types of closure, and the associated morbidity. METHODS: We employed a retrospective comparative study of open-abdomen patients treated with NPT using either AD or ABT. The indications for treatment were damage control surgery, abdominal compartment syndrome, or severe abdominal sepsis. RESULTS: The group of patients treated with ABT showed a higher percentage of primary closures (41 vs. 11%) and required fewer days of NPT (17 vs. 26 days) than the AD group. Differences were statistically significant. In addition, only 4% of patients in the ABT group exhibited enteroatmospheric fistulae, compared to 17% in the AD group. CONCLUSIONS: Compared to the AD system, ABT can achieve faster primary closure after open abdomen treatment with only minor complications.
Subject(s)
Abdomen/physiopathology , Abdomen/surgery , Abdominal Wound Closure Techniques , Negative-Pressure Wound Therapy , Aged , Female , Humans , Male , Middle Aged , Retrospective StudiesABSTRACT
Autochthonous microorganisms [a consortium of arbuscular-mycorrhizal (AM) fungi and Bacillus thuringiensis (Bt)] were assayed and compared to Rhizophagus intraradices (Ri), Bacillus megaterium (Bm) or Pseudomonas putida (Psp) and non-inoculation on Trifolium repens in a natural arid soil under drought conditions. The autochthonous bacteria Bt and the allochthonous bacteria Psp increased nutrients and the relative water content and decreased stomatal conductance, electrolyte leakage, proline and APX activity, indicating their abilities to alleviate the drought stress. Mycorrhizal inoculation significantly enhanced plant growth, nutrient uptake and the relative water content, particularly when associated with specific bacteria minimizing drought stress-imposed effects. Specific combinations of autochthonous or allochthonous inoculants also contributed to plant drought tolerance by changing proline and antioxidative activities. However, non-inoculated plants had low relative water and nutrients contents, shoot proline accumulation and glutathione reductase activity, but the highest superoxide dismutase activity, stomatal conductance and electrolyte leakage. Microbial activities irrespective of the microbial origin seem to be coordinately functioning in the plant as an adaptive response to modulated water stress tolerance and minimizing the stress damage. The autochthonous AM fungi with Bt or Psp and those allochthonous Ri with Bm or Psp inoculants increased water stress alleviation. The autochthonous Bt showed the greatest ability to survive under high osmotic stress compared to the allochthonous strains, but when single inoculated or associated with Ri or AM fungi were similarly efficient in terms of physiological and nutritional status and in increasing plant drought tolerance, attenuating and compensating for the detrimental effect of water limitation.
Subject(s)
Adaptation, Physiological , Bacteria/metabolism , Droughts , Mycorrhizae/physiology , Soil , Trifolium/microbiology , Trifolium/physiology , Biomass , Elements , Microbial Viability , Plant Roots/enzymology , Plant Roots/growth & development , Plant Roots/microbiology , Plant Shoots/enzymology , Plant Shoots/growth & development , Plant Shoots/microbiology , Plant Stomata/physiology , Proline/metabolism , Water/chemistryABSTRACT
American Cutaneous Leishmaniasis (ACL) is caused by protozoan parasites of the Leishmania genus, and transmitted by females of the Phlebotominae family. The role of wild and domestic hosts in the cycle of Leishmania is still unknown. ACL is endemic in the province of Formosa where Nyssomyia neivai was the most abundant species in several captures and 31 cumulative ACL human cases were reported between 2005 and 2011 in the province. The present report describes the detection, by PCR-RFLP and confirmed by sequencing, of subgenus Leishmania (Viannia) DNA in four free-ranging owl monkeys (Aotus azarai azarai) from Formosa Province. The sequence amplified was the mini-exon gene present in tandem repeats in all species of the Leishmania genus from buffy coat samples. The absence of inhibitors in the samples was checked by a ß-globin protocol originally designed to amplify the human ß-globin gene. However, other free-ranging primates were found with natural infections of L. (V) braziliensis complex and Leishmania (Viannia) subgenus by parasitological means in America. To the best of our knowledge, there are no published reports on detection of subgenus Leishmania (Viannia) DNA by PCR-RFLP in argentinean free-ranging primates. Additional eco-epidemiological and parasitological studies are necessary to confirm owl monkeys, or any other natural infected mammal species detected by PCR, as a reservoir, incidental host or to propose it as an animal model for research on this topic.
Subject(s)
Aotidae/parasitology , Leishmania/genetics , Leishmaniasis/veterinary , Monkey Diseases/parasitology , Polymerase Chain Reaction/veterinary , Polymorphism, Restriction Fragment Length , Animals , Argentina/epidemiology , Female , Leishmania/isolation & purification , Leishmaniasis/epidemiology , Leishmaniasis/parasitology , Male , Monkey Diseases/epidemiology , Polymerase Chain Reaction/methods , Psychodidae/parasitologyABSTRACT
BACKGROUND: Inguinal hernia is one of the most common pathologies in the surgical setting. The introduction of the Lichtenstein technique in 1989 (tension-free hernioplasty with polypropylene mesh) represented one of the most significant breakthroughs in the treatment of this condition since Bassini's hernia repair. The aim of this study was to know the most significant predictive variables of complications in Lichtenstein hernioplasty and if some changes introduced in the technique could reduce these complications. STUDY DESIGN: A prospective study of 2002 inguinal hernias in 1592 patients, operated on during 17 years, using the Lichtenstein tension-free technique for hernia repair with a heavy polypropylene mesh. The early and late complications and the outcome of modifications introduced in the technique over the study period have all been studied. RESULTS: The modification in the type of closure reduced the complications rate from 14.4% down to 2.7%. The introduction of antibiotic prophylaxis reduced the infection rate from 1.2% down to 0.2%. CONCLUSIONS: The most significant predictive variables of complications in Lichtenstein hernioplasty are the type of closure, antibiotic prophylaxis, ASA risk and the presence of previous recurrence.
Subject(s)
Hernia, Inguinal/surgery , Plastic Surgery Procedures/methods , Postoperative Complications/prevention & control , Surgical Mesh , Adult , Female , Follow-Up Studies , Humans , Incidence , Male , Middle Aged , Postoperative Complications/epidemiology , Prospective Studies , Prosthesis Design , Spain/epidemiology , Time Factors , Treatment Outcome , Wound HealingABSTRACT
INTRODUCTION: Laryngeal transplantation is a possibility for patients with irreversible laryngeal disease, such as complex trauma and larynx cancer. The objective of performing this procedure was to solve problems that these patients face with a laryngectomy. The medical literature has reviews about larynx transplantations, but almost nothing about the larynx donor. The following is our experience on management of these donors. MATERIALS AND METHODS: Selection criteria was as follows: (1) 18-50 years old; (2) gender and ABO blood type matched between donor and recipient; (3) No abuse of tobacco, cocaine, and marijuana, (4) tracheal intubation time <3 days; and (5) time in the intensive care unit <7 days. The preservation was simple hypothermia with larynx infusion via the carotid artery with University of Wisconsin solution. RESULTS: Between 2001 and 2006, we managed 25 donors, among whom 12 grafts were discarded. The 13 larynx donors were of average age 27.2 +- 7.9 years and their cause of death was head trauma. Each was of male and 12 were multiorgan donors. Three donors had previous consumption of tobacco and 2 donors of marijuana. There were 2 cases of acute rejection episodes. Graft survival rate at 2 years was 90%. DISCUSSION: These donors may have differences from other multiorgan donors: (1) they do not require strict fluid management; (2) vasoactive agents may be used in higher doses than in organ donors, and (3) the larynx tolerates hemodynamic instability. It was necessary to use some donors who had used addictive substances, showing that some selection criteria may be flexible. There was no conflict between thoracic surgeons and larynx surgeons. The priority always was for life-saving organs. Family consent was sometimes difficult because of the retrieval times and body donor reconstruction. The larynx surgery retrieval demanded an additional 2-5 hours during routine multiorgan donor surgery, and always the family asked about body reconstruction. The body appearance was always preserved.
Subject(s)
Larynx , Tissue Donors , Trachea , Adolescent , Adult , Colombia , Female , Histocompatibility Testing , Humans , Laryngeal Diseases/surgery , Larynx/transplantation , Male , Middle Aged , Patient Selection , Retrospective Studies , Trachea/transplantation , Tracheal Diseases/surgery , Treatment OutcomeABSTRACT
UNLABELLED: We performed a multivariate analysis to evaluate the importance of histologic parameters in donor kidney biopsies as predictors of graft outcome. METHODS: Wedge protocol biopsies from a single center were analyzed for glomerulosclerosis (GS), interstitial fibrosis (IF), tubular atrophy (TA), arteriosclerosis (AS), and arteriolar hyalinosis (AH). Alterations were quantified as percentage (GS, IF) or semiquantified according to Banff criteria (IF, TA, AS, AH). We calculated creatinine clearance (CrCl) at 1, 2, and 3 years posttransplant. Donor data included age, gender, and type: non-heart-beating donor or brain dead donors. Recipient data included age, gender, cold ischemia time, number of HLA mismatches, peak level of the panel reactive antibody (PRA), number of acute rejection episodes (ARE), and presence or absence of cytomegalovirus (CMV) disease. Univariate and multivariate analyses were performed. Follow-up range was 1 to 4.2 years. RESULTS: GS, IF, TA, and AH were associated with graft survival in the multivariate analysis. The histologic parameters were associated with CrCl at several posttransplant time intervals, but the significance of association was lost in the multivariate analysis. Donor age showed a better correlation with graft function. In the univariate analyses adjusting for donor age, only IF and AH were associated with graft function. CONCLUSIONS: Histologic parameters showed a modest association with graft function. In our study, donor age is the better predictor of graft function. IF and AH may be similar to or better than GS as predictors of graft outcome.
Subject(s)
Kidney Diseases/surgery , Kidney Transplantation/physiology , Kidney , Tissue Donors , Adolescent , Adult , Age Factors , Aged , Cadaver , Cause of Death , Child , Child, Preschool , Female , Follow-Up Studies , Humans , Kidney/cytology , Kidney/physiology , Kidney Diseases/classification , Male , Middle Aged , Multivariate Analysis , Time Factors , Treatment OutcomeABSTRACT
Recently in Latin America, there has been a strong influence of the "Spanish model" of organ procurement. In 2001, The "Punta Cana Group" was created by Latin American transplantation coordinators with the objective of registering and improving the system of donation and procurement. In many countries there is no universal financial support from the government for medical treatment, including dialysis and transplantation. In other countries there is complete financial support for all of the population, including immunosuppressive drugs. Practically all countries have transplantation laws that follow ethical concepts, such as brain death diagnosis criteria, forms of consent, criteria of allocation, and inhibition of commerce. The rate of potential donors notified in countries that perform transplantations with deceased donors varied from 6 to 47 per million population yearly (pmp/y); The rate of effective donors varied from 1 to 20 pmp. In 2004, the mean rate of effective donors in Latin America was 5.4 pmp. The family refusal rate for the donation of organs varied from 28% in Uruguay to 70% in Peru. In some countries, such as Puerto Rico, Uruguay, and Cuba, it was more than 15 pmp, whereas in others countries deceased donors were practically not used. The number of patients on the waiting list for solid organ transplants in 12 Latin American countries is 55,000. Although the donation rate has increased by 100% during the last 10 years, it is lower than that in Europe (15 pmm/y) or the United States (20 pmp/y).
Subject(s)
Tissue and Organ Procurement/statistics & numerical data , Brain Death , Cadaver , Cause of Death , Humans , Latin America , Living Donors/statistics & numerical data , Renal Replacement Therapy/statistics & numerical data , Tissue Donors/statistics & numerical data , Tissue and Organ Procurement/economics , Tissue and Organ Procurement/ethics , Waiting ListsABSTRACT
INTRODUCTION: We have observed expanded glomeruli in biopsies of kidneys from non-heart-beating donors (NHBDs). We sought to determine the differences in glomerular size between NHBDs and brain-dead donors and to assess whether glomerular size impacted graft outcome. METHODS: We estimated the glomerular area using the maximal planar area (MPA) method in 198 pretransplant biopsies from 119 donors: 54 (45.4%) NHBDs and 65 (54.6%) brain-dead donors. Donor data and graft outcomes were correlated with MPA and percentage of glomerulosclerosis. The range of follow-up was 1 to 3 years. Uni- and multivariate analyses were performed. RESULTS: MPA was larger among NHBDs. MPA and GS both significantly correlated with donor age. The association between MPA and age was independent of nephron loss (ie; GS). Increased glomerular size was only observed among donors younger than 50 years. Graft survival and function were not independently associated with MPA. Donor age was a better predictor of graft outcome. CONCLUSIONS: The perfusion pressure used in NHBDs may expand the glomeruli, but this maneuver does not have any effect on graft outcome. Among donors without severe changes, glomerular size increment shows a limit around the sixth decade of life. In our study, MPA was not an independent predictor of graft survival or function.
Subject(s)
Heart Arrest , Kidney Glomerulus/pathology , Kidney Transplantation , Tissue Donors , Adolescent , Adult , Aged , Biopsy , Cause of Death , Female , Humans , Male , Middle Aged , Perfusion , Pressure , Retrospective Studies , Tissue and Organ ProcurementABSTRACT
We described a case of allograft kidney dysfunction associated with renal parenchymal infection with amastigotes of Trypanosoma cruzi. The patient was diagnosed as being chronically infected prior to transplantation. The infection was probably acquired by blood transfusion. He could not complete antiparasitic treatment due to drug toxicity. He was transplanted from a cadaver who showed a negative test for Chagas' disease. One year after transplantation the serum creatinine progressively increased. Histological examination of renal biopsy revealed intracytoplasmic amastigotes of T cruzi. No evidence of other specific alterations in the graft was detected. It was unknown whether graft dysfunction was only due to parasitic infection. The present case confirmed that T cruzi can infect kidney grafts and that immunosuppression in kidney transplantation is potentially a cause of dissemination of Chagas' disease.
Subject(s)
Chagas Disease/pathology , Kidney Transplantation/pathology , Kidney/parasitology , Trypanosoma cruzi/isolation & purification , Adult , Animals , Humans , Kidney Failure, Chronic/surgery , Lupus Nephritis/surgery , Male , Treatment OutcomeABSTRACT
PURPOSE: The purpose of our study was to evaluate short- and long-term results of transplants from cadaver donors who have died of poisoning by various substances. MATERIALS AND METHODS: The actuarial survival rate of organs from intoxicated donors was calculated using the Kaplan-Meier method. RESULTS: Among the 507 donors between January 1998 and December 2002, 5 (0.98%) had a cause of brain death of poisoning, namely, organo-phosphates (n = 2), methanol (n = 1), cyanide (n = 1) and acetylsalicilic acid(n = 1), from whom were procured 10 kidneys, 1 liver, 2 corneas, and 1 set of bones. The follow up for patients receiving solid organs was 15.2 months (range, 0-48 months). At 3 months, 90% of kidneys had normal function. No delayed graft function rejection episodes or major complications were reported in any recipient. None showed evidence of acute or chronic poisoning. Two died, 1 early mortality was due to anesthetic complications and the other at 17 months to an unknown cause. Actuarial kidney survival rates were 90% and 80% at 12 and 24 months, respectively. The liver recipient was well at the end of follow up. CONCLUSION: Using organs of poisoned donors is feasible with comparable graft survival rates to other recipient.
Subject(s)
Poisoning/mortality , Tissue Donors/classification , Transplantation/physiology , Adolescent , Adult , Cause of Death , Female , Follow-Up Studies , Humans , Male , Reproducibility of Results , Survival Analysis , Transplantation/mortalityABSTRACT
OBJECTIVE: The goal of this article is to present the experience of a new liver transplant team. MATERIALS AND METHODS: This review includes all patients who received a liver transplant between March 15, 2000 and March 15, 2003. RESULTS: We performed 87 transplantations on 84 patients; 39 were females and 45 were males of average age 43.6 years, including 6 children. The majority of the patients were from Colombia with time on the waiting list of less than 1 month. The average donor age was 26.7 years. The preservation solutions included Wisconsin, HTK-Brettschneider (Custodiol), and Corpaúl (similar to Henn-Ross). In this study, 95.4% were whole livers, with 97.7% using the piggyback method. We placed 23 arterial grafts and 2 venous grafts for vascular reconstructions; 95.4% were duct-to-duct anastomosis (95.4%). Among the cohort, 8.3% experienced acute rejection and 1.2% experienced chronic rejection. Two patients required retransplantation due to hepatic artery thrombosis with biliary tree necrosis. CONCLUSIONS: We consider that we have passed the crisis of beginning a new program with a reduction in postoperative complications and improving patient and graft survival. At present, we are a center that performs liver transplantations in adults and children, with a good organ donation culture in our city that allows us to offer a waiting time on the list less than one month. Neither a veno-venous bypass nor a T-tube were necessary for our cases. We also have developed a new, less expensive form of perfusing the liver in the donor.
Subject(s)
Liver Transplantation/statistics & numerical data , Adult , Cause of Death , Child , Colombia , Graft Survival/immunology , Humans , Liver Transplantation/immunology , Liver Transplantation/methods , Liver Transplantation/mortality , Postoperative Complications/classification , Postoperative Complications/epidemiology , Retrospective Studies , Survival Analysis , Tissue Donors/statistics & numerical dataABSTRACT
Se pretende conocer los efectos de la terapia ortopédica maxilar temprana en niños menores de 1 año con síndrome de Down, empleando una placa de acetato blanco con estimulador palatino tipo Castillo-Morales, que busca mejorar la posición lingual por medio de un estímulo exteroceptivo. Se tomaron modelos de estudio, medidas antopológicas y registro fílmico a 34 niños, evaluando cambios craneales, faciales y de arcos dentales durante seis meses. Los niños que recibieron la terapia evidenciaron mejoría en la posición lingual, algunos incluso obtuvieron selle labial; además presentaron mayores dimensiones faciales en las medidas antropométricas: altura facial, diámetro biciogomético, diámetro bigonial e índice facial. Las mediciones de modelos no mostraron diferencias estadísticamente significativas entre el grupo con tratamiento y el grupo sin tratamiento. Los pacientes más pequeños se adaptaron mejor a la placa, lo cual indica que la terapia puede ser favorable si se inicia a edades tempranas. Se observaron cambios cuantitativos (evaluación antropométrica y de modelos) y cualitativos (video y opinión de los padres) con la terapia ortopédica maxilar temprana que favorece el mejoramiento en la función y desarrollo craneofacial de los niños con síndrome de Down.
Subject(s)
Humans , Male , Female , Infant, Newborn , Infant , Dental Care for Disabled/methods , Dental Care for Children , Orthodontics, Corrective , Orthotic Devices , Down Syndrome/therapy , Acetates , Age of Onset , Analysis of Variance , Anthropometry , Cephalometry , Colombia , Dental Arch , Models, Dental , Face , Jaw , Maxillofacial Development , Evaluation Studies as Topic , Quantitative Trait, Heritable , Dental Service, Hospital/statistics & numerical data , Splints , Data Interpretation, Statistical , TongueABSTRACT
In Pseudomonas putida DOT-T1E multidrug efflux pumps of the resistance-nodulation-division family make a major contribution to solvent resistance. Two pumps have been identified: TtgABC, expressed constitutively, and TtgDEF, induced by aromatic hydrocarbons. A double mutant lacking both efflux pumps was able to survive a sudden toluene shock if and only if preinduced with small amounts of toluene supplied via the gas phase. In this article we report the identification and characterization in this strain of a third efflux pump, named TtgGHI. The ttgGHI genes form an operon that is expressed constitutively at high levels from a single promoter. In the presence of toluene the operon is expressed at an even higher level from two promoters, the constitutive one and a previously unreported one that is inducible and that partially overlaps the constitutive promoter. By site-directed mutagenesis we constructed a single ttgH mutant which was shown to be unable to survive sudden 0.3% (vol/vol) toluene shocks regardless of the preculture conditions. The mutation was transferred to single and double mutants to construct mutant strains in which two or all three pumps are knocked out. Survival analysis of induced and noninduced cells revealed that the TtgABC and TtgGHI pumps extruded toluene, styrene, m-xylene, ethylbenzene, and propylbenzene, whereas the TtgDEF pump removed only toluene and styrene. The triple mutant was hypersensitive to toluene, as shown by its inability to grow with toluene supplied via the vapor phase.
Subject(s)
Bacterial Proteins , Biological Transport, Active/physiology , Genes, Bacterial , Membrane Transport Proteins , Pseudomonas putida/metabolism , Toluene/metabolism , Anti-Bacterial Agents/metabolism , Base Sequence , Benzene Derivatives/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cloning, Molecular , Drug Resistance, Microbial , Molecular Sequence Data , Mutagenesis, Insertional , Pseudomonas putida/drug effects , Toluene/pharmacologyABSTRACT
Pseudomonas putida DOT-T1E is a solvent-tolerant strain able to grow in the presence of 1% (vol/vol) toluene in the culture medium. Random mutagenesis with mini-Tn5-'phoA-Km allowed us to isolate a mutant strain (DOT-T1E-42) that formed blue colonies on Luria-Bertani medium supplemented with 5-bromo-4-chloro-3-indolylphosphate and that, in contrast to the wild-type strain, was unable to tolerate toluene shocks (0.3%, vol/vol). The mutant strain exhibited patterns of tolerance or sensitivity to a number of antibiotics, detergents, and chelating agents similar to those of the wild-type strain. The mutation in this strain therefore seemed to specifically affect toluene tolerance. Cloning and sequencing of the mutation revealed that the mini-Tn5-'phoA-Km was inserted within the fliP gene, which is part of the fliLMNOPQRflhBA cluster, a set of genes that encode flagellar structure components. FliP is involved in the export of flagellar proteins, and in fact, the P. putida fliP mutant was nonmotile. The finding that, after replacing the mutant allele with the wild-type one, the strain recovered the wild-type pattern of toluene tolerance and motility unequivocally assigned FliP a function in solvent resistance. An flhB knockout mutant, another gene component of the flagellar export apparatus, was also nonmotile and hypersensitive to toluene. In contrast, a nonpolar mutation at the fliL gene, which encodes a cytoplasmic membrane protein associated with the flagellar basal body, yielded a nonmotile yet toluene-resistant strain. The results are discussed regarding a possible role of the flagellar export apparatus in the transport of one or more proteins necessary for toluene tolerance in P. putida DOT-T1E to the periplasm.
Subject(s)
Bacterial Proteins/physiology , Cyclin-Dependent Kinases/physiology , Flagella/metabolism , Genes, Bacterial , Pseudomonas putida/drug effects , Solvents/pharmacology , Toluene/pharmacology , Alkaline Phosphatase , Alleles , Anti-Bacterial Agents/pharmacology , Bacterial Proteins/genetics , Biological Transport , Chelating Agents/pharmacology , Cloning, Molecular , Cyclin-Dependent Kinases/genetics , Detergents/pharmacology , Drug Resistance, Microbial , Gene Expression , Mutagenesis, Insertional , Pseudomonas putida/genetics , Pseudomonas putida/growth & development , Pseudomonas putida/physiology , Transcription, Genetic , cis-trans-Isomerases/physiologyABSTRACT
Lipopolysaccharides (LPS) are major components of the outer membrane of gram-negative bacteria and are considered a defense barrier. To determine if LPS play a role in resistance to solvents in the solvent-tolerant Pseudomonas putida DOT-T1E strain, we have generated mutants unable to synthesize the O-antigen side chain of LPS. The wbpL gene, encoding the enzyme that begins the synthesis of the O-antigen side chain of LPS of the solvent-tolerant strain, was cloned, sequenced, and knocked out in vitro with a cassette encoding kanamycin resistance, and a mutant called WbpL0 of the DOT-T1E strain was generated in vivo by site-directed mutagenesis. The WbpL mutant was compared with the wild-type strain with regard to tolerance to a number of toxic compounds, including chelating agents, organic acids, detergents, and aromatic hydrocarbons. It was found that the mutant was as tolerant as the wild-type strain to organic acids and aromatic hydrocarbons and more sensitive to ethylenediaminetetraacetic acid and deoxycholate.
Subject(s)
Bacterial Proteins , Glycosyltransferases/metabolism , O Antigens/immunology , Organic Chemicals/pharmacology , Pseudomonas putida/enzymology , Solvents/pharmacology , Amino Acid Sequence , Base Sequence , DNA Primers , DNA, Recombinant , Glycosyltransferases/genetics , Glycosyltransferases/immunology , Microbial Sensitivity Tests , Molecular Sequence Data , Mutagenesis, Site-Directed , Phenotype , Pseudomonas putida/drug effects , Pseudomonas putida/genetics , Sequence Homology, Amino AcidABSTRACT
Owl monkeys are small monogamous primates ranging over a wide area extending from Panama to the Chaco region of northern Argentina. The Chaco, an alluvial plain covering over one million km2 of Argentina, Bolivia, Brazil, and Paraguay, consists of a mosaic of grasslands, savannas, xeric thorn forests, and gallery forests. The region shows significant seasonal variation in climate, rainfall, and food availability. The goal of this study was to determine the density, size, and structure of a population of Aotus azarai in the seasonal gallery forests of the eastern Argentinean Chaco. Reported population density, as well as group size and composition are based on data collected from 11 groups contacted on approximately 900 occasions, and observed for over 2,000 hours during a three-year period. Group and individual densities were 16 groups/km2 and 64 individuals/km2, respectively. Approximately half of the groups (n = 5) were small groups which had three individuals most of the time and never more than four, whereas the remaining groups were large groups composed of four or five individuals, and sometimes even six or seven individuals. This is the first study of A. azarai based on monitoring of a relatively large number of distinct groups. Our data suggest that owl monkeys in the seasonal subtropical forests of Formosa live at a density as high as those reported for owl monkey populations observed in tropical forests. The data also show that the social groups in the owl monkey population are of comparable size and composition to those characteristic of populations in the tropics.
Subject(s)
Aotidae , Social Behavior , Animals , Female , Male , Population Dynamics , SeasonsABSTRACT
Pseudomonas putida DOT-T1E grows on a water-toluene double liquid phase. Toluene tolerance in this microorganism is mainly achieved by at least two efflux pumps that belong to the RND family. The TtgDEF efflux pump is induced by toluene, whereas the other efflux pump, called TtgABC, is expressed at a high level in cells not exposed to toluene and at a lower level in cells grown with toluene. The ttgR gene is adjacent to the ttgABC operon and is transcribed divergently from ttgA. The expression level of ttgR was fourfold higher in cells growing in the presence of toluene than in its absence. In a TtgR-deficient background, expression from the ttgA promoter increased about 20-fold, suggesting that TtgR represses expression from the ttgA promoter. In this mutant, background expression of the ttgR gene was also much higher than in the wild-type background; however, its level of expression increased in the presence of toluene. In a ttgR mutant background, expression from the ttgD promoter followed the same pattern of expression as in the wild type. Analysis of a P. putida pTn5cat mutant that exhibited increased sensitivity to a sudden toluene shock, regardless of whether or not it was previously exposed to low toluene concentrations, revealed that pTn5cat had interrupted an lrp-like gene. The ttgR gene was expressed at very high levels in this mutant, with concomitant repression of expression of the ttgABC operon. The second ttgDEF efflux pump was expressed at low levels in this mutant strain, suggesting that the Lrp-like protein is a global regulatory protein involved in the solvent-tolerant response of this strain.