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1.
Postgrad Med J ; 99(1168): 56-62, 2023 Mar 31.
Article in English | MEDLINE | ID: mdl-36828395

ABSTRACT

PURPOSE: This work aims to describe patients hospitalized in internal medicine wards in terms of nutrition and sarcopenia. It also seeks to evaluate short- and long-term mortality related to malnutrition and sarcopenia. METHODS: This cross-sectional study collected data on consecutive patients admitted to a single center's internal medicine ward. Patients were recruited in May and October 2021. Malnutrition was determined by the Mini-Nutritional Assessment-Short Form (MNA-SF) and sarcopenia by the Strength, Assistance in walking, Rise from a chair, Climb stairs, and Falls questionnaire (SARC-F scale) and handgrip strength test. Patients who were hospitalized for >48 hours were excluded. RESULTS: The sample included 619 patients with a mean ± SD age of 76.0 ± 14.8 years of which 50.6% were women. Patients were classified into three groups based on malnutrition: group 1 (MNA-SF 12-14 points) (no risk) included 158 patients, group 2 (MNA-SF 8-12 points) (high risk) included 233 patients, and group 3 (MNA-SF 0-7 points) (malnourished) included 228 patients. Malnourished patients had more dysphagia, significantly lower protein and albumin levels, and significantly higher inflammatory marker levels and pressure ulcers. In-hospital mortality was significantly higher in groups 2 and 3 (p < .00001). The worst outcome (mortality and readmissions or mortality) was more common among malnourished patients (p = .0001). Inflammation, comorbidity, and sarcopenia were most closely associated with negative outcomes. CONCLUSION: Malnutrition upon admission is associated with worse short- and long-term outcomes in internal medicine inpatients. Sarcopenia, multimorbidity, and inflammation-measured by albumin, C-reactive protein, or their ratios-are key risk factors. Early identification of malnutrition and sarcopenia through active screening is important in caring for internal medicine patients.


Subject(s)
Malnutrition , Sarcopenia , Humans , Female , Middle Aged , Aged , Aged, 80 and over , Male , Sarcopenia/epidemiology , Inpatients , Hand Strength , Cross-Sectional Studies , Malnutrition/epidemiology , Malnutrition/diagnosis , Nutritional Status , Nutrition Assessment , C-Reactive Protein , Inflammation , Geriatric Assessment
2.
Andes Pediatr ; 92(1): 104-109, 2021 Feb.
Article in English, Spanish | MEDLINE | ID: mdl-34106190

ABSTRACT

INTRODUCTION: Meckel's diverticulum (MD) is the remnant of the vitelline duct (VD) also called omphalomesente ric duct and it is considered the most frequent gastrointestinal malformation. Most of the cases are asymptomatic and the diagnosis of this type is always a challenge. OBJECTIVE: To describe 3 sympto matic presentations of MD and to discuss its symptoms, signs, and possible diagnostic-therapeutic tools. CLINICAL CASES: Case 1: A six-month-old patient with obstructive bowel syndrome. In explo ratory laparotomy, an MD was identified with a mesodiverticular band causing an internal hernia. Case 2: A three-year-old patient presenting with digestive hemorrhage and severe anemia requiring blood transfusion. Upper gastrointestinal endoscopy did not show bleeding origin. Due to persis tent melena, the patient required a new blood transfusion. An Abdomen/pelvis tomography scan was performed, showing a suspicious image of MD which was confirmed by laparotomy. Case 3: A newborn with prenatal anencephaly and omphalocele diagnosis. In immediate care of the newborn, meconium evacuation from the umbilical defect was noticed. It was managed as ruptured omphalo cele, installing a bowel silo bag. In primary closure, the permeability of the omphalomesenteric duct was confirmed. An intestinal en bloc resection and anastomosis were performed in all 3 cases. The last one developed an anastomosis leakage resulting in a terminal ileostomy. CONCLUSION: MD, frequently asymptomatic, is often overlooked as a differential diagnosis of abdominal emergencies in children. When suspecting DM with gastric ectopic mucosa, Tc-99m pertechnetate scintigraphy should be performed as a diagnostic procedure of choice, according to each case.


Subject(s)
Meckel Diverticulum/diagnosis , Blood Transfusion , Child, Preschool , Diagnosis, Differential , Gastrointestinal Hemorrhage/diagnosis , Gastrointestinal Hemorrhage/therapy , Hernia, Umbilical/therapy , Humans , Infant , Infant, Newborn , Intestinal Obstruction/etiology , Intestinal Obstruction/surgery , Meckel Diverticulum/complications , Meckel Diverticulum/surgery , Photography
3.
J. oral res. (Impresa) ; 9(6): 457-465, dic. 31, 2020. ilus, tab
Article in English | LILACS | ID: biblio-1178939

ABSTRACT

Soft tissue calcifications can indicate the presence of more serious, potentially life-threatening pathologies. Therefore, their study can lead to an early diagnosis of those conditions that have not yet become clinically apparent. Main objective: To determine the prevalence of calcifications in soft tissues of the head and neck in cone beam computed tomography images obtained from the Oral and Maxillofacial Radiology Service at Universidad Andrés Bello (UNAB), Viña del Mar, Chile. Material and Methods: Retrospective, cross-sectional, quantitative study. A total of 288 images of cone beam computed tomography (CBCT) were used. Images were obtained at random from the database of the Oral and Maxillofacial Radiology Service at UNAB, Viña Del Mar, between 2014 and 2019. Results: A prevalence of 59.72% of soft tissue calcifications was obtained. The most prevalent were: tonsilloliths and calcified stylohyoid ligament, accounting for 30.65% and 45.56%, respectively. Conclusion: A high prevalence of soft tissue calcifications was found in a population that has not been studied previously; therefore, it is important that the dentist perform a detailed analysis of the cone beam computed tomography.


Introducción: Las calcificaciones en tejidos blandos pueden indicar patologías más graves, que incluso pueden comprometer la vida. Por lo tanto, investigarlas puede conducir a un diagnóstico temprano de aquellas que aún no se han manifestado clínicamente. Objetivo principal: determinar la prevalencia de calcificaciones en tejidos blandos de cabeza y cuello en tomografía computarizada de haz cónico del Servicio de Radiología Oral y Maxilofacial de la UNAB, Viña del Mar, Chile. Material y Métodos: Estudio retrospectivo, transversal, cuantitativo. Se utilizaron 288 volúmenes de tomografía computarizada de haz cónico (CBCT, por las iniciales en inglés de Cone Beam Computed Tomography), obtenidas al azar, de la base de datos del Servicio de Radiología Oral y Maxilofacial de la Universidad Andrés Bello (UNAB), Viña del Mar entre 2014 y 2019. Resultados: Se obtuvo una prevalencia de 59.72% de calcificaciones en tejidos blandos. Las más prevalentes fueron: tonsilolitos, con un 30,65% y ligamento estilohioídeo calcificado, con un 45,56%. Conclusión: Se encontró una alta prevalencia de calcificaciones en tejidos blandos en una población que no ha sido estudiada previamente, por ello es importante que el odontólogo realice un análisis detallado de la tomografía computarizada de haz cónico.


Subject(s)
Humans , Male , Female , Calcinosis/diagnostic imaging , Cone-Beam Computed Tomography/methods , Head/diagnostic imaging , Neck/diagnostic imaging , Palatine Tonsil/diagnostic imaging , Calcinosis/epidemiology , Chile , Prevalence , Retrospective Studies , Ligaments
4.
Animals (Basel) ; 10(11)2020 Nov 11.
Article in English | MEDLINE | ID: mdl-33187104

ABSTRACT

Kennelled dogs are at risk of suffering chronic stress due to long-term spatial, social and feeding restrictions. Chronic stress may decrease the dogs' capacity to feel pleasure when facing hedonic experiences, modifying their perception for palatable ingredients. However, different abilities to cope with environmental stressors could prevent the onset of anhedonia. Fourteen kennelled Beagle dogs were used to study the acceptability and preference for different dilute sucrose and monosodium glutamate (MSG) solutions. Coping style of animals was previously evaluated through a human approach test (HAT) and classified as close dogs (CD; proactive) or distant dogs (DD; reactive) according to whether or not they approached an unfamiliar human when a feeding opportunity was presented. Consumption results were analysed taking into account the sucrose/MSG concentrations, HAT (CD or DD), age, and weight of the animals. DD presented a lower intake of sucrose (p = 0.041) and MSG (p = 0.069) solutions compared with CD. However, DD exhibited a higher consumption of MSG than CD at its highest concentrations, supporting that their intake depends on solution palatability. Finally, DD did not prefer sucrose or MSG solutions over water at any dilute solution offered. Together, these results suggest that dogs that are categorized as reactive animals could diminish their ability to perceive dilute palatable solutions, reflecting depressive-like behaviours as anhedonia.

5.
Medwave ; 20(9): e8053, 2020 Nov 02.
Article in Spanish | MEDLINE | ID: mdl-33231574

ABSTRACT

Alopecia areata is a common type of non-scarring alo¬pecia. Although the exact pathogenesis remains elusive, alopecia areata is thought to have a multifactorial etiology described as an interplay of genetic predisposition and environmental exposures. In patients with genetic susceptibility, stress, infection, and microtrauma have been documented to decrease immunosuppressive cytokines that generally maintain the hair follicle's immune privilege. There is currently no curative therapy for alopecia areata, although some treatments can induce hair growth in a percentage of patients. It has been postulated that simvastatin reestablishes the immune privilege, and ezetimibe would provide an immunomodulatory and anti-inflammatory effect. We report a case of a 23 years-old woman with alopecia areata successfully treated with simvastatin/ezetimibe.


La alopecia areata es un tipo común de alopecia no cicatricial. Aunque la patogénesis exacta permanece sin dilucidar, se piensa que la alopecia areata tiene una etiología multifactorial en donde se interrelacionan predisposición genética y factores ambientales. En pacientes susceptibles, se han documentado que el estrés, infecciones y microtraumas disminuyen las citoquinas inmunosupresoras que normalmente mantienen el privilegio inmune del folículo piloso. Actualmente no hay terapia curativa para la alopecia areata, aunque ciertos tratamientos pueden inducir el crecimiento del cabello en un porcentaje de pacientes. Se postula que la simvastatina restablece el privilegio inmune y ezetimibe aportaría un efecto inmunomodulador y antiinflamatorio. Se presenta el caso de una mujer de 23 años con alopecia areata, exitosamente tratada con simvastatina y ezetimibe.


Subject(s)
Alopecia Areata , Ezetimibe , Immunosuppressive Agents , Simvastatin , Adult , Alopecia Areata/drug therapy , Alopecia Areata/genetics , Ezetimibe/therapeutic use , Female , Genetic Predisposition to Disease , Humans , Immunosuppressive Agents/therapeutic use , Simvastatin/therapeutic use , Young Adult
6.
Medwave ; 20(10): e8053, 18 nov. 2020.
Article in Spanish | LILACS | ID: biblio-1145818

ABSTRACT

La alopecia areata es un tipo común de alopecia no cicatricial. Aunque la patogénesis exacta permanece sin dilucidar, se piensa que la alopecia areata tiene una etiología multifactorial en donde se interrelacionan predisposición genética y factores ambientales. En pacientes susceptibles, se han documentado que el estrés, infecciones y microtraumas disminuyen las citoquinas inmunosupresoras que normalmente mantienen el privilegio inmune del folículo piloso. Actualmente no hay terapia curativa para la alopecia areata, aunque ciertos tratamientos pueden inducir el crecimiento del cabello en un porcentaje de pacientes. Se postula que la simvastatina restablece el privilegio inmune y ezetimibe aportaría un efecto inmunomodulador y antiinflamatorio. Se presenta el caso de una mujer de 23 años con alopecia areata, exitosamente tratada con simvastatina y ezetimibe.


Alopecia areata is a common type of non-scarring alo¬pecia. Although the exact pathogenesis remains elusive, alopecia areata is thought to have a multifactorial etiology described as an interplay of genetic predisposition and environmental exposures. In patients with genetic susceptibility, stress, infection, and microtrauma have been documented to decrease immunosuppressive cytokines that generally maintain the hair follicle's immune privilege. There is currently no curative therapy for alopecia areata, although some treatments can induce hair growth in a percentage of patients. It has been postulated that simvastatin reestablishes the immune privilege, and ezetimibe would provide an immunomodulatory and anti-inflammatory effect. We report a case of a 23 years-old woman with alopecia areata successfully treated with simvastatin/ezetimibe.


Subject(s)
Humans , Female , Adult , Young Adult , Simvastatin/therapeutic use , Alopecia Areata/genetics , Alopecia Areata/drug therapy , Ezetimibe/therapeutic use , Immunosuppressive Agents/therapeutic use , Genetic Predisposition to Disease
7.
Nucleic Acids Res ; 48(14): 7991-8005, 2020 08 20.
Article in English | MEDLINE | ID: mdl-32621607

ABSTRACT

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.


Subject(s)
Bacterial Proteins/metabolism , DNA Helicases/metabolism , DNA, Single-Stranded/metabolism , Deoxyribonuclease I/metabolism , Geobacillus stearothermophilus/enzymology , Adenosine Triphosphatases/chemistry , Adenosine Triphosphatases/isolation & purification , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/isolation & purification , DNA , DNA Helicases/chemistry , DNA Helicases/isolation & purification , Deoxyribonuclease I/chemistry , Deoxyribonuclease I/isolation & purification
8.
Nucleic Acids Res ; 48(4): 2013-2025, 2020 02 28.
Article in English | MEDLINE | ID: mdl-31930301

ABSTRACT

The rolling-circle replication is the most common mechanism for the replication of small plasmids carrying antibiotic resistance genes in Gram-positive bacteria. It is initiated by the binding and nicking of double-stranded origin of replication by a replication initiator protein (Rep). Duplex unwinding is then performed by the PcrA helicase, whose processivity is critically promoted by its interaction with Rep. How Rep and PcrA proteins interact to nick and unwind the duplex is not fully understood. Here, we have used magnetic tweezers to monitor PcrA helicase unwinding and its relationship with the nicking activity of Staphylococcus aureus plasmid pT181 initiator RepC. Our results indicate that PcrA is a highly processive helicase prone to stochastic pausing, resulting in average translocation rates of 30 bp s-1, while a typical velocity of 50 bp s-1 is found in the absence of pausing. Single-strand DNA binding protein did not affect PcrA translocation velocity but slightly increased its processivity. Analysis of the degree of DNA supercoiling required for RepC nicking, and the time between RepC nicking and DNA unwinding, suggests that RepC and PcrA form a protein complex on the DNA binding site before nicking. A comprehensive model that rationalizes these findings is presented.


Subject(s)
Bacterial Proteins/genetics , DNA Helicases/genetics , DNA Replication/genetics , Drug Resistance, Bacterial/genetics , DNA Breaks, Single-Stranded/drug effects , DNA-Binding Proteins/genetics , Geobacillus stearothermophilus/drug effects , Geobacillus stearothermophilus/genetics , Geobacillus stearothermophilus/pathogenicity , Plasmids/drug effects , Plasmids/genetics , Protein Binding/genetics , Protein Interaction Domains and Motifs/genetics , Staphylococcus aureus/drug effects , Staphylococcus aureus/genetics , Staphylococcus aureus/pathogenicity , Tetracycline/pharmacology , Trans-Activators/genetics
9.
Int. j. morphol ; 37(4): 1493-1497, Dec. 2019. tab
Article in Spanish | LILACS | ID: biblio-1040159

ABSTRACT

Uno de los principales factores genéticos que influenciarían el rendimiento muscular humano es el gen ACTN3, que codifica la proteína estructural del sarcómero α-actinina-3. El polimorfismo R577X (rs1815739) del gen ACTN3 ha sido asociado con varios indicadores de rendimiento muscular y físico en deportistas y población general, pero este fenómeno ha sido escasamente descrito en poblaciones de Latinoamérica y Chile. Por lo tanto, el objetivo del presente estudio fue describir la frecuencia genotípica y distribución alélica de los genotipos de ACTN3 R577X en deportistas universitarios chilenos. 129 deportistas universitarios chilenos representantes de diferentes selecciones deportivas (halterofilia, balonmano, voleibol, rugby, basquetbol, futbol y futsal) participaron como voluntarios. Los análisis moleculares del polimorfismo R577X del gen ACTN3 fueron realizados mediante reacción en cadena de la polimerasa (PCR) y restricción enzimática (RFLP). La distribución de genotipos del polimorfismo ACTN3 R577X fue RR: 34,8 % (n=45), RX: 50,4 % (n=65), XX: 14,7 % (n=19), y la frecuencia relativa de alelos fue R: 0,601 y X: 0,399. Además, se encontró asociación entre distribución de genotipos (c2= 12,26; 2 gl; p=0,002) y frecuencia relativa de alelos (c2= 11.02; 1 gl; p=0.0009) con el sexo de los participantes. Sin embargo, no hubo asociación al realizar análisis por tipo de deporte practicado. Los hallazgos de la presente investigación sugieren que el polimorfismo R577X del gen ACTN3 está asociado con el sexo en deportistas universitarios chilenos. Además, estos resultados describen de forma inédita la distribución genotípica y frecuencia alélica de esta variante genética en población chilena, mostrando una distribución similar a otros estudios realizados en poblaciones de deportistas en Brasil, Rusia, Estados Unidos y Turquía. No obstante, también muestra diferencias con otras poblaciones generales y de deportistas.


One of the main genetic factors that influence the muscular performance is the gene that encodes the structural protein α-actinin-3 (ACTN3). The R577X polymorphism (rs1815739) of ACTN3 has been associated with indicators of muscle and physical performance in athletes and general population, but this has been scarcely described in the Latin American and Chilean population. Thus, the aim of the present study was to describe the genotypic frequency and allelic distribution of ACTN3 R577X genotypes in college athletes. A total of 129 unrelated Chilean college athletes representing various sport disciplines (weightlifting, handball, volleyball, rugby, basketball, soccer and futsal) were volunteered for the study. ACTN3 R577X gene polymorphism was analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). For the total sample the genotypes distribution for R577X polymorphism was RR: 34.8 % (n=45), RX: 50.4 % (n=65), XX: 14.7 % (n=19), and the relative frequency of alleles was R: 0,601 and X: 0,399. Moreover, an association was found between genotype distribution (c2= 12.26; 2 df; p=0.002) and allele frequencies (c2= 11.02; 1 df; p=0.0009) with the sex of the participants. However, there were no associations when performing analysis by type of sports. These findings suggest that the R577X polymorphism of the ACTN3 gene is associated with sex in Chilean college athletes. Furthermore, these results describe in an unprecedented manner, the genotypic distribution and allelic frequency of this genetic variant in Chilean population, showing a similar distribution to other studies conducted in populations of athletes in Brazil, Russia, the United States and Turkey. However, it also shows differences with other general and athletes populations.


Subject(s)
Humans , Male , Female , Adolescent , Adult , Young Adult , Polymorphism, Genetic , Students , Actins/genetics , Athletes , Universities , Chile , Athletic Performance/physiology
10.
Exp Parasitol ; 200: 42-47, 2019 May.
Article in English | MEDLINE | ID: mdl-30922888

ABSTRACT

In order to develop a method of identification and discrimination of Echinococcus granulosus sensu stricto from faecal samples of dogs infected with taeniid eggs (Echinococcus spp., Taenia spp.), a combined strategy of Polymerase Chain Reaction (PCR) and Restriction Fragments of Lenght Polymorphisms (RFLP) was proposed. Initially, a pair of primers was designed to amplify a fragment of the 12 Subunit of ribosomal RNA gene (12SrRNA) from mitochondrial DNA. The amplified product was digested by SspI restriction enzyme, which in E. granulosus kept the intact fragment of 160 basis pairs (bp), while in Taenia spp. produced two fragments (62 bp and another of 98 bp). The method was tested using positive controls of DNA, in faecal samples experimentally contaminated with eggs of E. granulosus and Taenia spp. and in dogs naturally infected. In all of them, reproducible results were obtained and the primers were specific to amplify only Taeniidae DNA. The sensitivity of the technique was tested, achieving amplification of DNA extractions with a single egg. In conclusion, the technique developed was optimal and easy to identify patent infections by E. granulosus s.s., constituting a possible alternative for epidemiological studies in dogs, especially in endemic areas where this infection occurs.


Subject(s)
Dog Diseases/parasitology , Echinococcosis/veterinary , Echinococcus granulosus/isolation & purification , Feces/parasitology , Taenia/isolation & purification , Taeniasis/veterinary , Animals , Base Sequence , Consensus Sequence , DNA, Helminth/chemistry , Dogs , Echinococcosis/parasitology , Echinococcus granulosus/genetics , Polymerase Chain Reaction/veterinary , Polymorphism, Restriction Fragment Length , Sensitivity and Specificity , Sequence Alignment/veterinary , Taenia/genetics , Taeniasis/parasitology
11.
Nucleic Acids Res ; 46(11): 5704-5716, 2018 06 20.
Article in English | MEDLINE | ID: mdl-29762781

ABSTRACT

In prokaryotes, the centromere is a specialized segment of DNA that promotes the assembly of the segrosome upon binding of the Centromere Binding Protein (CBP). The segrosome structure exposes a specific surface for the interaction of the CBP with the motor protein that mediates DNA movement during cell division. Additionally, the CBP usually controls the transcriptional regulation of the segregation system as a cell cycle checkpoint. Correct segrosome functioning is therefore indispensable for accurate DNA segregation. Here, we combine biochemical reconstruction and structural and biophysical analysis to bring light to the architecture of the segrosome complex in Type III partition systems. We present the particular features of the centromere site, tubC, of the model system encoded in Clostridium botulinum prophage c-st. We find that the split centromere site contains two different iterons involved in the binding and spreading of the CBP, TubR. The resulting nucleoprotein complex consists of a novel double-ring structure that covers part of the predicted promoter. Single molecule data provides a mechanism for the formation of the segrosome structure based on DNA bending and unwinding upon TubR binding.


Subject(s)
Centromere/chemistry , Centromere/ultrastructure , DNA-Binding Proteins/metabolism , Binding Sites , Centromere/metabolism , Clostridium botulinum/genetics , DNA, Bacterial/chemistry , Operon , Promoter Regions, Genetic , Prophages/genetics
12.
J Biol Phys ; 44(2): 225-235, 2018 06.
Article in English | MEDLINE | ID: mdl-29654426

ABSTRACT

We use the nano-dissection capabilities of atomic force microscopy to induce structural alterations on individual virus capsids in liquid milieu. We fracture the protein shells either with single nanoindentations or by increasing the tip-sample interaction force in amplitude modulation dynamic mode. The normal behavior is that these cracks persist in time. However, in very rare occasions they self-recuperate to retrieve apparently unaltered virus particles. In this work, we show the topographical evolution of three of these exceptional events occurring in T7 bacteriophage capsids. Our data show that single nanoindentation produces a local recoverable fracture that corresponds to the deepening of a capsomer. In contrast, imaging in dynamic mode induced cracks that separate the virus morphological subunits. In both cases, the breakage patterns follow intratrimeric loci.


Subject(s)
Bacteriophage T7/metabolism , Microscopy, Atomic Force , Bacteriophage T7/physiology , Biomechanical Phenomena , Capsid/chemistry , Capsid/metabolism , Time Factors , Virion/chemistry , Virion/metabolism
13.
Nucleic Acids Res ; 44(18): 8885-8896, 2016 Oct 14.
Article in English | MEDLINE | ID: mdl-27488190

ABSTRACT

Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5'-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate.


Subject(s)
DNA Breaks, Single-Stranded , DNA Helicases/metabolism , DNA Replication , Trans-Activators/metabolism , DNA Helicases/chemistry , Models, Biological , Plasmids/genetics , Protein Binding , Protein Multimerization , Recombinant Proteins , Trans-Activators/chemistry
14.
Nucleic Acids Res ; 44(6): 2727-41, 2016 Apr 07.
Article in English | MEDLINE | ID: mdl-26762979

ABSTRACT

In bacteria, the repair of double-stranded DNA breaks is modulated by Chi sequences. These are recognised by helicase-nuclease complexes that process DNA ends for homologous recombination. Chi activates recombination by changing the biochemical properties of the helicase-nuclease, transforming it from a destructive exonuclease into a recombination-promoting repair enzyme. This transition is thought to be controlled by the Chi-dependent opening of a molecular latch, which enables part of the DNA substrate to evade degradation beyond Chi. Here, we show that disruption of the latch improves Chi recognition efficiency and stabilizes the interaction of AddAB with Chi, even in mutants that are impaired for Chi binding. Chi recognition elicits a structural change in AddAB that maps to a region of AddB which resembles a helicase domain, and which harbours both the Chi recognition locus and the latch. Mutation of the latch potentiates the change and moderately reduces the duration of a translocation pause at Chi. However, this mutant displays properties of Chi-modified AddAB even in the complete absence of bona fide hotspot sequences. The results are used to develop a model for AddAB regulation in which allosteric communication between Chi binding and latch opening ensures quality control during recombination hotspot recognition.


Subject(s)
Bacillus subtilis/enzymology , Bacterial Proteins/chemistry , DNA Helicases/chemistry , DNA, Bacterial/chemistry , Exodeoxyribonucleases/chemistry , Recombinational DNA Repair , Bacillus subtilis/genetics , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Breaks, Double-Stranded , DNA Helicases/genetics , DNA Helicases/metabolism , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , Exodeoxyribonucleases/genetics , Exodeoxyribonucleases/metabolism , Gene Expression , Models, Molecular , Mutation , Protein Structure, Secondary , Protein Structure, Tertiary
15.
Small ; 11(11): 1273-84, 2015 Mar 18.
Article in English | MEDLINE | ID: mdl-25400244

ABSTRACT

Motor protein functions like adenosine triphosphate (ATP) hydrolysis or translocation along molecular substrates take place at nanometric scales and consequently depend on the amount of available thermal energy. The associated rates can hence be investigated by actively varying the temperature conditions. In this article, a thermally controlled magnetic tweezers (MT) system for single-molecule experiments at up to 40 °C is presented. Its compact thermostat module yields a precision of 0.1 °C and can in principle be tailored to any other surface-coupled microscopy technique, such as tethered particle motion (TPM), nanopore-based sensing of biomolecules, or super-resolution fluorescence imaging. The instrument is used to examine the temperature dependence of translocation along double-stranded (ds)DNA by individual copies of the protein complex AddAB, a helicase-nuclease motor involved in dsDNA break repair. Despite moderately lower mean velocities measured at sub-saturating ATP concentrations, almost identical estimates of the enzymatic reaction barrier (around 21-24 k(B)T) are obtained by comparing results from MT and stopped-flow bulk assays. Single-molecule rates approach ensemble values at optimized chemical energy conditions near the motor, which can withstand opposing loads of up to 14 piconewtons (pN). Having proven its reliability, the temperature-controlled MT described herein will eventually represent a routinely applied method within the toolbox for nano-biotechnology.


Subject(s)
DNA Helicases/chemistry , DNA/chemistry , Magnetics/instrumentation , Micromanipulation/instrumentation , Microscopy/instrumentation , Molecular Probe Techniques/instrumentation , DNA/ultrastructure , DNA Helicases/ultrastructure , Equipment Design , Equipment Failure Analysis , Heating/instrumentation , Protein Binding , Stress, Mechanical , Temperature
16.
DNA Repair (Amst) ; 20: 119-129, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24569169

ABSTRACT

The fate of a cell depends on its ability to repair the many double-stranded DNA breaks (DSBs) that occur during normal metabolism. Improper DSB repair may result in genomic instability, cancer, or other genetic diseases. The repair of a DSB can be initiated by the recognition and resection of a duplex DNA end to form a 3'-terminated single-stranded DNA overhang. This task is carried out by different single-strand exonucleases, endonucleases, and helicases that work in a coordinated manner. This manuscript reviews the different single-molecule approaches that have been employed to characterize the structural features of these molecular machines, as well as the intermediates and products formed during the process of DSB repair. Imaging techniques have unveiled the structural organization of complexes involved in the tethering and recognition of DSBs. In addition to that static picture, single molecule studies on the dynamics of helicase-nuclease complexes responsible for the processive resection of DSBs have provided detailed mechanistic insights into their function.


Subject(s)
DNA Breaks, Double-Stranded , Microscopy, Atomic Force/methods , Microscopy, Electron, Scanning/methods , Recombinational DNA Repair , Animals , Humans , Microscopy, Fluorescence/methods
17.
Proc Natl Acad Sci U S A ; 110(28): E2562-71, 2013 Jul 09.
Article in English | MEDLINE | ID: mdl-23798400

ABSTRACT

Double-stranded DNA break repair by homologous recombination is initiated by resection of free DNA ends to produce a 3'-ssDNA overhang. In bacteria, this reaction is catalyzed by helicase-nuclease complexes such as AddAB in a manner regulated by specific recombination hotspot sequences called Crossover hotspot instigator (Chi). We have used magnetic tweezers to investigate the dynamics of AddAB translocation and hotspot scanning during double-stranded DNA break resection. AddAB was prone to stochastic pausing due to transient recognition of Chi-like sequences, unveiling an antagonistic relationship between DNA translocation and sequence-specific DNA recognition. Pauses at bona fide Chi sequences were longer, were nonexponentially distributed, and resulted in an altered velocity upon restart of translocation downstream of Chi. We propose a model for the recognition of Chi sequences to explain the origin of pausing during failed and successful hotspot recognition.


Subject(s)
DNA Damage , DNA/genetics , Recombination, Genetic
18.
J Am Chem Soc ; 135(1): 122-31, 2013 Jan 09.
Article in English | MEDLINE | ID: mdl-23214411

ABSTRACT

Double-stranded (ds) RNA is the genetic material of a variety of viruses and has been recently recognized as a relevant molecule in cells for its regulatory role. Despite that the elastic response of dsDNA has been thoroughly characterized in recent years in single-molecule stretching experiments, an equivalent study with dsRNA is still lacking. Here, we have engineered long dsRNA molecules for their individual characterization contrasting information with dsDNA molecules of the same sequence. It is known that dsRNA is an A-form molecule unlike dsDNA, which exhibits B-form in physiological conditions. These structural types are distinguished at the single-molecule level with atomic force microscopy (AFM) and are the basis to understand their different elastic response. Force-extension curves of dsRNA with optical and magnetic tweezers manifest two main regimes of elasticity, an entropic regime whose end is marked by the A-form contour-length and an intrinsic regime that ends in a low-cooperative overstretching transition in which the molecule extends to 1.7 times its A-form contour-length. DsRNA does not switch between the A and B conformations in the presence of force. Finally, dsRNA presents both a lower stretch modulus and overstretching transition force than dsDNA, whereas the electrostatic and intrinsic contributions to the persistence length are larger.


Subject(s)
DNA/chemistry , RNA/chemistry , Microscopy, Atomic Force
19.
Proc Natl Acad Sci U S A ; 109(30): 12028-33, 2012 Jul 24.
Article in English | MEDLINE | ID: mdl-22797893

ABSTRACT

In this study we test the hypothesis that mechanically elastic regions in a virus particle (or large biomolecular complex) must coincide with conformationally dynamic regions, because both properties are intrinsically correlated. Hypothesis-derived predictions were subjected to verification by using 19 variants of the minute virus of mice capsid. The structural modifications in these variants reduced, preserved, or restored the conformational dynamism of regions surrounding capsid pores that are involved in molecular translocation events required for virus infectivity. The mechanical elasticity of the modified capsids was analyzed by atomic force microscopy, and the results corroborated every prediction tested: Any mutation (or chemical cross-linking) that impaired a conformational rearrangement of the pore regions increased their mechanical stiffness. On the contrary, any mutation that preserved the dynamics of the pore regions also preserved their elasticity. Moreover, any pseudo-reversion that restored the dynamics of the pore regions (lost through previous mutation) also restored their elasticity. Finally, no correlation was observed between dynamics of the pore regions and mechanical elasticity of other capsid regions. This study (i) corroborates the hypothesis that local mechanical elasticity and conformational dynamics in a viral particle are intrinsically correlated; (ii) proposes that determination by atomic force microscopy of local mechanical elasticity, combined with mutational analysis, may be used to identify and study conformationally dynamic regions in virus particles and large biomolecular complexes; (iii) supports a connection between mechanical properties and biological function in a virus; (iv) shows that viral capsids can be greatly stiffened by protein engineering for nanotechnological applications.


Subject(s)
Capsid Proteins/chemistry , Elasticity , Minute Virus of Mice , Models, Molecular , Protein Conformation , Virion/chemistry , Capsid Proteins/ultrastructure , Microscopy, Atomic Force , Microscopy, Scanning Probe , Mutagenesis, Site-Directed , Nanotechnology/methods , Plasmids/genetics , Protein Engineering/methods , Spectrometry, Fluorescence , Thermodynamics , Virion/ultrastructure
20.
PLoS One ; 7(1): e30204, 2012.
Article in English | MEDLINE | ID: mdl-22295076

ABSTRACT

Structural Biology (SB) techniques are particularly successful in solving virus structures. Taking advantage of the symmetries, a heavy averaging on the data of a large number of specimens, results in an accurate determination of the structure of the sample. However, these techniques do not provide true single molecule information of viruses in physiological conditions. To answer many fundamental questions about the quickly expanding physical virology it is important to develop techniques with the capability to reach nanometer scale resolution on both structure and physical properties of individual molecules in physiological conditions. Atomic force microscopy (AFM) fulfills these requirements providing images of individual virus particles under physiological conditions, along with the characterization of a variety of properties including local adhesion and elasticity. Using conventional AFM modes is easy to obtain molecular resolved images on flat samples, such as the purple membrane, or large viruses as the Giant Mimivirus. On the contrary, small virus particles (25-50 nm) cannot be easily imaged. In this work we present Frequency Modulation atomic force microscopy (FM-AFM) working in physiological conditions as an accurate and powerful technique to study virus particles. Our interpretation of the so called "dissipation channel" in terms of mechanical properties allows us to provide maps where the local stiffness of the virus particles are resolved with nanometer resolution. FM-AFM can be considered as a non invasive technique since, as we demonstrate in our experiments, we are able to sense forces down to 20 pN. The methodology reported here is of general interest since it can be applied to a large number of biological samples. In particular, the importance of mechanical interactions is a hot topic in different aspects of biotechnology ranging from protein folding to stem cells differentiation where conventional AFM modes are already being used.


Subject(s)
Mechanical Phenomena , Microscopy, Atomic Force/methods , Nanostructures , Parvovirus , Aluminum Silicates/chemistry , Animals , Biomechanical Phenomena , Mice , Surface Properties , Virion
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