Regulatory effects of microRNAs on monocytic HLA-DR surface expression.

Decreased monocytic HLA-DR expression is the most studied biomarker of immune competency in critically ill and autoimmune disease patients. However, the underlying regulatory mechanisms remain largely unknown. One probable HLA-DR dysregulation is through microRNAs. The aim of this study was to investigate the effects of specific microRNAs on HLA-DR expression in human monocytic cells. Four up- and four down-HLA-DR-regulating microRNAs were identified, with hsa-miR-let-7f-2-3p showing the most significant upregulation and hsa-miR-567 and hsa-miR-3972 downregulation. Anti-inflammatory glucocorticoid medication Dexamethasone-decreased HLA-DR was significantly restored by hsa-miR-let-7f-2-3p and hsa-miR-5693. Contrarily, proinflammatory cytokines IFN-γ and TNF-α-increased HLA-DR were significantly reversed by hsa-miR-567. Clinically, paired plasma samples from patients before and one day after cardiac surgery revealed up-regulated expression of hsa-miR-5693, hsa-miR-567, and hsa-miR-3972, following the major surgical trauma. In silico approaches were applied for functional microRNA-mRNA interaction prediction and candidate target genes were confirmed by qPCR analysis. In conclusion, novel monocytic HLA-DR microRNA modulators were identified and validated in vitro. Moreover, both the interaction between the microRNAs and anti- and proinflammatory molecules and the up-regulated microRNAs identified in cardiac surgery highlight the potential clinical relevance of our findings.

Scrofuloderma of the chest with mediastinal TB.

Cutaneous tuberculosis is a rare manifestation of extrapulmonary tuberculosis caused by Mycobacterium tuberculosis in most cases and rarely by Mycobacterium bovis. Diagnosis may be challenging due to a wide range of clinical findings and similarities to other chronic dermatoses, leading to delayed treatment. We present a case of scrofuloderma in a 4-year-old girl that arose from a contiguous spread from the anterior mediastinum with associated pulmonary involvement.

Characterization of two novel proteins involved in mitochondrial DNA anchoring in Trypanosoma brucei.

Trypanosoma brucei is a single celled eukaryotic parasite in the group of the Kinetoplastea. The parasite harbors a single mitochondrion with a singular mitochondrial genome that is known as the kinetoplast DNA (kDNA). The kDNA consists of a unique network of thousands of interlocked circular DNA molecules. To ensure proper inheritance of the kDNA to the daughter cells, the genome is physically linked to the basal body, the master organizer of the cell cycle in trypanosomes. The connection that spans, cytoplasm, mitochondrial membranes and the mitochondrial matrix is mediated by the Tripartite Attachment Complex (TAC). Using a combination of proteomics and RNAi we test the current model of hierarchical TAC assembly and identify TbmtHMG44 and TbKAP68 as novel candidates of a complex that connects the TAC to the kDNA. Depletion of TbmtHMG44 or TbKAP68 each leads to a strong kDNA loss but not missegregation phenotype as previously defined for TAC components. We demonstrate that the proteins rely on both the TAC and the kDNA for stable localization to the interface between these two structures. In vitro experiments suggest a direct interaction between TbmtHMG44 and TbKAP68 and that recombinant TbKAP68 is a DNA binding protein. We thus propose that TbmtHMG44 and TbKAP68 are part of a distinct complex connecting the kDNA to the TAC.

High-risk twin pregnancy: case report of an adolescent patient with cystic fibrosis and systemic lupus erythematosus.

BACKGROUND: We present the first case to our knowledge of a spontaneous twin pregnancy in a 16-year-old Caucasian patient with cystic fibrosis and systemic lupus erythematosus. Cystic fibrosis is one of the most common autosomal recessive genetic disorders and primarily affects the respiratory and digestive systems. Systemic lupus erythematosus is a chronic inflammatory disease of unknown cause that affects nearly every organ. Patients with cystic fibrosis or systemic lupus erythematosus are progressively having longer life expectancy and better quality of life, which has led a greater number of female patients reporting the desire to become mothers. CASE PRESENTATION: We present a case of a Caucasian 16-year-old pregnant with twins being treated for both cystic fibrosis and systemic lupus erythematosus. She has two CFTR mutations: p.F508del and 1812_1G>A. In the second trimester, she was admitted for possible preterm labor, which was successfully stopped. The patient's nutritional status worsened, and she had a pulmonary exacerbation as well as a flare of systemic lupus erythematosus. At the 28th gestational week, she presented with a massive hemoptysis episode. The cesarean delivery had no complications, and there were no serious immediate postpartum complications. DISCUSSION AND CONCLUSIONS: While adolescent pregnancies in and of themselves are considered high risk for both the young mothers and their children, they are further complicated when the mother has two chronic diseases and a twin pregnancy. We achieved positive results using a multidisciplinary approach; however, the risks involved were so high that major efforts are to be taken by our medical community to prevent unplanned pregnancies in all patients with cystic fibrosis, especially when a serious comorbidity like the one in this case is present.

Flow cytometry-based high-throughput RNAi screening for miRNAs regulating MHC class II HLA-DR surface expression.

HLA-DR isotype is a MHC-II cell-surface receptor found on APCs and plays a key role in initiating immune responses. In severely immunocompromised patients with conditions like sepsis, the number of HLA-DR molecules expressed on leukocytes is considered to correlate with infectious complications and patients' probability of survival. The underlying regulatory mechanisms of HLA-DR expression remain largely unknown. One probable path to regulation is through microRNAs (miRNAs), which have been implicated as regulatory elements of both innate and adaptive immune system development and function. In our study, flow cytometry-based high-throughput miRNA screening was performed in a stable HLA-DR-expressing human melanoma cell line, MelJuSo, for either up- or downregulating miRNAs of the surface HLA-DR expression. By the end of the screening, the top ten upregulators and top five downregulators were identified, and both the HLA-DR protein and mRNA regulations were further verified and validated. In-silico approaches were applied for functional miRNA-mRNA interaction prediction. The potential underlying gene regulations of different miRNAs were proposed. Our results promote the study of miRNA-mediated HLA-DR regulation under both physiological and pathological conditions, and may pave the way for potential clinical applications.

Molecular model of the mitochondrial genome segregation machinery in Trypanosoma brucei.

In almost all eukaryotes, mitochondria maintain their own genome. Despite the discovery more than 50 y ago, still very little is known about how the genome is correctly segregated during cell division. The protozoan parasite Trypanosoma brucei contains a single mitochondrion with a singular genome, the kinetoplast DNA (kDNA). Electron microscopy studies revealed the tripartite attachment complex (TAC) to physically connect the kDNA to the basal body of the flagellum and to ensure correct segregation of the mitochondrial genome via the basal bodies movement, during the cell cycle. Using superresolution microscopy, we precisely localize each of the currently known TAC components. We demonstrate that the TAC is assembled in a hierarchical order from the base of the flagellum toward the mitochondrial genome and that the assembly is not dependent on the kDNA itself. Based on the biochemical analysis, the TAC consists of several nonoverlapping subcomplexes, suggesting an overall size of the TAC exceeding 2.8 mDa. We furthermore demonstrate that the TAC is required for correct mitochondrial organelle positioning but not for organelle biogenesis or segregation.

The nuclear RNA binding protein RBP33 influences mRNA and spliced leader RNA abundance in Trypanosoma brucei.

RNA recognition motif (RRM) containing proteins are important regulators of gene expression in trypanosomes. Here we expand our current knowledge on the exclusively nuclear localized RRM domain containing protein RBP33 of Trypanosoma brucei. Overexpression of RBP33 leads to a quick growth arrest in G2/M in bloodstream form cells likely due to an overall mRNA- and spliced leader abundance decrease while the ribosomal RNAs remain unaffected. The recombinant RBP33 binds to poly(A) and random sequence RNA in vitro confirming its role as a RNA binding protein. Finally super-resolution microscopy detects RBP33 in small punctae throughout the nucleus and surrounding the nucleolus, however the signal is depleted inside the nucleolus.

Mitochondrial growth during the cell cycle of Trypanosoma brucei bloodstream forms.

Mitochondrial organelles need to be replicated during cell division. Many aspects of this process have been studied in great detail, however the actual size increase and the position of organelle growth are less well understood. We use the protozoan parasite Trypanosoma brucei that contains a single mitochondrion to study organelle biogenesis by fluorescence microscopy. From the analysis of more than 1000 T. brucei bloodstream form cells of a nonsynchronous population we conclude that the mitochondrial network mostly grows from two areas along the main organelle axis, posterior and anterior of the nucleus. Loops and branches from these two areas eventually fuse to build a complex network. Together with the appearance of the division fold in the posterior part of the cell, pruning of the mitochondrial network and finally separation into the two daughter cells occurs. Overall organelle biogenesis is not continuous during cell growth and occurs mostly in the last part of the cell cycle. Furthermore, using 3D STED super resolution microscopy we reconstruct the volume of the organelle and characterize the region where the mitochondrial genome is positioned by serial block face scanning electron microscopy.

Correction: TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes.

[This corrects the article DOI: 10.1371/journal.ppat.1005586.].

TAC102 Is a Novel Component of the Mitochondrial Genome Segregation Machinery in Trypanosomes.

Trypanosomes show an intriguing organization of their mitochondrial DNA into a catenated network, the kinetoplast DNA (kDNA). While more than 30 proteins involved in kDNA replication have been described, only few components of kDNA segregation machinery are currently known. Electron microscopy studies identified a high-order structure, the tripartite attachment complex (TAC), linking the basal body of the flagellum via the mitochondrial membranes to the kDNA. Here we describe TAC102, a novel core component of the TAC, which is essential for proper kDNA segregation during cell division. Loss of TAC102 leads to mitochondrial genome missegregation but has no impact on proper organelle biogenesis and segregation. The protein is present throughout the cell cycle and is assembled into the newly developing TAC only after the pro-basal body has matured indicating a hierarchy in the assembly process. Furthermore, we provide evidence that the TAC is replicated de novo rather than using a semi-conservative mechanism. Lastly, we demonstrate that TAC102 lacks an N-terminal mitochondrial targeting sequence and requires sequences in the C-terminal part of the protein for its proper localization.

A novel component of the mitochondrial genome segregation machinery in trypanosomes.

We recently described a new component (TAC102) of the mitochondrial genome segregation machinery (mtGSM) in the protozoan parasite Trypanosoma brucei. T. brucei belongs to a group of organisms that contain a single mitochondrial organelle with a single mitochondrial genome (mt-genome) per cell. The mt-genome consists of 5000 minicircles (1 kb) and 25 maxicircles (23 kb) that are catenated into a large network. After replication of the network its segregation is driven by the separating basal bodies, which are homologous structures to the centrioles organizing the spindle apparatus in many eukaryotes. The structure connecting the basal body to the mt-genome was named the Tripartite Attachment Complex (TAC) owing its name to the distribution across three areas in the cell including the two mitochondrial membranes.

Sleep-disordered breathing in patients with cystic fibrosis.

OBJECTIVE: To test the hypothesis that disease severity in patients with cystic fibrosis (CF) is correlated with an increased risk of sleep apnea. METHODS: A total of 34 CF patients underwent clinical and functional evaluation, as well as portable polysomnography, spirometry, and determination of IL-1ß levels. RESULTS: Mean apnea-hypopnea index (AHI), SpO2 on room air, and Epworth Sleepiness Scale score were 4.8 ± 2.6, 95.9 ± 1.9%, and 7.6 ± 3.8 points, respectively. Of the 34 patients, 19 were well-nourished, 6 were at nutritional risk, and 9 were malnourished. In the multivariate model to predict the AHI, the following variables remained significant: nutritional status (ß = -0.386; p = 0.014); SpO2 (ß = -0.453; p = 0.005), and the Epworth Sleepiness Scale score (ß = 0.429; p = 0.006). The model explained 51% of the variation in the AHI. CONCLUSIONS: The major determinants of sleep apnea were nutritional status, SpO2, and daytime sleepiness. This knowledge not only provides an opportunity to define the clinical risk of having sleep apnea but also creates an avenue for the treatment and prevention of the disease.

Sleep-disordered breathing in patients with cystic fibrosis / Distúrbios respiratórios do sono em pacientes com fibrose cística

AbstractObjective: To test the hypothesis that disease severity in patients with cystic fibrosis (CF) is correlated with an increased risk of sleep apnea.Methods: A total of 34 CF patients underwent clinical and functional evaluation, as well as portable polysomnography, spirometry, and determination of IL-1β levels.Results: Mean apnea-hypopnea index (AHI), SpO2 on room air, and Epworth Sleepiness Scale score were 4.8 ± 2.6, 95.9 ± 1.9%, and 7.6 ± 3.8 points, respectively. Of the 34 patients, 19 were well-nourished, 6 were at nutritional risk, and 9 were malnourished. In the multivariate model to predict the AHI, the following variables remained significant: nutritional status (β = −0.386; p = 0.014); SpO2 (β = −0.453; p = 0.005), and the Epworth Sleepiness Scale score (β = 0.429; p = 0.006). The model explained 51% of the variation in the AHI.Conclusions: The major determinants of sleep apnea were nutritional status, SpO2, and daytime sleepiness. This knowledge not only provides an opportunity to define the clinical risk of having sleep apnea but also creates an avenue for the treatment and prevention of the disease.

ResumoObjetivo: Testar a hipótese de que a gravidade da doença em pacientes com fibrose cística (FC) correlaciona-se com maior risco de apneia do sono.Métodos: Um total de 34 pacientes com FC foram submetidos a avaliação clínica e funcional, polissonografia portátil, espirometria e dosagem de IL-1β.Resultados: As médias do índice de apneia e hipopneia (IAH), da SpO2 em ar ambiente e da pontuação na Escala de Sonolência de Epworth foram de 4,8 ± 2,6, 95,9 ± 1,9% e 7,6 ± 3,8, respectivamente. Dos 34 pacientes, 19 eram eutróficos, 6 apresentavam risco nutricional e 9 apresentavam desnutrição. No modelo multivariado para prever o IAH, permaneceram significativos o estado nutricional (β = −0,386; p = 0,014), a SpO2 (β = −0,453; p = 0,005) e a pontuação na Escala de Sonolência de Epworth (β = 0,429; p = 0,006). O modelo explicou 51% da variação do IAH.Conclusões: Os maiores determinantes de apneia do sono foram o estado nutricional, a SpO2 e a sonolência diurna. Esse conhecimento representa não somente uma oportunidade de definir o risco clínico de apresentar apneia do sono, mas também de atuar na prevenção e tratamento da doença.

Defining regulatory and phosphoinositide-binding sites in the human WIPI-1 ß-propeller responsible for autophagosomal membrane localization downstream of mTORC1 inhibition.

BACKGROUND: Autophagy is a cytoprotective, lysosomal degradation system regulated upon induced phosphatidylinositol 3-phosphate (PtdIns(3)P) generation by phosphatidylinositol 3-kinase class III (PtdIns3KC3) downstream of mTORC1 inhibition. The human PtdIns(3)P-binding ß-propeller protein WIPI-1 accumulates at the initiation site for autophagosome formation (phagophore), functions upstream of the Atg12 and LC3 conjugation systems, and localizes at both the inner and outer membrane of generated autophagosomes. In addition, to a minor degree WIPI-1 also binds PtdIns(3,5)P2. By homology modelling we earlier identified 24 evolutionarily highly conserved amino acids that cluster at two opposite sites of the open Velcro arranged WIPI-1 ß-propeller. RESULTS: By alanine scanning mutagenesis of 24 conserved residues in human WIPI-1 we define the PtdIns-binding site of human WIPI-1 to critically include S203, S205, G208, T209, R212, R226, R227, G228, S251, T255, H257. These amino acids confer PtdIns(3)P or PtdIns(3,5)P2 binding. In general, WIPI-1 mutants unable to bind PtdIns(3)P/PtdIns(3,5)P2 lost their potential to localize at autophagosomal membranes, but WIPI-1 mutants that retained PtdIns(3)P/PtdIns(3,5)P2 binding localized at Atg12-positive phagophores upon mTORC1 inhibition. Both, downregulation of mTOR by siRNA or cellular PtdIns(3)P elevation upon PIKfyve inhibition by YM201636 significantly increased the localization of WIPI-1 at autophagosomal membranes. Further, we identified regulatory amino acids that influence the membrane recruitment of WIPI-1. Exceptional, WIPI-1 R110A localization at Atg12-positive membranes was independent of autophagy stimulation and insensitive to wortmannin. R112A and H185A mutants were unable to bind PtdIns(3)P/PtdIns(3,5)P2 but localized at autophagosomal membranes, although in a significant reduced number of cells when compared to wild-type WIPI-1. CONCLUSIONS: We identified amino acids of the WIPI-1 ß-propeller that confer PtdIns(3)P or PtdIns(3,5)P2 binding (S203, S205, G208, T209, R212, R226, R227, G228, S251, T255, H257), and that regulate the localization at autophagosomal membranes (R110, R112, H185) downstream of mTORC1 inhibition.

Infecção respiratória na fibrose cística e tratamento / Cystic fibrosis respiratory infection and treatment

Fibrose cística é uma doença multissistêmica e complexa, que exige tratamento contínuo. A doença pulmonar é o principal determinante da sua evolução. Alterações na composição da secreção das vias aéreas levam ao desenvolvimento de doença pulmonar progressiva com infecção respiratória crônica por germes peculiares à doença. Esquemas de antibioticoterapia para tratamento das exacerbações pulmonares, erradicação de bactérias ou de supressão da infecção crônica constituem parte importante do tratamento, com repercussão significativa na melhora dos sintomas, da função pulmonar e da qualidade de vida dos pacientes com fibrose cística.

Cystic fibrosis is a complex multisystemic disease that requires lifelong treatment. Pulmonary disease is the major prognostic determinant. The altered airway surface liquid leads to the development of progressive pulmonary disease with chronic respiratory infection by specific germs. Antibiotic strategies for treatment of pulmonary disease exacerbations, bacterial eradication or chronic infection suppression play an important role in the treatment, contributing to the improvement of symptoms, lung function and the quality of life of Cystic Fibrosis patients.

Evolução do estado nutricional de pacientes com fibrose cística em uso de suplementação nutricional por gastrostomia / Nutritional status of patientes with cystic fibrosis in use of nutritional supplementation by gastrostomy

Introdução: A nutrição tem papel essencial na sobrevida e qualidade de vida de pacientes com Fibrose Cística (FC). O objetivo desse estudo foi acompanhar a evolução do estado nutricional de pacientes com FC em uso de suplementação nutricional por gastrostomia. Métodos: Estudo longitudinal retrospectivo com todos os pacientes com FC e em uso de suplementação nutricional com dieta hipercalórica por gastrostomia em um centro de referência no sul do Brasil. Os pacientes foram acompanhados nos períodos de 6 meses antes, no momento da instalação da gastrostomia, 6 meses, 1 ano e 2 anos após o procedimento. Os parâmetros nutricionais utilizados foram escore Z para peso/idade e estatura/idade, percentil de índice de massa corpórea (PIMC), além de medidas de composição corporal. Foram obtidos dados de função pulmonar e de ingestão alimentar. Resultados: Foram avaliados dez pacientes, sendo sete (70%) do sexo masculino. A idade média da instalação da gastrostomia foi de 9,8 ± 3,8 anos. O ganho de peso foi de 8,35 Kg (P =0,007) e o de estatura de 16,2 cm (P <0,001). O PIMC passou de 14,2 para 27,1 no período de 2 anos e meio (P =0,282). Com relação à composição corporal houve aumento da reserva muscular e de gordura. Houve queda não significativa na função pulmonar ao longo do tempo. A média de calorias ofertadas comparada com a recomendação alimentar diária passou de 111,6 ± 24,6% para 157,7 ± 37,86% (P =0,048). Conclusão: A suplementação nutricional por gastrostomia resultou em significativo ganho de peso e estatura, bem como aumento da reserva muscular e de gordura, no entanto não houve melhora ou estabilização da função pulmonar nesse grupo de pacientes.

Background: Nutrition plays an essential role in the survival and quality of life of cystic fibrosis (CF) patients. The aim was to follow nutritional status of patients with CF in use of nutritional supplementation by gastrostomy. Methods: Retrospective longitudinal study including all patients with CF on nutritional supplementation by gastrostomy at a referral center in southern Brazil. Patients were followed up in periods of 6 months before the installation of gastrostomy, and then, 6 months, 1 year and 2 years after the procedure. The nutritional parameters used were Z scores for weight/age and height/age, percentile of body mass index (PBMI), and body composition measurements. Data of pulmonary function and food intake were recorded. Results: Ten patients were evaluated, of which seven (70%) were male. The average age when gastrostomy was made was 9.8 ±3.8 years. The weight earning was 8.35 Kg (P =0.007) and the height was 16.2 cm (P <0.001). The PBMI increased from 14.2 to 27.1 over two and a half years (P =0.282). In relation to body composition an increase of muscular and fat reserve was observed. There was no significant decline in pulmonary function over the time. Average calories offered compared with the Recommended Dietary Allowance increased from 111.6 ±24.6% to 157.7 ±37.86% (P =0.048). Conclusion: nutritional supplementation by gastrostomy resulted in significant weight and height gain, as well as increased muscle and fat reserves, however there wasn’t improvement or stabilization of pulmonary function in this group of patients.

Cystic fibrosis patient with Burkholderia pseudomallei infection acquired in Brazil.

Burkholderia pseudomallei is rarely isolated from cystic fibrosis patients outside known areas of endemicity. We report the recovery of B. pseudomallei from the sputum of a cystic fibrosis patient who lives in Brazil. We highlight the importance of careful attention to unusual nonfermentative gram-negative rods in cystic fibrosis patients.