Development, synthesis, and application of magnetic layered double hydroxides (Fe3O4@SiO-LDH/DS-) as an efficient adsorbent for the removal of tetracyclines from milk samples.

This work presents the development, synthesis, and application of a layered double hydroxide (LDH) coupled to magnetic particles for the removal of antibiotics as tetracyclines (TC´s): tetracycline (TC), chlortetracycline (CT), oxytetracycline (OT), and doxycycline (DT) from milk samples. The LDH synthesis conditions, reaction time (30-90 min), molar ratios Mg2+/Al3+ (7:1-1:7), interlayer anion (NO3-, Cl-, CO32-, and dodecyl sulphate (DS-)) were evaluated. Under synthesis conditions (reaction time of 30 min, Mg2+/Al3+ molar ratio of 7:1, and DS- as interlayer anion), the LDH was coupled in a magnetic solid phase microextraction (MSPµE) methodology. At the optimal extraction conditions (pH 6, 5 min of contact time, 10 mg of adsorbent), a removal percentage of 99.0 % was obtained for each tetracycline. FTIR, TGA, SEM, and adsorption isotherms were employed to characterize the optimal adsorbent. Each experiment was corroborated by large-volume sample stacking capillary electrophoresis (LVSS-CE). The adsorbent was applied directly to positive milk samples (previously tested) for TC´s removal.

Evolution of anti-SARS-CoV-2 immune response in a cohort of French healthcare workers followed for 7 months.

OBJECTIVES: We aimed to understand the immune response among healthcare workers (HCWs) following SARS-CoV-2 infection, and to determine the infection prevalence during the first wave of the pandemic among workers in our hospital. METHODS: Determination of the serological status against SARS-CoV-2 (nucleocapsid) was offered to all HCWs. All HCWs with positive SARS-CoV-2 serology were proposed to be included in a longitudinal medical and serological follow-up (anti-spike) for 7months. RESULTS: We included 3062 HCWs; 256 (8.4%) were positive for anti-SARS-CoV-2 nucleocapsid IgG. Among them, early decrease in the anti-nucleocapsid antibody index was observed between the first (S1) and second (S2) serology samplings in 208 HCWs (84.2%). The initial anti-nucleocapsid IgG index seemed to be related to the HCWs' age. Seventy-four HCWs were included in the 7-month cohort study. Among them, 69 (90.5%) had detectable anti-spike IgG after 7months and 24 (32.4%) reported persistent symptoms consistent with post-acute COVID-19 syndrome diagnosis. CONCLUSION: The prevalence of serological positivity among HCWs was 6.7%. Infection should be followed by vaccination because of antibody decrease.

TGFß induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy.

PurposeOne of the earliest hallmarks of diabetic retinopathy is the loss of retinal pericytes. However, the mechanisms that promote pericyte dropout are unknown. In the present study, we propose a novel pathway in which pericyte apoptosis is mediated by macrophages, TGFß and pro-apoptotic BIGH3 (TGFß-induced Gene Human Clone 3) protein.Patients and methodsTo elucidate this pathway, we assayed human retinal pericyte (HRP) apoptosis by TUNEL assay, BIGH3 mRNA expression by qPCR, and BIGH3 protein expression by western blot analysis. HRP were treated with BIGH3 protein, TGFß1 and TGFß2 and inhibition assays were carried out by blocking with antibodies against BIGH3. The distribution of BIGH3 and CD68+ macrophages were compared in a post-mortem donor eye with 7-year history of Type II diabetes and histopathogically confirmed non-proliferative diabetic retinopathy (NPDR).ResultsTGFß induced a significant increase in BIGH3 mRNA and protein expression, and HRP apoptosis. BIGH3 treatment showed HRP undergo apoptosis in a dose-dependent manner. At 5 µg/ml, BIGH3 induced 3.5-times more apoptosis in HRP than in retinal endothelial cells. TGFß induced apoptosis was inhibited by blocking with antibodies against BIGH3. In an example of NPDR, BIGH3 accumulated within the walls of the inner retina arterioles. Macrophage infiltrates were frequently associated with these vessels and the inner nuclear layer.ConclusionTogether with our previously published results on macrophage-induced retinal endothelial cell apoptosis, the present study supports a novel inflammatory pathway mediated by macrophages and the BIGH3 protein leading to HRP apoptosis. As shown in human post-mortem globes, these observations are clinically relevant, suggesting a new mechanism underlying pericyte dropout during NPDR.

Abnormal glucose tolerance and insulin secretion in pancreas-specific Tcf7l2-null mice.

AIMS/HYPOTHESIS: Individuals carrying type 2 diabetes risk alleles in TCF7L2 display decreased beta cell levels of T cell factor 7 like-2 (TCF7L2) immunoreactivity, and impaired insulin secretion and beta cell sensitivity to glucagon-like peptide 1 (GLP-1). Here, we sought to determine whether selective deletion of Tcf7l2 in mouse pancreas impairs insulin release and glucose homeostasis. METHODS: Pancreas-specific Tcf7l2-null (pTcf7l2) mice were generated by crossing mice carrying conditional knockout alleles of Tcf7l2 (Tcf7l2-flox) with mice expressing Cre recombinase under the control of the Pdx1 promoter (Pdx1.Cre). Gene expression was assessed by real-time quantitative PCR and beta cell mass by optical projection tomography. Glucose tolerance, insulin secretion from isolated islets, and plasma insulin, glucagon and GLP-1 content were assessed by standard protocols. RESULTS: From 12 weeks of age, pTcf7l2 mice displayed decreased oral glucose tolerance vs control littermates; from 20 weeks they had glucose intolerance upon administration of glucose by the intraperitoneal route. pTcf7l2 islets displayed impaired insulin secretion in response to 17 (vs 3.0) mmol/l glucose (54.6 ± 4.6%, p < 0.01) or to 17 mmol/l glucose plus 100 nmol/l GLP-1 (44.3 ± 4.9%, p < 0.01) compared with control islets. Glp1r (42 ± 0.08%, p < 0.01) and Ins2 (15.4 ± 4.6%, p < 0.01) expression was significantly lower in pTcf7l2 islets than in controls. Maintained on a high-fat (but not on a normal) diet, pTcf7l2 mice displayed decreased expansion of pancreatic beta cell volume vs control littermates. No differences were observed in plasma insulin, proinsulin, glucagon or GLP-1 concentrations. CONCLUSIONS/INTERPRETATION: Selective deletion of Tcf7l2 in the pancreas replicates key aspects of the altered glucose homeostasis in human carriers of TCF7L2 risk alleles, indicating the direct role of this factor in controlling beta cell function.

[Ectopic testicular parenchyma in albugineal tunic]. / Parénquima testicular ectópico en la túnica albugínea.

We introduce a male patient with bilateral chryptorchidism submitted to surgery; during the intervention, macroscopically round macules were seen in the outer surface of both testes. In the round macules histological study, groups of seminiferous tubules were observed, including stroma and Leydig's intestitial cells surrounded of tunica albuginea, isolated from the rest of testicular parenchymal tissue; the diagnosis "Ectopic testicular parenchyma tissue" was confirmed by histopathological study. Due to the seminiferous tubules are surrounded by the tunica albuginea, with no apparent connection to the rest of the testicular parenchyma, a long-term assessment is required, both during puberty as well as in adulthood; owing to that can mislead and make unnecessary laparotomies and orchiectomies in the future.

Exceptional points and non-Hermitian degeneracy of resonances in a two-channel model.

We study the mixing and degeneracy of two unbound energy eigenstates (resonances) in a two coupled channel model of scattering and reactions. We derive the necessary and sufficient conditions for existence of an exceptional point in the extended spectrum of bound and resonance energy eigenvalues in this model and show that these are not the same as in the single channel case. When these conditions are satisfied, in the complex energy plane, the two simple resonance poles of the scattering matrix merge into one double pole at the exceptional point. In parameter space, the surface of the eigenenergies has a branch point of square root type and branch cuts in its real and imaginary parts that start at the exceptional point and extend in opposite directions. The rich phenomenology of crossings and anticrossings of energies and widths of the doublet of unbound states, as well as the changes of identity of the poles of the scattering matrix observed when one control parameter is varied while the other is kept constant, is fully explained in terms of sections of the eigenenergy surfaces.

Trichinella spiralis: intranasal immunization with attenuated Salmonella enterica carrying a gp43 antigen-derived 30mer epitope elicits protection in BALB/c mice.

Trichinellosis is a public health problem and is considered an emergent/re-emergent disease in various countries. The etiological agent of trichinellosis is the nematode Trichinella, which infects domestic animals such as pigs and horses, as well as wild animals and humans. A veterinary vaccine could be an option to control the disease in domestic animals. Although several vaccine candidates have shown promising results, a vaccine against trichinellosis remains unavailable to date. Attenuated Salmonella strains are especially attractive live vectors because they elicit mucosal immunity, which is known to be important for the control of Trichinella spiralis infection at the intestinal level and can be administered by oral or intranasal routes. In this study, the autotransporter ShdA was used to display, on the surface of the Salmonella enterica serovar Typhimurium SL3261, the 210-239 amino acid epitope, (designated as Ag30) derived from the 43 kDa glycoprotein of T. spiralis muscle larvae. The fusion protein elicited antibodies in BALB/c mice that were able to recognize the native epitope on the surface of T. spiralis muscle larvae. Mice immunized by intranasal route with the recombinant Salmonella induced a protective immune response against the T. spiralis challenge, reducing by 61.83% the adult burden at day eight postinfection. This immune response was characterized by the induction of antigen-specific IgG1 and of IL-5 production. This study demonstrates the usefulness of Salmonella as a carrier of nematode epitopes providing a surface display system for intestinal parasite vaccine applications.

Survival of a large cohort of HIV-infected tuberculosis patients in the era of highly active antiretroviral treatment.

OBJECTIVES: To determine the survival rate, predictive factors and causes of death in a cohort of human immunodeficiency virus (HIV) infected tuberculosis (TB) patients in the era of highly active antiretroviral treatment (HAART). DESIGN: This retrospective cohort study included all HIV-infected TB patients reported in Barcelona between 1996 and 2006. A survival analysis was conducted based on the Kaplan-Meier estimator and Cox proportional hazards; hazard ratios (HRs) and their 95% confidence intervals (CIs) were calculated. Causes of death were classified using the International Classification of Diseases (ICD) 9 and ICD-10, and defined as acquired immune-deficiency syndrome (AIDS) related, non-AIDS-related or unknown. RESULTS: Of the 792 patients included, 341 (43.1%) died. Survival at 10 years was 47.4% (95%CI 45.2-49.6). Poorer survival was observed in patients aged >30 years (HR 1.6, 95%CI 1.1-2.1), inner-city residents (HR 1.3, 95%CI 1.1-1.7), injecting drug users (HR 1.4, 95%CI 1.1-1.8), those with a non-cavitary radiological pattern (HR 1.5, 95%CI 1.0-2.2), those with <200 CD4/microl (HR 1.8, 95%CI 1.2-2.7) and those diagnosed with AIDS prior to their TB episode (HR 1.85, 95%CI 1.4-2.2). No differences were found for TB treatment (6 vs. 9 months) or for anti-tuberculosis drug resistance; 64.8% of the deaths were non-AIDS-related. CONCLUSIONS: Poor survival was observed despite the availability of HAART, and non-AIDS-related mortality was high. Earlier HAART could help address AIDS and non-AIDS-related mortality.

Optimización del proceso de compostaje de productos post-cosecha (cereza) del café con la aplicación de microorganismos nativos / Composting process optimization of post-harvest product (cherry) coffee with the implementation of native micoorganisms

El cultivo del café trae consigo la producción de grandes cantidades de residuos pos-cosecha, como la cereza. Este desecho causa problemas ambientales debido a que contamina fuentes hídricas y genera alteraciones en el ecosistema cuando no es tratado adecuadamente. Una alternativa a este problema es la utilización de la cereza para la producción de abono orgánico, reduciendo los costos de producción. Este estudio tiene como objetivo disminuir el tiempo de compostaje de la cereza del café utilizando 3 consorcios bacterianos de cepas aisladas de la cereza, a través de la bio-aumentación. En estos estudios se obtuvo compost que cumple con los parámetros exigidos por la NTC 5167 del 2004 y la resolución 00150 de enero de 2003 del ICA en 40 días. Se observaron diferencias significativas entre la biopila testigo en los parámetros fisicoquímicos y los obtenidos en la pruebas bajo tratamiento con los consorcios bacterianos en 40 días, pH (P= 0,00552), en cuanto a la relación C/N (P= 0,00197)demostrando una madurez a los 40 días del estudio, Potasio (P= 0,01213), Fosforo total (P= 0,09547), Hierro total (P= 0,04502), Nitrógeno orgánico (P= 0,00421) elementos de gran importancia que contribuyen al desarrollo radicular y crecimiento de las plantas.

Prevalence and antimicrobial resistance patterns of Salmonella from different raw foods in Mexico.

The presence of Salmonella was determined in 116 samples of poultry meat, 81 samples of pork, 73 samples of beef, 33 samples of cheese, 61 samples of fish, and 78 samples of vegetables collected from retail stores and supermarkets in Hidalgo State (Mexico). Ninety-three Salmonella strains isolated from raw foods were characterized, and MICs were determined for 10 antimicrobials. Salmonella was detected in 35.3% of poultry meat, 30.3% of cheese, 21.8% of vegetable, 17.3% of pork, and 15.1% of beef samples, but no Salmonella was detected in fish samples. Significantly higher counts were obtained in chicken meat (P = 0.0001), pork (P = 0.0116), cheese (P = 0.0228), and vegetables (P = 0.0072) obtained from retail stores compared with those samples obtained from supermarkets. Salmonella isolates had high levels of resistance to ampicillin (66.7% of isolates), tetracycline (61.3%), and chloramphenicol (64.5%) and low levels of resistance to cefotaxime (0%), gentamicin (3.2%), and kanamycin (4.3%). Higher levels of quinolone resistance were found in isolates from poultry meat and vegetables compared with that in other foods tested. High levels of multiresistant strains were found in all foods tested except fish, ranging from 100% of pork samples to 47.1% of vegetable samples. The present study revealed that Salmonella prevalence was higher in foods from retail stores than in foods from supermarkets. Resistance rates observed for Salmonella were largely comparable to those reported in other countries for most antimicrobials, although resistance to chloramphenicol tended to be higher.

Influence of farming methods on microbiological contamination and prevalence of resistance to antimicrobial drugs in isolates from beef.

The presence of Escherichia coli, Staphylococcus aureus, Listeria monocytogenes and Salmonella spp. was determined in 75 samples of conventional beef and in 75 samples of organic beef. All samples came from cattle slaughtered and processed in the same slaughterhouse and quartering room. A total of 180 E. coli, 180 S. aureus and 98 L. monocytogenes strains were analyzed by an agar disk diffusion assay for their resistance to 11 antimicrobials, for the case of E. coli and S. aureus, or 9 antimicrobials, for the case of L. monocytogenes. Salmonella spp. were not isolated from any of the beef samples. No significant differences in prevalence were obtained for any of the bacterial species tested between organic and conventional beef. E. coli isolated from organic beef exhibited significant differences in antimicrobial resistance against 5 of the 11 antimicrobials tested as compared to isolates recovered from conventional beef. In the case of S. aureus, these differences were only found for 3 of the 11 antimicrobials tested and for L. monocytogenes, no differences were obtained between isolates obtained from organic or conventional beef. Although no significant differences were obtained in microbiological contamination, E. coli and S. aureus isolates from organically farmed beef samples showed significantly lower rates of antimicrobial resistance in E. coli and S. aureus isolates.

[Tooth loss in diabetic patients with and without chronic kidney disease and dialysis]. / Pérdida de dientes en pacientes diabéticos con y sin insuficiencia renal crónica y diálisis.

AIM: To compare tooth loss (TL) in ESRD (ESRD DM) and non-ESRD (DM) type 2 diabetic patients. METHODS: Teeth loss was quantified, and dentition classified as: Non-Compromised (NCD) with > or = 25 teeth, partially compromised (PCD) with 9 to 24, and compromised (CD) with 0 to 8 teeth. RESULTS: ESRD DM and DM: n> or = 103 and 130, mean age 57.9 and 58.5 yr (p> or =0.716), and at diabetes diagnosis 38.5 and 47.8 yr (p<0.001). Edentulous 23.5% and 13.8% (p> or =0.057), NCD 24.5% and 35.4% (p> or =0.074). TL was strongly associated mainly to periodontal disease (p<0.001). For ESRD DM, a low serum albumin (<3.5 g/dl) was more prevalent in peritoneal dialysis cases (p> or =0.0014), women (p> or =0.0100), people reporting unpleasant taste (UT) (p> or =0.0174), and those with a CD (p> or =0.0242). CONCLUSIONS: There was a clear trend for more severe TL in ESRD DM cases, but no statistical difference was found. The association between low serum albumin, UT and CD imply a need for treatment of these conditions as a part of nutritional intervention in ESRD DM cases.

A fusogenic peptide expressed on the surface of Salmonella enterica elicits CTL responses to a dengue virus epitope.

Attenuated Salmonella strains are used widely as live carriers of antigens because they elicit both mucosal and systemic immunity against passenger antigens. However, they generally evoke poor cytotoxic T cell (CTL) responses because Salmonella resides within vacuolar compartments and the passenger antigens must travel to the cytosol and be processed through the MHC class I-dependent pathway to simulate CTLs. To address this problem, we designed a fusion protein to destabilize the phagosome membrane and allow a dengue epitope to reach the cytosol. The fusion protein was displayed on the bacterial surface of Salmonella enterica serovar Typhimurium SL3261 through the beta domain of the autotransporter MisL. The passenger alpha domain contained, from the N-terminus, a fusogenic sequence, the NS3 protein 298-306-amino acid CTL epitope from the dengue virus type 2, a molecular tag, and a recognition site for the protease OmpT to release it to the milieu. Display of the fusion protein on the bacterial surface was demonstrated by IFA and flow cytometry using antibodies against the molecular tag. Cleavage of the fusogenic protein-dengue peptide was demonstrated by flow cytometry using OmpT+ Escherichia coli strains. The recombinant Salmonella strains displaying the fusogenic-dengue peptide were able to lyse erythrocytes, induced specific proliferative responses, and elicited CTL responses. These results suggest that the recombinant fusion proteins containing fusogenic sequences provide a promising system to induce CTLs by live vector vaccines.

Antimicrobial resistance in Enterococcus spp. strains isolated from organic chicken, conventional chicken, and turkey meat: a comparative survey.

The mean counts of Enterococcus spp. were determined for 30 samples each of organic chicken meat, conventional chicken meat, and turkey meat, and differences for Enterococcus contamination in meat were determined. Two enterococci strains from each sample were isolated to obtain a total of 180 strains, and resistance to ampicillin, chloramphenicol, doxycycline, ciprofloxacin, erythromycin, gentamicin, nitrofurantoin, and vancomycin was determined by a disk diffusion method. Average counts obtained showed that Enterococcus mean counts from organic chicken meat (3.18 log CFU/g) were significantly higher than those obtained from conventional chicken meat (2.06 log CFU/g) or conventional turkey meat (1.23 log CFU/g). However, the resistance data obtained showed that isolates from organic chicken meat were less resistant than enterococci isolates from conventional chicken meat to ampicillin (P = 0.0067), chloramphenicol (P = 0.0154), doxycycline (P = 0.0277), ciprofloxacin (P = 0.0024), erythromycin (P = 0.0028), and vancomycin (P = 0.0241). In addition, isolates from organic chicken were less resistant than conventional turkey meat isolates to ciprofloxacin (P = 0.001) and erythromycin (P = 0.0137). Multidrug-resistant isolates were found in every group tested, but rates of multidrug-resistant strains were significantly higher in conventional chicken and turkey than those obtained from organic chicken meat. Enterococcus faecalis was the most common species isolated from organic chicken (36.67%), whereas Enterococcus durans was the most common species isolated from conventional chicken (58.33%) and turkey (56.67%). The rates obtained for antimicrobial resistance suggest that although organic chicken meat may have higher numbers of Enterococcus, these bacteria present a lower level of antimicrobial resistance.

Increasing incidence of type 1 diabetes in population from Santiago of Chile: trends in a period of 18 years (1986-2003).

UNLABELLED: STUDY OBJECTIVE AND SETTING: The aim of this study is to determine the incidence rate of type 1 diabetes in the metropolitan region of Santiago, Chile from 1 January 1986 to 31 December 2003. DESIGN: Population-based incidence study. PARTICIPANTS: A case must fulfill the following requirements to be included in this study: age at onset: 0 to 14 years, diagnosed with diabetes and placed on insulin, diagnosed within the defined time period, and to be a resident of the metropolitan region of Santiago at the time of the diagnosis. The population 'at risk' is the population less than 15 years of age. RESULT: The overall rate of type 1 diabetes was estimated as 4.02 cases per 100 000 children per year (95% confidence interval: 2.98-4.83). CONCLUSION: The incidence of type 1 diabetes in Santiago, Chile has increased during the last years. This data are concordant with the observation that the incidence of type 1 diabetes is increasing in Latin America and worldwide.

Energy eigenvalue surfaces close to a degeneracy of unbound states: crossings and anticrossings of energies and widths.

The rich phenomenology of crossings and anticrossings of energies and widths, as well as the sudden change in shape of the S(E)-matrix pole trajectories, observed in an isolated doublet of resonances when one control parameter is varied, is fully explained in terms of sections of the energy eigenvalue surfaces in parameter space close to a degeneracy point.

[Celiac disease in adolescents and young adults. A challenge for gastroenterologists of children and adults]. / Enfermedad celíaca en el adolescente y adulto joven. Un desafío para gastroenterólogos de niños y adultos.

BACKGROUND: Follow-up of celiac patients in Chile is often interrupted when adolescents are referred to adult gastroenterologists. AIM: To study the evolution of patients with celiac disease when they reach adolescence or young adulthood. PATIENTS AND METHODS: Current adherence to gluten-free diet and its relation to symptoms and circulating antiendomysial antibodies were evaluated in the 58 confirmed celiac patients older than 12 years of age controlled at 3 hospitals in Santiago. RESULTS: Mean age at the moment of this assessment was 17.8 +/- 5 years, 65.5% were women, 12.5% were at nutritional risk (-IDS) while 20% were overweight/obese. Although all patients declared themselves asymptomatic, a focused questionnaire revealed that 26% suffered some symptoms. Only 24.1% followed a strict gluten-free diet. Eight of 20 patients who ate gluten-containing diets had negative antiendomysial antibodies (EMA), three of whom turned positive within 6 to 9 months. In three of four (asymptomatic) cases that accepted a new jejunal biopsy, histology was abnormal. One patient who followed a strict diet had EMA (+) and normal histology. CONCLUSIONS: These results confirm that after childhood, symptoms abate significantly in celiac patients. The observed sensitivity and specificity of EMA makes necessary to maintain small intestinal biopsies as the gold standard for diagnosis and confirmation of the disease.

Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule.

A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes-DNA topoisomerases-alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 A resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.

Structure and mechanism of the RNA triphosphatase component of mammalian mRNA capping enzyme.

The 5' capping of mammalian pre-mRNAs is initiated by RNA triphosphatase, a member of the cysteine phosphatase superfamily. Here we report the 1.65 A crystal structure of mouse RNA triphosphatase, which reveals a deep, positively charged active site pocket that can fit a 5' triphosphate end. Structural, biochemical and mutational results show that despite sharing an HCxxxxxR(S/T) motif, a phosphoenzyme intermediate and a core alpha/beta-fold with other cysteine phosphatases, the mechanism of phosphoanhydride cleavage by mammalian capping enzyme differs from that used by protein phosphatases to hydrolyze phosphomonoesters. The most significant difference is the absence of a carboxylate general acid catalyst in RNA triphosphatase. Residues conserved uniquely among the RNA phosphatase subfamily are important for function in cap formation and are likely to play a role in substrate recognition.

The mechanism of type IA topoisomerase-mediated DNA topological transformations.

Type IA DNA topoisomerases possess several domains forming a toroidal molecule with a central hole large enough to accommodate single- or double-stranded DNA. The sign inversion model predicts several protein-DNA intermediates, including those in which DNA is trapped within the hole. Opposing cysteine residues were incorporated into two independent domains surrounding the putative DNA binding cavity of E. coli topoisomerase III, creating a molecule that can be covalently closed or opened by oxidizing or reducing the disulfide bond. The formation of the disulfide bond allowed the trapping of single- and double-stranded DNA within the cavity of the enzyme and the identification of other intermediates proposed by the sign inversion model.