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AIM: To evaluate the change in diagnosis rates, disease severity at presentation, and treatment of acute appendicitis and diverticulitis during the COVID-19 shutdown. MATERIALS AND METHODS: Following institutional review board approval, 6,002 CT examinations performed at five hospitals for suspected acute appendicitis and/or diverticulitis over the 12 weeks preceding and following the shutdown were reviewed retrospectively. Semi-automated language analysis (SALA) of the report classified 3,676 CT examinations as negative. Images of the remaining 2,326 CT examinations were reviewed manually and classified as positive or negative. Positive cases were graded as non-perforated; perforated, contained; and perforated, free. RESULTS: CT examinations performed for suspected appendicitis and/or diverticulitis decreased from 3,558 to 2,200 following the shutdown. The rates of positive diagnoses before and after shutdown were 4% (144) and 4% (100) for appendicitis and 8% (284) and 7% (159) for diverticulitis (p>0.2 for both). For positive CT examinations, the rates of perforation, hospitalisation, surgery, and catheter drainage changed by -2%, -3%, -2%, and -3% for appendicitis (n=244, p>0.3 for all) and +6% (p=0.2) +9% (p=0.06), +4% (p=0.01) and +1% (p=0.6) for diverticulitis (n=443). CONCLUSION: CT examinations performed for suspected appendicitis or diverticulitis declined after the shutdown, likely reflecting patients leaving urban centres and altered triage of non-COVID-19 patients. The diagnosis rates, disease severity at presentation, and treatment approach otherwise remained mostly unchanged.
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Apendicitis , COVID-19 , Diverticulitis , Enfermedad Aguda , Apendicitis/diagnóstico por imagen , Apendicitis/cirugía , COVID-19/diagnóstico por imagen , Diverticulitis/diagnóstico por imagen , Diverticulitis/cirugía , Humanos , Estudios Retrospectivos , Tomografía Computarizada por Rayos X/métodosAsunto(s)
Adenina/análogos & derivados , Antineoplásicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Compuestos Bicíclicos Heterocíclicos con Puentes/uso terapéutico , Linfoma de Células del Manto/tratamiento farmacológico , Linfoma de Células del Manto/inmunología , Piperidinas/uso terapéutico , Sulfonamidas/uso terapéutico , Adenina/uso terapéutico , Humanos , Inmunofenotipificación , Linfoma de Células del Manto/diagnóstico , Linfoma de Células del Manto/patología , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
Asparaginyl endopeptidases (AEPs) are proteases that have crucial roles in plant defense and seed storage protein maturation. Select plant AEPs, however, do not function as proteases but as transpeptidases (ligases) catalyzing the intra-molecular ligation of peptide termini, which leads to peptide cyclization. These ligase-type AEPs have potential biotechnological applications ranging from in vitro peptide engineering to plant molecular farming, but the structural features enabling these enzymes to catalyze peptide ligation/cyclization rather than proteolysis are currently unknown. Here, we compare the sequences, structures, and functions of diverse plant AEPs by combining molecular modeling, sequence space analysis, and functional testing in planta. We find that changes within the substrate-binding pocket and an adjacent loop, here named the "marker of ligase activity", together play a key role for AEP ligase efficiency. Identification of these structural determinants may facilitate the discovery of more ligase-type AEPs and the engineering of AEPs with tailored catalytic properties.
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Cisteína Endopeptidasas/metabolismo , Péptidos Cíclicos/biosíntesis , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Cisteína Endopeptidasas/genética , Modelos Moleculares , Proteínas de Plantas/genética , Plantas/genética , Plantas Modificadas Genéticamente , ARN de Planta/genética , Análisis de Secuencia de ARNRESUMEN
To survive, Escherichia coli must be able to survive in rapidly changing environmental conditions including changes in temperature and osmolarity. We have studied the role of the inner membrane protein YhiM in changing environmental conditions. Our data indicate that YhiM is required for normal growth at 37 and 41 °C but not 21 °C. YhiM-deficient cells grown at high temperatures spend more time in lag phase and stop growing at lower cell densities in comparison with their wild-type counterparts. They also have growth defects in low NaCl medium at 37 °C and do not grow at all at 41 °C. The effects of low NaCl can be rescued by addition of KCl or sucrose to the low salt medium. Finally, YhiM-deficient cells fail to grow in dilute medium at 41 °C. These data suggest that YhiM may be important in protecting the cells from changes in temperature and osmolarity.
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Proteínas de Escherichia coli/metabolismo , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , Proteínas de la Membrana/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Calor , Proteínas de la Membrana/genética , Concentración Osmolar , Cloruro de Sodio/metabolismoRESUMEN
A membrane-less organic-inorganic flow battery based on zinc and quinone species is proposed. By virtue of the slow dissolution rate of the deposited anode (<11.5 mg h-1 cm-2), the battery has a cell voltage of ca. 1.52 V with an average energy efficiency of ca. 73% at 30 mA cm-2 over 12 cycles.
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OBJECTIVE: Colocation of mental health screening, assessment, and treatment in primary care reduces stigma, improves access, and increases coordination of care between mental health and primary care providers. However, little information exists regarding older adults' attitudes about screening for mental health problems in primary care. The objective of this study was to evaluate older primary care patients' acceptance of and satisfaction with screening for depression and anxiety. METHODS: The study was conducted at an urban, academically affiliated primary care practice serving older adults. Study patients (N = 107) were screened for depression/anxiety and underwent a post-screening survey/interview to assess their reactions to the screening experience. RESULTS: Most patients (88.6%) found the length of the screening to be "just right." A majority found the screening questions somewhat or very acceptable (73.4%) and not at all difficult (81.9%). Most participants did not find the questions stressful (84.9%) or intrusive (91.5%); and a majority were not at all embarrassed (93.4%), upset (93.4%), or uncomfortable (88.8%) during the screening process. When asked about frequency of screening, most patients (72.4%) desired screening for depression/anxiety yearly or more. Of the 79 patients who had spoken with their physicians about mental health during the visit, 89.8% reported that it was easy or very easy to talk with their physicians about depression/anxiety. Multivariate results showed that patients with higher anxiety had a lower positive reaction to the screen when controlling for gender, age, and patient-physician communication. CONCLUSIONS: These results demonstrate strong patient support for depression and anxiety screening in primary care.
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Trastornos de Ansiedad/diagnóstico , Prestación Integrada de Atención de Salud/normas , Trastorno Depresivo/diagnóstico , Servicios de Salud para Ancianos/organización & administración , Tamizaje Masivo/organización & administración , Servicios de Salud Mental/organización & administración , Satisfacción del Paciente , Atención Primaria de Salud/organización & administración , Adulto , Anciano , Femenino , Evaluación Geriátrica/métodos , Servicios de Salud para Ancianos/normas , Humanos , Masculino , Tamizaje Masivo/normasRESUMEN
Overexpression of the prosurvival protein Bcl-2 marks many B-lymphoid malignancies and contributes to resistance to many commonly used chemotherapeutic agents. The first effective BH3 mimetic inhibitors of Bcl-2, ABT-737 and navitoclax, also target Bcl-xL, causing dose-limiting thrombocytopenia. This prompted the development of the Bcl-2-selective antagonist, ABT-199. Here we show that in lymphoid cells, ABT-199 specifically causes Bax/Bak-mediated apoptosis that is triggered principally by the initiator BH3-only protein Bim. As expected, malignant cells isolated from patients with chronic lymphocytic leukaemia are highly sensitive to ABT-199. However, we found that normal, untransformed mature B cells are also highly sensitive to ABT-199, both in vitro and in vivo. By contrast, the B-cell precursors are largely spared, as are cells of myeloid origin. These results pinpoint the probable impact of the pharmacological inhibition of Bcl-2 by ABT-199 on the normal mature haemopoietic cell lineages in patients, and have implications for monitoring during ABT-199 therapy as well as for the clinical utility of this very promising targeted agent.
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Apoptosis/efectos de los fármacos , Linfocitos B/efectos de los fármacos , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Sulfonamidas/farmacología , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Proteínas Reguladoras de la Apoptosis/fisiología , Linfocitos B/metabolismo , Proteína 11 Similar a Bcl2 , Western Blotting , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Citometría de Flujo , Voluntarios Sanos , Humanos , Leucemia Linfocítica Crónica de Células B/metabolismo , Leucemia Linfocítica Crónica de Células B/patología , Proteínas de la Membrana/fisiología , Ratones , Ratones Noqueados , Células Mieloides/efectos de los fármacos , Células Mieloides/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/fisiología , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto , Proteína Destructora del Antagonista Homólogo bcl-2/fisiología , Proteína X Asociada a bcl-2/fisiologíaRESUMEN
BACKGROUND: The 2001 National Health and Medical Research Council/Australasian Society of Blood Transfusion Clinical Practice Guidelines for cryoprecipitate are being updated, and cryoprecipitate has been incorporated into new Patient Blood Management modules. AIMS: This clinical audit sought to clarify current cryoprecipitate use in Victoria, Tasmania and the Australian Capital Territory; assess adherence to guidelines; and gain insights into deviations from recommended practice. This information can be utilised in updating guidelines to make them more relevant, to identify areas for clinician education and to form a baseline of practice prior to release of the 2011 guidelines. METHODS: Participating institutions were invited to audit up to 30 consecutive episodes of cryoprecipitate transfusion over an 11-month period in 2008. The audits were conducted using a standardised pro forma and involved review of patient records. These were collated electronically using algorithms to determine alignment versus non-alignment with guidelines. RESULTS: Cryoprecipitate is used in a variety of situations with surgery accounting for the highest volume. Twenty-six per cent (26%) of transfusions were aligned with 2001 guidelines rising to 61% with a modified fibrinogen trigger. Fibrinogen levels did not appear to dictate all clinical decisions regarding cryoprecipitate use perhaps owing to the acuity of many cases. Additional bleeding risk together with low fibrinogen levels (e.g. thrombocytopenic patients) may contribute to empiric cryoprecipitate use. CONCLUSIONS: These results highlight discrepancies between guidelines and practice, providing rationale for the update of the guidelines that is currently underway. Cryoprecipitate has attendant risks, and it is appropriate that transfusion be restricted to situations with good evidence or sound principles to underpin use.
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Transfusión Sanguínea/normas , Bases de Datos Factuales/normas , Factor VIII/administración & dosificación , Fibrinógeno/administración & dosificación , Fibronectinas/administración & dosificación , Adhesión a Directriz/normas , Guías de Práctica Clínica como Asunto/normas , Transfusión Sanguínea/tendencias , Bases de Datos Factuales/tendencias , Factor VIII/efectos adversos , Fibrinógeno/efectos adversos , Fibronectinas/efectos adversos , Adhesión a Directriz/tendencias , HumanosRESUMEN
The flowers of the ornamental tobacco produce high levels of a series of 6 kDa serine protease inhibitors (NaPIs) that are effective inhibitors of trypsins and chymotrypsins from lepidopteran species. These inhibitors have a negative impact on the growth and development of lepidopteran larvae and have a potential role in plant protection. Here we investigate the effect of NaPIs on the activity and levels of serine proteases in the gut of Helicoverpa armigera larvae and explore the adaptive mechanisms larvae employ to overcome the negative effects of NaPIs in the diet. Polyclonal antibodies were raised against a Helicoverpa punctigera trypsin that is a target for NaPIs and two H. punctigera chymotrypsins; one that is resistant and one that is susceptible to inhibition by NaPIs. The antibodies were used to optimize procedures for extraction of proteases for immunoblot analysis and to assess the effect of NaPIs on the relative levels of the proteases in the gut and frass. We discovered that consumption of NaPIs did not lead to over-production of trypsins or chymotrypsins but did result in excessive loss of proteases to the frass.
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Quimotripsina/metabolismo , Proteínas de Insectos/metabolismo , Mariposas Nocturnas/enzimología , Extractos Vegetales/metabolismo , Solanum tuberosum/química , Inhibidores de Tripsina/metabolismo , Tripsina/metabolismo , Secuencia de Aminoácidos , Animales , Quimotripsina/antagonistas & inhibidores , Quimotripsina/genética , Clonación Molecular , Tracto Gastrointestinal/enzimología , Control de Insectos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Datos de Secuencia Molecular , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/genética , Mariposas Nocturnas/metabolismo , Extractos Vegetales/química , Alineación de Secuencia , Tripsina/química , Tripsina/genética , Inhibidores de Tripsina/químicaRESUMEN
Small RNA regulatory pathways are used to control the activity of transposons, regulate gene expression and resist infecting viruses. We examined the biogenesis of mRNA-derived endogenous short-interfering RNAs (endo-siRNAs) in the disease vector mosquito Aedes aegypti. Under standard conditions, mRNA-derived endo-siRNAs were produced from the bidirectional transcription of tail-tail overlapping gene pairs. Upon infection with the alphavirus, Sindbis virus (SINV), another class of mRNA-derived endo-siRNAs was observed. Genes producing SINV-induced endo-siRNAs were not enriched for overlapping partners or nearby genes, but were enriched for transcripts with long 3' untranslated regions. Endo-siRNAs from this class derived uniformly from the entire length of the target transcript, and were found to regulate the transcript levels of the genes from which they were derived. Strand-specific quantitative PCR experiments demonstrated that antisense strands of targeted mRNA genes were produced to exonic, but not intronic regions. Finally, small RNAs mapped to both sense and antisense strands of exon-exon junctions, suggesting double-stranded RNA precursors to SINV-induced endo-siRNAs may be synthesized from mature mRNA templates. These results suggest additional complexity in small RNA pathways and gene regulation in the presence of an infecting virus in disease vector mosquitoes.
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Aedes/genética , Aedes/virología , ARN Interferente Pequeño/biosíntesis , Virus Sindbis/fisiología , Regiones no Traducidas 3'/genética , Infecciones por Alphavirus/genética , Animales , Exones/genética , Femenino , Regulación de la Expresión Génica , Genes de Insecto/genética , Genoma Viral/genética , Interferencia de ARN , Empalme del ARN/genética , ARN sin Sentido/genética , ARN Bicatenario/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Virus Sindbis/genéticaRESUMEN
BACKGROUND: Temporary prophylactic pancreatic duct stenting effectively reduces post-endoscopic retrograde cholangiopancreatography (ERCP) pancreatitis (PEP) in high-risk patients, but the optimal stent remains unclear. We compared rate of spontaneous passage, and technical difficulty of placement for 3-Fr and 5-Fr stents. METHODS: A randomized controlled trial at a single academic medical center. Patients deemed high risk for PEP randomly received 5-Fr or 3-Fr pancreatic duct stents. Primary outcome was spontaneous stent passage by 2 weeks. Secondary outcomes were ease and time for stent placement, and number of guide wires required for the entire procedure. RESULTS: Patients (69 female [89 %]; mean age 44.9 years, standard deviation [SD] 16.8) were randomly assigned to receive 5-Fr (n = 38) and 3-Fr (n = 40) stents. Indications for stenting were similar. Seven patients in the 3-Fr group actually received a 5-Fr stent, and two in the 5-Fr group had a 3-Fr stent. Spontaneous passage or non-passage was confirmed in 64 (83 %). No statistically significant difference in spontaneous passage rates was seen (5-Fr group, 68.4 %; 3-Fr group 75.0 %; P = 0.617). Non-passage rates were 10.5 % (5-Fr group) and 10.0 % (3-Fr group) ( P = 1.00). The study was stopped after a futility analysis for the primary end point. Placement of 5-Fr stents was rated easier, at a mean score of 1.8 (5-Fr) vs. 3.4 (3-Fr), P < 0.001, with a trend towards being faster, 9.2 vs. 11.1 minutes ( P = 0.355). Fewer guide wires were required for 5-Fr stent placement, 1.5 vs. 1.9 ( P = 0.002). PEP rates did not differ ( P = 0.519). CONCLUSION: Placement of 5-Fr compared to 3-Fr pancreatic duct stents for PEP prophylaxis is easier, faster, and requires fewer wires. No statistically significant difference in spontaneous passage was found between the two sizes.
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Colangiopancreatografia Retrógrada Endoscópica/efectos adversos , Conductos Pancreáticos/cirugía , Pancreatitis/etiología , Pancreatitis/prevención & control , Stents , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Factores de Riesgo , Stents/efectos adversosRESUMEN
BACKGROUND: Effectiveness of medical therapies in chronic pancreatitis has been described in small studies of selected patients. AIM: To describe frequency and perceived effectiveness of non-analgesic medical therapies in chronic pancreatitis patients evaluated at US referral centres. METHODS: Using data on 516 chronic pancreatitis patients enrolled prospectively in the NAPS2 Study, we evaluated how often medical therapies [pancreatic enzyme replacement therapy (PERT), vitamins/antioxidants (AO), octreotide, coeliac plexus block (CPB)] were utilized and considered useful by physicians. RESULTS: Oral PERT was commonly used (70%), more frequently in the presence of exocrine insufficiency (EI) (88% vs. 61%, P < 0.001) and pain (74% vs. 59%, P < 0.002). On multivariable analyses, predictors of PERT usage were EI (OR 5.14, 95% CI 2.87-9.18), constant (OR 3.42, 95% CI 1.93-6.04) or intermittent pain (OR 1.98, 95% CI 1.14-3.45). Efficacy of PERT was predicted only by EI (OR 2.16, 95% CI 1.36-3.42). AO were tried less often (14%) and were more effective in idiopathic and obstructive vs. alcoholic chronic pancreatitis (25% vs. 4%, P = 0.03). Other therapies were infrequently used (CPB - 5%, octreotide - 7%) with efficacy generally <50%. CONCLUSIONS: Pancreatic enzyme replacement therapy is commonly utilized, but is considered useful in only subsets of chronic pancreatitis patients. Other medical therapies are used infrequently and have limited efficacy.
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Dolor Abdominal/terapia , Antioxidantes/uso terapéutico , Fármacos Gastrointestinales/uso terapéutico , Octreótido/uso terapéutico , Vitaminas/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Bloqueo Nervioso Autónomo/métodos , Terapia de Reemplazo Enzimático , Femenino , Humanos , Masculino , Persona de Mediana Edad , Páncreas/patología , Pancreatitis Crónica , Estudios Prospectivos , Encuestas y Cuestionarios , Resultado del Tratamiento , Estados Unidos , Adulto JovenRESUMEN
AnWj is a high-incidence antigen present on the red blood cells (RBCs) of greater than 99 percent of the general population. A 58-year-old man underwent autologous hematopoietic stem cell transplantation (HSCT) for stage IVa mantle cell lymphoma. This procedure was complicated by failure to engraft, necessitating ongoing support with blood components. After a 2-month period of uneventful transfusion support, the patient experienced increasingly severe reactions with fever and evidence of intravascular hemolysis, including hemoglobinuria. Testing revealed a complement-dependent anti-AnWj. Phenotyping confirmed the AnWj- phenotype. Anti-AnWj was persistent despite immunosuppression, including treatment with allogeneic HSCT. Of interest, the pathogenesis of the downregulation of the graft AnWj in this patient is unclear.
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Antígenos de Grupos Sanguíneos/inmunología , Proteínas del Sistema Complemento/inmunología , Eritrocitos/inmunología , Rechazo de Injerto/inmunología , Trasplante de Células Madre Hematopoyéticas , Linfoma de Células del Manto/terapia , Anticuerpos/metabolismo , Citotoxicidad Celular Dependiente de Anticuerpos , Transfusión de Eritrocitos , Hemoglobinuria/inmunología , Hemólisis/inmunología , Humanos , Terapia de Inmunosupresión , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias/inmunologíaRESUMEN
Potato type I and II serine protease inhibitors are produced by solanaceous plants as a defense mechanism against insects and microbes. Nicotiana alata proteinase inhibitor (NaPI) is a multidomain potato type II inhibitor (pin II) that is produced at high levels in the female reproductive tissues of the ornamental tobacco, Nicotiana alata. The individual inhibitory domains of NaPI target the major classes of digestive enzymes, trypsin and chymotrypsin, in the gut of lepidopteran larval pests. Although consumption of NaPI dramatically reduced the growth and development of a major insect pest, Helicoverpa punctigera, we discovered that surviving larvae had high levels of chymotrypsin activity resistant to inhibition by NaPI. We found a potato type I inhibitor, Solanum tuberosum potato type I inhibitor (StPin1A), was a strong inhibitor of the NaPI-resistant chymotrypsin activity. The combined inhibitory effect of NaPI and StPin1A on H. armigera larval growth in the laboratory was reflected in the increased yield of cotton bolls in field trials of transgenic plants expressing both inhibitors. Better crop protection thus is achieved using combinations of inhibitors in which one class of proteinase inhibitor is used to match the genetic capacity of an insect to adapt to a second class of proteinase inhibitor.
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Gossypium/metabolismo , Gossypium/parasitología , Mariposas Nocturnas/patogenicidad , Control Biológico de Vectores/métodos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Inhibidores de Proteasas/metabolismo , Solanum tuberosum/metabolismo , Animales , Quimotripsina/antagonistas & inhibidores , Expresión Génica , Genes de Plantas , Gossypium/genética , Proteínas de Insectos/antagonistas & inhibidores , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Datos de Secuencia Molecular , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/crecimiento & desarrollo , Enfermedades de las Plantas/parasitología , Enfermedades de las Plantas/prevención & control , Proteínas de Plantas/farmacología , Plantas Modificadas Genéticamente , Inhibidores de Proteasas/farmacología , Solanum tuberosum/genéticaRESUMEN
Plants produce a variety of proteinase inhibitors (PIs) that have a major function in defense against insect herbivores. In turn, insects have developed strategies to minimize the effect of dietary PIs on digestion. We have discovered that Helicoverpa larvae that survive consumption of a multidomain serine PI from Nicotiana alata (NaPI) contain high levels of a chymotrypsin that is not inhibited by NaPI. Here we describe the isolation of this NaPI-resistant chymotrypsin and an NaPI-susceptible chymotrypsin from Helicoverpa larvae, together with their corresponding cDNAs. We investigated the mechanism of resistance by mutating selected positions of the NaPI-susceptible chymotrypsin using the corresponding amino acids of the NaPI-resistant chymotrypsin. Four critical residues that conferred resistance to NaPI were identified. Molecular modeling revealed that a Phe-->Leu substitution at position 37 in the chymotrypsin results in the loss of important binding contacts with NaPI. Identification of the molecular mechanisms that contribute to PI resistance in insect digestive proteases will enable us to develop better inhibitors for the control of lepidopteran species that are major agricultural pests worldwide.
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Quimotripsina/antagonistas & inhibidores , Quimotripsina/genética , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/genética , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/enzimología , Proteínas de Plantas/metabolismo , Inhibidores de Proteasas/metabolismo , Inhibidores de Proteasas/farmacología , Solanum tuberosum/metabolismo , Secuencia de Aminoácidos , Animales , Arginina/química , Sitios de Unión/genética , Quimotripsina/química , Proteínas de Insectos/química , Larva/efectos de los fármacos , Larva/enzimología , Larva/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mariposas Nocturnas/genética , Mariposas Nocturnas/patogenicidad , Proteínas de Plantas/farmacología , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homología de Secuencia de AminoácidoRESUMEN
To date, only a limited number of promoter sequences have been described to drive transgene expression in the disease vector Aedes aegypti. We sought to increase this repertoire by characterizing the ability of upstream sequences derived from the Ae. aegypti Ub(L40) and polyubiquitin genes to drive the expression of marker proteins. Both genomic fragments were able to drive robust expression of luciferase in cultured mosquito cells. Following Mos1-transformation, the Ub(L40) promoter drove strong expression of a fluorescent marker in early larvae and in ovaries, while the polyubiquitin promoter drove robust EGFP expression in all stages of development, including constitutive expression throughout the midgut. These promoter fragments provide two new expression profiles for future Ae. aegypti genetic experiments.
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Aedes/genética , Genes de Insecto/genética , Regiones Promotoras Genéticas/genética , Ubiquitina/genética , Aedes/citología , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Regulación de la Expresión Génica , Genes Reporteros/genética , Proteínas Fluorescentes Verdes/metabolismo , Poliubiquitina/genética , Reproducibilidad de los Resultados , Transformación GenéticaRESUMEN
Aedes aegypti is a major vector of arthropod-borne viruses such as yellow fever virus and dengue viruses. Efforts to discern the function of genes involved in important behaviours, such as vector competence and host seeking through reverse genetics, would greatly benefit from the ability to generate targeted gene disruptions. Homing endonucleases are selfish elements which catalyze double-stranded DNA (dsDNA) breaks in a sequence-specific manner. In this report we demonstrate that the homing endonucleases I-PpoI, I-SceI, I-CreI and I-AniI are all able to induce dsDNA breaks in adult female Ae. aegypti chromosomes as well as catalyze the somatic excision of a transgene. These experiments provide evidence that homing endonucleases can be used to manipulate the genome of this important disease vector.
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Aedes/enzimología , Aedes/genética , Biocatálisis , Roturas del ADN de Doble Cadena , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Transgenes/genética , Aedes/virología , Animales , Secuencia de Bases , Bioensayo , Southern Blotting , Huella de ADN , Reparación del ADN , ADN Recombinante/genética , Datos de Secuencia Molecular , Plásmidos/genética , Transporte de Proteínas , Virus Sindbis/fisiologíaRESUMEN
A previously published hydrothermal brine-river water mixing model driven by ocean crust production suggests that the molar Mg/Ca ratio of seawater (mMg/Ca(sw)) has varied significantly (approximately 1.0-5.2) over Precambrian time, resulting in six intervals of aragonite-favouring seas (mMg/Ca(sw) > 2) and five intervals of calcite-favouring seas (mMg/Ca(sw) < 2) since the Late Archaean. To evaluate the viability of microbial carbonates as mineralogical proxy for Precambrian calcite-aragonite seas, calcifying microbial marine biofilms were cultured in experimental seawaters formulated over the range of Mg/Ca ratios believed to have characterized Precambrian seawater. Biofilms cultured in experimental aragonite seawater (mMg/Ca(sw) = 5.2) precipitated primarily aragonite with lesser amounts of high-Mg calcite (mMg/Ca(calcite) = 0.16), while biofilms cultured in experimental calcite seawater (mMg/Ca(sw) = 1.5) precipitated exclusively lower magnesian calcite (mMg/Ca(calcite) = 0.06). Furthermore, Mg/Ca(calcite )varied proportionally with Mg/Ca(sw). This nearly abiotic mineralogical response of the biofilm CaCO3 to altered Mg/Ca(sw) is consistent with the assertion that biofilm calcification proceeds more through the elevation of , via metabolic removal of CO2 and/or H+, than through the elevation of Ca2+, which would alter the Mg/Ca ratio of the biofilm's calcifying fluid causing its pattern of CaCO3 polymorph precipitation (aragonite vs. calcite; Mg-incorporation in calcite) to deviate from that of abiotic calcification. If previous assertions are correct that the physicochemical properties of Precambrian seawater were such that Mg/Ca(sw) was the primary variable influencing CaCO3 polymorph mineralogy, then the observed response of the biofilms' CaCO3 polymorph mineralogy to variations in Mg/Ca(sw), combined with the ubiquity of such microbial carbonates in Precambrian strata, suggests that the original polymorph mineralogy and Mg/Ca(calcite )of well-preserved microbial carbonates may be an archive of calcite-aragonite seas throughout Precambrian time. These results invite a systematic evaluation of microbial carbonate primary mineralogy to empirically constrain Precambrian seawater Mg/Ca.