High prevalence of dengue, Zika, and chikungunya viruses in blood donors during a dengue outbreak and an endemic period in Colombia.

Objective: Arboviruses pose a challenge in ensuring the supply of pathogen-free blood components because they are not routinely screened in blood banks, and blood components from infected asymptomatic donors could be transfused. This study aimed to detect and characterize arboviral infections in Colombian blood donors. Methods: In a cross-sectional study, the prevalence of dengue (DENV), Zika (ZIKV), and chikungunya (CHIKV) viruses and co-infections of blood donors were compared between an epidemic period (November 2019-February 2020, n = 462) and an endemic period (November 2021-August 2022, n = 1,119). Viral RNA from each donor serum was purified, and the viruses were detected using a previously standardized multiplex hemi-nested RT-PCR protocol. Subsequently, donors who tested positive were surveyed 15 days after the detection of the virus to identify clinical characteristics related to the arboviral infection. The prevalences of each virus were presented as percentages and compared between epidemic and endemic periods. Results: Significantly higher prevalences were found in the epidemic period compared with the endemic period for DENV (14.5 vs. 1.9%), ZIKV (7.8 vs. 0.3%), CHIKV (8 vs. 3.3%), and co-infections (4.3 vs. 0.2%). The survey response rate of positive donors in the two periods was 83/175 (47%). In total, 57% of the donors surveyed were asymptomatic. Symptomatic donors most frequently reported headache (31%), malaise (13%), arthralgia (10%), and fever/chills (8%). Conclusion: The prevalence observed in epidemic and endemic periods was higher than that reported in other studies in the Americas. The high proportion of asymptomatic cases found, in addition to the mild and nonspecific manifestations among the symptomatic, may limit the effectiveness of the donor selection criteria used to mitigate the risk of transfusion-transmitted arboviruses.

Development and Optimization of a Multiplex Real-Time RT-PCR to Detect SARS-CoV-2 in Human Samples.

PCR and its variants (RT-PCR and qRT-PCR) are valuable and innovative molecular techniques for studying nucleic acids. qPCR has proven to be highly sensitive, efficient, and reproducible, generating reliable results that are easy to analyze. During the COVID-19 pandemic, qPCR became the gold standard technique for detecting the SARS-CoV-2 virus that allowed to confirm the infection event, and those asymptomatic ones, and thus save millions of lives. In-house multiplex qPCR tests were developed worldwide to detect different viral targets and ensure results, follow the infections, and favor the containment of a pandemic. Here, we present the detailed fundamentals of the qPCR technique based on fluorogenic probes and processes to develop and optimize a successful multiplex RT-qPCR test for detecting SARS-CoV-2 that could be used to diagnose COVID-19 accurately.

A strategy for entomo-virological surveillance of yellow fever, dengue, zika, and chikungunya viruses in field-collected mosquitoes.

Arboviruses transmitted by Culicidae insects are significant threats to human health, presenting dynamic transmission cycles and involving different vectors and hosts. The surveillance and characterization of the vectors involved in these cycles are crucial for understanding and preventing potential outbreaks. Therefore, we propose a strategy that we used for entomological surveillance of urban, rural, and sylvatic mosquitoes and to characterize natural infection by four major arboviruses.•Immature and adult mosquitoes were collected intra, peri and extradomicilie of urban and rural households, using different collection methodologies.•Mosquitoes were pooled or separated in head-thorax and abdomen, according to the species.•A multiplex nested RT-PCR (Reverse transcription polymerase chain reaction) method was used for the simultaneous detection of dengue virus (DENV), zika virus (ZIKV), chikungunya virus (CHIKV), and yellow fever virus (YFV).Overall, this strategy proved helpful for vectors surveillance at different ecosystems, as well as for implementing a low-cost molecular surveillance system that allows the early detection of potential outbreaks, and identify other potential vectors involved in viral transmission.

Deubiquitinating Enzyme Inhibitors Block Chikungunya Virus Replication.

Ubiquitination and deubiquitination processes are widely involved in modulating the function, activity, localization, and stability of multiple cellular proteins regulating almost every aspect of cellular function. Several virus families have been shown to exploit the cellular ubiquitin-conjugating system to achieve a productive infection: enter the cell, promote genome replication, or assemble and release viral progeny. In this study, we analyzed the role of deubiquitinating enzymes (DUBs) during chikungunya virus (CHIKV) infection. HEK293T, Vero-E6, and Huh-7 cells were treated with two DUB inhibitors (PR619 or WP1130). Then, infected cells were evaluated by flow cytometry, and viral progeny was quantified using the plaque assay method. The changes in viral proteins and viral RNA were analyzed using Western blotting and RT-qPCR, respectively. Results indicate that treatment with DUB inhibitors impairs CHIKV replication due to significant protein and viral RNA synthesis deregulation. Therefore, DUB activity may be a pharmacological target for blocking CHIKV infection.

Performance of Invasive Mode in Different Heated Humidification Systems With High-Flow Nasal Cannula.

BACKGROUND: Oxygen therapy via high-flow nasal cannula generates physiologic changes that impact ventilatory variables of patients. However, we know that there are detrimental effects on airway mucosa related to inhalation of gases. The objective of this study was to evaluate the performance in terms of absolute humidity, relative humidity, and temperature of different brands of heated humidifiers and circuits in the invasive mode during the use of high-flow oxygen therapy in flows between 30 and 100 L/min. METHODS: A prospective observational study conducted at the Sanatorio Anchorena equipment analysis laboratory; September 5 to October 20, 2019. RESULTS: A statistically significant interaction was found among the programmed flows and the different combinations of devices and circuits for the delivery of absolute humidity (P < .001). An effect of flow on delivered absolute humidity was found, regardless of the equipment and circuit combination (P < .001). However, in the invasive mode, the combination of the Fisher&Paykel MR850 heated humidifier with the Medtronic-Dar circuit, the Intersurgical circuit, and the AquaVENT circuit always reached or achieved absolute humidity values > 33 mg/L, even at flows up to 100 L/min. The combination of the Flexicare FL9000 heated humidifier with the Fisher&Paykel RT202 circuit, the Fisher&Paykel Evaqua 2 circuit, the Flexicare circuit, the AquaVENT circuit, and the GGM circuit achieved similar results. The mean (SD) of absolute humidity delivered in the invasive mode (36.2 ± 5.9 mg/L) was higher compared with the noninvasive mode (26.8 ± 7.2 mg/L) (P < .001), regardless of circuit and programmed flows. CONCLUSIONS: When heated humidifiers were used in the invasive mode for high-flow oxygen therapy, absolute humidity depended not only on the heated humidifiers and the combination of circuits but also on the programmed flow, especially at flows > 50 L/min. Moreover, the heated humidifiers exhibited different behaviors, in some cases inefficient, in delivering adequate humidification. However, some equipment improved performance when set to the invasive mode.

[Predictors of weaning and mortality in prolonged mechanical ventilation ­ surviving survival]. / Predictores de desvinculación y mortalidad en ventilación mecánica prolongada-Sobrevivir al sobrevivir.

Objetive: to describe a sample of tracheostomized patients requiring prolonged mechanical ventilation who were admitted to a weaning center, to analyze their evolution and to determine the predictors of weaning failure from mechanical ventilation and of mortality. Design: Design: an observational and retrospective cohort study was carried out, in the period between January 2005 and December 2017. Setting: weaning and rehabilitation center. Patients: all tracheostomized patients requiring mechanical ventilation who were admitted during the study period were included. Of 1027 patients admitted to the respiratory care service, a consecutive sample of 677 patients was analyzed. Main variables of interest: the main variables were mortality and weaning failure from mechanical ventilation. Results: : age older than 70 years (OR 1,461 95% CI 1,016-2,099), a cardiovascular history (OR 1,533 95% CI 1,050-2,237), admission due to respiratory disease (OR 1,538 95% CI 1,001-2,364) and presenting more than 105 days of hospitalization (OR 0,408 95% CI 0,261-0,637) were found as predictors of weaning failure. The predictors of mortality were, age over 70 years (OR 2,116 95% CI 1,491-3,004), history of cerebrovascular accident (OR 1,991 95% CI 1,255-3,158), admission to intensive care due to cardiorespiratory arrest (OR 5,821 95% CI 2,193-15,445) and presenting more than 64 days of hospitalization (OR 1, 63 95% CI 1,116-2,292). Conclusions: The data found in this study manage to describe factors associated with weaning and mortality of patients in a weaning and rehabilitation center.

Objetivos: describir una muestra de pacientes traqueostomizados con requerimiento de ventilación mecánica prolongada que ingresaron a un centro de desvinculación, analizar su evolución y determinar los predictores de fracaso de desvinculación de la ventilación mecánica y de mortalidad. Diseño: se realizó un estudio de cohorte observacional y retrospectivo, en el período comprendido entre enero del 2005 y diciembre del 2017. Ámbito: centro de desvinculación de la ventilación mecánica y de rehabilitación. Pacientes: se incluyeron a todos los pacientes traqueostomizados con requerimiento de ventilación mecánica, que ingresaron durante el periodo de estudio. De 1027 pacientes ingresados al servicio de cuidados respiratorios, se analizó una muestra consecutiva de 677 pacientes. Variables de interés principales: las variables principales fueron mortalidad y falla en la desvinculación de la ventilación mecánica. Resultados: Se encontraron como predictores del fracaso de la desvinculación la edad mayor a 70 años (OR 1.461 IC95% 1.016-2.099), tener antecedentes cardiovasculares (OR 1.533 IC95% 1.050-2.237), motivo de ingreso por afección respiratoria (OR 1.538 IC95% 1.001-2.364) y presentar más de 105 días de internación (OR 0.408 IC95% 0.261-0.637). Los predictores de mortalidad fueron, la edad mayor a 70 años (OR 2.116 IC95% 1.491-3.004), tener como antecedente un accidente cerebro-vascular (OR 1.991 IC95% 1.255-3.158), motivo de ingreso a terapia intensiva por paro cardiorrespiratorio (OR 5.821 IC95% 2.193-15.445) y presentar más de 64 días de internación (OR1.63 IC95% 1.116-2.292). Conclusión: Los datos hallados en este estudio logran describir factores asociados a la desvinculación y mortalidad de los pacientes en un centro de desvinculación.

Molecular and biological characterization of an Asian-American isolate of Chikungunya virus.

Chikungunya virus is an arthropod-transmitted virus that causes chikungunya fever, a disease characterized by severe muscle and joint pain. In 2013, the virus was introduced to the Americas and caused approximately 2.7 million cases of infection during the subsequent two years. The lack of knowledge regarding the biological behavior of the viral strains circulating during the outbreak motivated the characterization of an isolate from the Colombian outbreak, starting from analysis of the complete genome to the biological behavior in vitro. The full genome was retrieved using next-generation sequencing. The infective and replicative capacities were evaluated in HEK293T, Huh-7, and MRC-5 cell lines. The infection rates were determined by flow cytometry, and the cytopathic effect was assessed by a resazurin fluorescent metabolic assay. The viral yield was quantified using the virus plaque formation assay, while the viral proteins and genomic RNA kinetics were subsequently evaluated by western-blot and RT-qPCR. The COL7624 isolate clustered with other American and Caribbean sequences in the Asian American lineage. The T669A substitution in E2 protein distinguished it from other Colombian sequences reported in 2014. After 48 h post infection (hpi), the three cell lines analyzed reached infection percentages exceeding 65%, generating a high load of infectious viral progeny. The infection kinetics indicated that the replication peak of this CHIKV isolate is around 24 hpi, although gRNA is detectable in the culture supernatant from 4 hpi onwards. The infection caused the overexpression of interferon and pro-inflammatory cytokines, such as IL-1ß, TNF-α, and IL-8. The COL7624 CHIKV isolate exhibited a high infective and replicative capacity as well as activation of cellular immune responses, similar to isolates belonging to the other genotypes.

Activity of the Giardia intestinalis proteasome during encystation and its connection with the expression of the cyst wall protein 1 (CWP1).

Giardia is a parasite whose life cycle is composed of two stages: replicative trophozoites, responsible for the symptoms of the disease, and infective cysts, resistant to adverse environments outside of hosts. Proteasomes are multicatalytic peptidase complexes responsible for the specific degradation of proteins in eukaryotic cells. This study assessed the proteasome activity in the trophozoite and during encystation. Strong activation of the proteasome was observed during the differentiation of trophozoites into cysts, reaching its maximum level 24 h after the stimulus. We also found that the Giardia proteasome presents unusual characteristics related to higher eukaryotic proteasomes, making it an eventual therapeutic target. Here we tested the effects on the synthesis of a cyst wall protein by chemical inactivation of the proteasome and by overexpression or partial inhibition of the deubiquitinating protein RPN11 in transfected cells. Moreover, an analysis of the intracellular localization of RPN11 (an integral part of the proteasome regulatory particle) revealed major changes associated with the differentiation of trophozoites into cysts. This evidence further supports the important role of the proteasome in Giardia encystation.

Rediscovering the chikungunya virus / [Reconociendo el virus del chikunguña].

The chikungunya virus (CHIKV) is an Alphavirus that belongs to the Old World group. These arthritogenic viruses cause a febrile illness characterized by arthralgias and myalgias. Although fatal cases during CHIKV infection are rare, the disease may be disabling and generate a broad spectrum of atypical manifestations, such as cardiovascular, respiratory, eye, kidney, and skin complications, among others. When joint pain persists for three or more months, it results in the chronic form of the disease called post-chikungunya chronic inflammatory rheumatism, which constitutes the main disease sequel. CHIKV is not considered a neurotropic virus; however, it can affect the central nervous system, especially in children and the elderly, causing severe and permanent sequelae. CHIKV outbreaks had been previously reported in Africa, Asia, and Europe, but the virus introduction to the American continent was documented until the end of 2013. Since then, the irus has spread to 45 countries and territories causing near two million cases in just two years. This review describes the molecular biology, clinical manifestations, pathogenesis, and significant post-infection complications of CHIKV. Additionally, it collects published information about the outbreak in Colombia and the American continent between 2014 and 2020.

El virus de chikunguña (CHIKV) es un Alfavirus perteneciente al grupo denominado del Viejo Mundo; estos son virus artritogénicos que causan una enfermedad febril caracterizada por artralgias y mialgias. Aunque la muerte por CHIKV es poco frecuente, la enfermedad puede llegar a ser incapacitante y generar un amplio espectro de manifestaciones atípicas, como complicaciones cardiovasculares, respiratorias, oculares, renales y dérmicas, entre otras. Cuando el dolor articular persiste por tres o más meses, da lugar a la forma crónica de la enfermedad denominada reumatismo inflamatorio crónico poschikunguña, el cual es la principal secuela de la enfermedad. Se considera que este virus no es neurotrópico, sin embargo, puede afectar el sistema nervioso central y generar secuelas graves y permanentes, principalmente, en niños y ancianos. En África, Asia y Europa se habían reportado anteriormente brotes epidémicos por CHIKV, pero solo hasta finales del 2013 se documentó la introducción del virus a las Américas; desde entonces, el virus se ha propagado a 45 países o territorios del continente y el número de casos acumulados ascendió a cerca de dos millones en dos años. Esta revisión describe de manera general la biología molecular del virus, sus manifestaciones clínicas, su patogénesis y las principales complicaciones posteriores a la infección. Además, reúne la información de la epidemia en Colombia y el continente americano publicada entre el 2014 y el 2020.

A Case Series of Severe Dengue with Neurological Presentation in Children from a Colombian Hyperendemic Area.

Dengue transmission is sustained in Colombia with increasing prevalence mainly in children. This work aimed to describe a case series of children diagnosed with dengue presenting neurological disease in Huila Province of Colombia. Eleven pediatric febrile patients confirmed for dengue disease and presenting neurological signs were studied in the University Hospital of Neiva, Huila Province. Clinical and laboratory findings, CSF cytochemical analysis, neurology images, and serology and molecular studies were performed. Viral RNA was detected in all patients' sera by RT-PCR. Nine out of 11 were primary infections. Tonic-clonic seizures (73%), consciousness alterations (27%), irritability (27%), and ataxia (18%) were the most frequent neurological signs. None of the patients had plasma leakage, hypovolemic shock, or liver disease, confirming the encephalitis diagnosis. Diagnostic images did not show abnormal findings, but neither bacterial nor fungal infections were detected in CSF analysis. All patients survived without sequelae except for one patient that presented ataxia for months. In conclusion, we described a group of children with neurological signs during severe dengue disease as the main finding, indicating the importance to including dengue as a differential diagnosis in neurological patients from endemic areas.

Reconociendo el virus del chikunguña / Rediscovering the chikungunya virus

Resumen | El virus de chikunguña (CHIKV) es un Alfavirus perteneciente al grupo denominado del Viejo Mundo; estos son virus artritogénicos que causan una enfermedad febril caracterizada por artralgias y mialgias. Aunque la muerte por CHIKV es poco frecuente, la enfermedad puede llegar a ser incapacitante y generar un amplio espectro de manifestaciones atípicas, como complicaciones cardiovasculares, respiratorias, oculares, renales y dérmicas, entre otras. Cuando el dolor articular persiste por tres o más meses, da lugar a la forma crónica de la enfermedad denominada reumatismo inflamatorio crónico poschikunguña, el cual es la principal secuela de la enfermedad. Se considera que este virus no es neurotrópico, sin embargo, puede afectar el sistema nervioso central y generar secuelas graves y permanentes, principalmente, en niños y ancianos. En África, Asia y Europa se habían reportado anteriormente brotes epidémicos por CHIKV, pero solo hasta finales del 2013 se documentó la introducción del virus a las Américas; desde entonces, el virus se ha propagado a 45 países o territorios del continente y el número de casos acumulados ascendió a cerca de dos millones en dos años. Esta revisión describe de manera general la biología molecular del virus, sus manifestaciones clínicas, su patogénesis y las principales complicaciones posteriores a la infección. Además, reúne la información de la epidemia en Colombia y el continente americano publicada entre el 2014 y el 2020.

Abstract | The chikungunya virus (CHIKV) is an Alphavirus that belongs to the Old World group. These arthritogenic viruses cause a febrile illness characterized by arthralgias and myalgias. Although fatal cases during CHIKV infection are rare, the disease may be disabling and generate a broad spectrum of atypical manifestations, such as cardiovascular, respiratory, eye, kidney, and skin complications, among others. When joint pain persists for three or more months, it results in the chronic form of the disease called post-chikungunya chronic inflammatory rheumatism, which constitutes the main disease sequel. CHIKV is not considered a neurotropic virus; however, it can affect the central nervous system, especially in children and the elderly, causing severe and permanent sequelae. CHIKV outbreaks had been previously reported in Africa, Asia, and Europe, but the virus introduction to the American continent was documented until the end of 2013. Since then, the virus has spread to 45 countries and territories causing near two million cases in just two years. This review describes the molecular biology, clinical manifestations, pathogenesis, and significant post-infection complications of CHIKV. Additionally, it collects published information about the outbreak in Colombia and the American continent between 2014 and 2020.

Dengue-chikungunya coinfection outbreak in children from Cali, Colombia in 2018-2019.

OBJECTIVE: To identify the arbovirus involved in febrile cases identified in a pediatric clinic in Cali, Valle del Cauca province, Colombia, and study the clinical characteristics. METHODS: A descriptive, prospective study enrolled 345 febrile children for 12 months in a pediatric clinic. Medical record registers documenting signs and symptoms, and serum samples were analyzed to detect DENV, CHIKV, and ZIKV by reverse transcription-polymerase chain reaction and serology methods. Diagnosis at the time of admission and discharge were compared based on laboratory test results. RESULTS: All patients were diagnosed as severe dengue at admission. Molecular detection and serology tests identified 143 CHIKV-positive (41.4%), 20 DENV-positive (5.8%), and 123 DENV-CHIKV coinfection patients (35.7%). DENV or CHIKV serology test results of these double-infected patients yield poor performance to confirm patient cases. ZIKV infection was detected in 5 patients (1.4%), every time as double or triple infections. CONCLUSION: . A sustained CHIKV circulation and transmission was confirmed causing febrile illness in children and indicating that this virus spreads even during the regular DENV season, leading to double infections and altering clinical symptoms. Specific clinical tests are necessary to closely identify the arbovirus involved in causing infectious diseases that can help in better treatment and mosquito-transmitted virus surveillance.

Performance of Different Active Humidification Systems in High-Flow Oxygen Therapy.

BACKGROUND: We sought to evaluate the performance in terms of absolute humidity (AH), relative humidity (RH), and temperature of different heated humidifiers (HH) and circuits that are commonly used to deliver high-flow oxygen therapy in conventional ranges (30-60 L/min) and unconventional ranges (70-100 L/min). METHODS: In this prospective, observational study, an electronic thermohygrometer was used to obtain the required measurements. A mechanical ventilator was used as a source for high-flow nasal cannula oxygen therapy. For active humidification, the following equipment was used: a HH with standard disposable water trap circuit, 3 servo-controlled HH, and 7 circuits with a heated wire. Data on environmental conditions (ie, temperature, RH, AH) were collected from the laboratory during each measurement; the temperature, RH, and AH resulting from the application of 8 flows (30-100 L/min) were also recorded. Variables were compared with analysis of variance for repeated measurements with Tukey post hoc tests. A value of P < .05 was assumed to be significant. RESULTS: During the study, a statistically significant difference was found in the average AH for each flow for the different devices (P < .005). The highest AH values were recorded with the Fisher & Paykel MR850 and the Medtronic-DAR circuit (AH = 40.8 mg/L with flow of 50 L/min, P < .005), and the lowest AH values were recorded with the Flexicare FL9000 HH and the Flexicare circuit (AH = 11.4 mg/L with 100 L/min flow, P < .005). For flows > 50 L/min, the best performance for all flows in terms of AH was found with the Fisher & Paykel MR850 HH, regardless of the circuit used. CONCLUSIONS: During oxygen therapy with very high gas flows, HH devices behave differently and in many cases are inefficient in delivering adequate humidification, even at conventional flows. Caution is therefore recommended when selecting the device and flow settings for the implementation of high-flow nasal cannula oxygen therapy.

Minimal detectable change and reliability of the star excursion balance test in patients with lateral ankle sprain.

OBJECTIVE: Lateral ankle sprain (LAS) is one of the most prevalent musculoskeletal injuries in the general population and athletes. Dynamic postural control deficits and somatosensory alterations are common signs after an episode of LAS. It is important to detect these deficits to prevent a recurrent sprain and the development of chronic ankle instability. The Star Excursion Balance Test (SEBT) is a tool used to assess dynamic postural control in patients with LAS. SEBT test-retest reliability has been evaluated in several populations. However, no data on patients with LAS are available and we do not know the minimal detectable change (MDC). The primary objective of our study was to obtain the MDC for normalized reach distances of the eight SEBT directions in patients with LAS. The secondary objective was to determine test-retest reliability. METHODS: Cross-sectional study. Thirty-one patients (between 18 and 40 years old) diagnosed with a Grade I or II LAS. Participants were evaluated by two raters at two time-points separated by an interval of 24-72 hrs. In each assessment, four practice trials were allowed, then three test trials were performed in a randomized order. Normalized reaching distances were analyzed. RESULTS: From the initial 31 patients, two were eliminated, so 29 patients were considered for the final analysis. The MDC values obtained were 6.73-13.36%, and the medial and posteromedial directions showed the lowest and highest values, respectively. A statistically significant increase was found in lateral direction between T0 and T1. Intraclass correlation coefficients ranged from 0.72 to 0.93. CONCLUSION: The SEBT is an accurate and reliable tool to assess dynamic postural control in patients with LAS.

A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis.

BACKGROUND: Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE: To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS: We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS: We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the ß-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS: Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

A new gene inventory of the ubiquitin and ubiquitin-like conjugation pathways in Giardia intestinalis

BACKGROUND Ubiquitin (Ub) and Ub-like proteins (Ub-L) are critical regulators of complex cellular processes such as the cell cycle, DNA repair, transcription, chromatin remodeling, signal translation, and protein degradation. Giardia intestinalis possesses an experimentally proven Ub-conjugation system; however, a limited number of enzymes involved in this process were identified using basic local alignment search tool (BLAST). This is due to the limitations of BLAST's ability to identify homologous functional regions when similarity between the sequences dips to < 30%. In addition Ub-Ls and their conjugating enzymes have not been fully elucidated in Giardia. OBJETIVE To identify the enzymes involved in the Ub and Ub-Ls conjugation processes using intelligent systems based on the hidden Markov models (HMMs). METHODS We performed an HMM search of functional Pfam domains found in the key enzymes of these pathways in Giardia's proteome. Each open reading frame identified was analysed by sequence homology, domain architecture, and transcription levels. FINDINGS We identified 118 genes, 106 of which corresponded to the ubiquitination process (Ub, E1, E2, E3, and DUB enzymes). The E3 ligase group was the largest group with 82 members; 71 of which harbored a characteristic RING domain. Four Ub-Ls were identified and the conjugation enzymes for NEDD8 and URM1 were described for first time. The 3D model for Ub-Ls displayed the β-grasp fold typical. Furthermore, our sequence analysis for the corresponding activating enzymes detected the essential motifs required for conjugation. MAIN CONCLUSIONS Our findings highlight the complexity of Giardia's Ub-conjugation system, which is drastically different from that previously reported, and provides evidence for the presence of NEDDylation and URMylation enzymes in the genome and transcriptome of G. intestinalis.

Cross-cultural adaptation and validation of the Argentine "American Shoulder and elbow surgeons, patient self-report section" questionnaire.

BACKGROUND: American Shoulder and Elbow Surgeons questionnaire (ASES-p) has been translated into Spanish, but it has not been adapted to the Argentine population yet. Although Spain and Argentina speak the same language, linguistic differences between Spanish-speaking countries may affect the interpretation of the different items included in the questionnaire. OBJECTIVE: To conduct the translation, cross-cultural adaptation and validation of the self-report section of the ASES-p into Argentine Spanish for patients with musculoskeletal shoulder disorders, and to assess its psychometric properties. DESIGN: Study of diagnostic accuracy/assessment scale. METHOD: The study was carried out in three consecutive phases: translation, cross-cultural adaptation and validation for its use in Argentina. In the third phase, we used the ASES-p, Short Form 36 (SF-36), EuroQol-5D (EQ-5D), and Disabilities of the Arm, Shoulder and Hand (DASH) questionnaires, and the Global Rating of Change (GROC) scale. RESULTS: One hundred three participants completed a set of questionnaires on two occasions and were included in the final analysis. The time taken to answer and score the questionnaire was 118 and 52 s, respectively. Neither a ceiling nor a floor effect was observed. Cronbach's alpha coefficient was 0.85. Intraclass correlation coefficient was 0.83. A significant correlation was found between the DASH, the GROC and various SF-36 subscales. There were strong indices of concurrent-cross validation, longitudinal validity, and construct validity. The ASES-p questionnaire showed a minimal clinically important difference (MCID) value of 7.88 points. CONCLUSION: Some psychometric properties in reliability and validity were acceptable in the Argentine version of the ASES-p questionnaire.

Description of respiratory syncytial virus genotypes circulating in Colombia.

INTRODUCTION: Respiratory syncytial virus (RSV) is the most common cause of acute respiratory infections in children younger than two years but also produces infection in older children and even reinfection in people of any age, a characteristic related to the existence of different infecting subtypes and genotypes. Although Colombia has established the surveillance of classical respiratory viruses, there is no information about the RSV genotypes circulating in Colombian patients. METHODOLOGY: A subgroup of 227 previously RSV positive respiratory secretion samples were taken from a nationwide surveillance study, amplified and sequenced to define the circulation pattern of RSV subtypes and genotypes during 2000-2009 period in Colombia. RESULTS: RSV exhibited seasonal behavior with an A subtype more prevalent. Both RSV subtypes had low nucleotide variability. During the study period, the GA2 and GA5 genotypes from RSV subtype A and the BA genotype from RSV subtype B were found. CONCLUSION: In this report, for the first time RSV genotypes circulating in Colombia were described, this information adds valuable information about virus epidemiology helping to understand the RSV epidemic and prepare our country for the introduction of new vaccines.

Experimental and bioinformatic characterization of CaBP2933 an EF-Hand protein of Giardia intestinalis.

Giardia intestinalis is a parasite that inhabits the small intestine of humans. This parasite is a divergent eukaryote with a compact genome. The calcium ion is an essential messenger in cell signaling. Calcium's role as a messenger is mediated through calcium-binding proteins (CaBPs) that decode the message. The most important family of CaBPs is the EF-Hand protein family. In this study we have explored the role of EF-Hand protein CaBP2933. We analyzed its location, confirmed its ability to bind calcium and identified some of its interacting proteins. Take together our results suggest that CaBP2933 is involved in vesicular trafficking during encystation, via an interaction with kinesin-3 motor protein.

Production of recombinant proteins from Plasmodium falciparum in Escherichia coli / Producción de proteínas recombinantes de Plasmodium falciparum en Escherichia coli

Introduction: The production of recombinant proteins is essential for the characterization and functional study of proteins from Plasmodium falciparum . However, the proteins of P . falciparum are among the most challenging to express, and when expression is achieved, the recombinant proteins usually fold incorrectly and lead to the formation of inclusion bodies. Objective: To obtain and purify four recombinant proteins and to use them as antigens to produce polyclonal antibodies. The production efficiency and solubility were evaluated as the proteins were expressed in two genetically modified strains of Escherichia coli to favor the production of heterologous proteins (BL21-CodonPlus (DE3)-RIL and BL21-pG-KJE8). Materials and methods: The four recombinant P. falciparum proteins corresponding to partial sequences of PfMyoA (Myosin A) and PfGAP50 (gliding associated protein 50), and the complete sequences of PfMTIP (myosin tail interacting protein) and PfGAP45 (gliding associated protein 45), were produced as glutathione S-transferase-fusion proteins, purified and used for immunizing mice. Results: The protein expression was much more efficient in BL21-CodonPlus, the strain that contains tRNAs that are rare in wild-type E. coli , compared to the expression in BL21-pG-KJE8. In spite of the fact that BL21-pG-KJE8 overexpresses chaperones, this strain did not minimize the formation of inclusion bodies. Conclusion: The use of genetically modified strains of E . coli was essential to achieve high expression levels of the four evaluated P . falciparum proteins and lead to improved solubility of two of them. The approach used here allowed us to obtain and purify four P . falciparum proteins in enough quantity to produce polyclonal antibodies in mice, and a fair amount of two pure and soluble recombinant proteins for future assays.

Introducción. La producción de proteínas recombinantes es fundamental para el estudio funcional de las proteínas de Plasmodium falciparum . Sin embargo, las proteínas recombinantes de P . falciparum están entre las más difíciles de expresar y, cuando lo hacen, usualmente se agregan dentro de cuerpos de inclusión insolubles. Objetivo. Evaluar la producción de cuatro proteínas de P. falciparum usando como sistema de expresión dos cepas de Escherichia coli genéticamente modificadas para favorecer la producción de proteínas heterólogas y establecer una reserva de proteínas recombinantes puras y solubles, y producir anticuerpos policlonales a partir de ellas. Materiales y métodos. Las proteínas recombinantes, las cuales correspondían a secuencias parciales de PfMyoA (Miosina-A) y PfGAP50 (proteína-asociada a glideosoma de 50 kDa) y a las secuencias completas de PfMTIP (proteína de interacción con miosina-A) y PfGAP45 (proteína asociada a glideosoma de 45 kDa), fueron expresadas como proteínas de fusión con la glutatión S-transferasa y luego purificadas y usadas para producir anticuerpos policlonales en ratón. Resultados. La expresión de las proteínas recombinantes fue mucho más eficiente en la cepa BL21-CodonPlus (la cual expresa tRNAs escasos en las bacterias silvestres), que en la cepa BL21-pG-KJE8. Por el contrario, aunque la cepa BL21-pG-KJE sobreexpresa chaperonas, no redujo la formación de cuerpos de inclusión. Conclusión. El uso de cepas de E . coli genéticamente modificadas fue fundamental para alcanzar altos niveles de expresión de las cuatro proteínas recombinantes evaluadas y permitió obtener dos de ellas en forma soluble. La estrategia utilizada permitió expresar cuatro proteínas recombinantes de P . falciparum en cantidad suficiente para inmunizar ratones y producir anticuerpos policlonales y, además, conservar proteína pura y soluble de dos de ellas para ensayos futuros.