Selection of Anti-gluten DNA Aptamers in a Deep Eutectic Solvent.

Herein, we show the feasibility of using deep eutectic solvents as a faster way of selecting aptamers targeting poorly water-soluble species. This unexplored concept is illustrated for gluten proteins. In this way, aptamer-based gluten detection can be performed directly in the extraction media with improved detectability. We envision deep implications for applications not only in food safety control but also in biomedicine.

[When should gestation of a gastroschisis be terminated?] / ¿Cuándo debe terminarse la gestación de una gastrosquisis?

OBJECTIVES: In gastroschisis pregnancies, a) to correlate prenatal ultrasound variables with postnatal outcome and b) to determine the ideal time for setting the delivery in order to achieve the best neonatal outcome. MATERIAL AND METHODS: Retrospective review (2000-2015) of all available gastroschisis whose prenatal findings could be correlated with the neonatal outcome. Two study groups have been defined according to the complications present after birth: favorable gastrosquisis and complicated. Prenatal variables were compared by groups using McWhitney or Chi tests as needed. RESULTS: Twenty-two gastroschisis fulfilled the requirement. Twelve cases had uneventful outcomes. Ten patients experienced complications, including death in five. In the complicated group there were 15 episodes of sepsis and 17 reoperations. Any single ultrasound parameter could predict a bad follow up. In thirteen cases, delivery was forced due to sudden changes on ultrasound bowel appearance. Nine of these patients had very good neonatal outcome. CONCLUSIONS: Finishing pregnancy when sudden changes on the fetal bowel were identified was the only strategy that leaded us to diminish the complication rate in gastroschisis.

OBJETIVOS: En las gestaciones con gastrosquisis, a) valorar la presencia de algún dato ecográfico prenatal que pueda predecir la evolución postnatal de la gastrosquisis, y b) determinar el momento ideal del nacimiento de los pacientes con gastrosquisis que se relacione con una mejor evolución postnatal. MATERIAL Y METODOS: Revisión retrospectiva (2000-2015) de las gastrosquisis cuyos datos ecográficos prenatales hemos podido relacionar con las características de los pacientes y su evolución clínica posterior. Se han determinado dos grupos en función de la evolución favorable o complicada de la gastrosquisis. Todas las variables ecográficas prenatales se han comparado entre grupos según los test de McWitney o Chi cuadrado. RESULTADOS: Veintidós gastrosquisis cumplieron el requisito anterior. Doce casos tuvieron una evolución sin incidencias significativas. Diez pacientes tuvieron una evolución complicada, de los cuales cinco fueron exitus. En este grupo hubo 15 episodios de sepsis y 17 reintervenciones. Ningún parámetro ecográfico prenatal predijo con fiabilidad una evolución desfavorable. En 13 casos se finalizó la gestación porque aparecieron cambios súbitos en la ecografía. Nueve de estos pacientes evolucionaron sin ninguna complicación. CONCLUSIONES: Terminar la gestación cuando se produce un cambio súbito de la apariencia ecográfica de los intestinos fetales es la única estrategia que nos ha permitido disminuir la incidencia de complicaciones en los pacientes con gastrosquisis.

Comparison of isothermal helicase-dependent amplification and PCR for the detection of Mycobacterium tuberculosis by an electrochemical genomagnetic assay.

Methods for the early and sensitive detection of pathogenic bacteria suited to low-resource settings could impact diagnosis and management of diseases. Helicase-dependent isothermal amplification (HDA) is an ideal tool for this purpose, especially when combined with a sequence-specific detection method able to improve the selectivity of the assay. The implementation of this approach requires that its analytical performance is shown to be comparable with the gold standard method, polymerase chain reaction (PCR). In this study, we optimize and compare the asymmetric amplification of an 84-base-long DNA sequence specific for Mycobacterium tuberculosis by PCR and HDA, using an electrochemical genomagnetic assay for hybridization-based detection of the obtained single-stranded amplicons. The results indicate the generalizability of the magnetic platform with electrochemical detection for quantifying amplification products without previous purification. Moreover, we demonstrate that under optimal conditions the same gene can be amplified by either PCR or HDA, allowing the detection of as low as 30 copies of the target gene sequence with acceptable reproducibility. Both assays have been applied to the detection of M. tuberculosis in sputum, urine, and pleural fluid samples with comparable results. Simplicity and isothermal nature of HDA offer great potential for the development of point-of-care devices. Graphical Abstract Comparative evaluation of isothermal helicase-dependent amplification and PCR for electrochemical detection of Mycobacterium tuberculosis.

Fetoscopic laser surgery to decompress distal urethral obstruction caused by prolapsed ureterocele.

We report on the successful use of fetoscopic surgery to treat a case of prolapsed ureterocele in a female fetus. At 21 weeks' gestation, a double renal system with an intravesical ureterocele obstructing the bladder outlet was diagnosed, causing severe megacystis, bilateral hydronephrosis and progressive oligohydramnios. Ultrasound evaluation following referral to our center confirmed severe bilateral hydronephrosis with pelvic and calyceal dilatation, but amniotic fluid volume was normal and the ureterocele was not visualized in the bladder. Instead, a cystic mass within the external genitalia was observed, suggestive of a prolapsed ureterocele, causing intermittently severe obstruction of the urethra. The parents were counseled about the uncertain prognosis and fetal surgery to decompress the urinary system was proposed. The procedure involved firing a contact diode laser until perforation of the ureterocele was achieved. Following laser surgery, resolution of megacystis, reduction of hydronephrosis and normalization of amniotic fluid volume were observed. Our report demonstrates that fetoscopic decompression of a distal urethral obstruction is feasible in the rare event of congenital prolapsed ureterocele.

Aptamer binding to celiac disease-triggering hydrophobic proteins: a sensitive gluten detection approach.

Celiac disease represents a significant public health problem in large parts of the world. A major hurdle in the effective management of the disease by celiac sufferers is the sensitivity of the current available methods for assessing gluten contents in food. In response, we report a highly sensitive approach for gluten analysis using aptamers as specific receptors. Gliadins, a fraction of gluten proteins, are the main constituent responsible for triggering the disease. However, they are highly hydrophobic and large molecules, regarded as difficult targets for in vitro evolution of aptamers without nucleobase modification. We describe the successful selection of aptamers for these water insoluble prolamins that was achieved choosing the immunodominant apolar peptide from α2-gliadin as a target for selection. All aptamers evolved are able to bind the target in its native environment within the natural protein. The best nonprotein receptor is the basis for an electrochemical competitive enzyme-linked assay on magnetic particles, which allows the measurement of as low as 0.5 ppb of gliadin standard (0.5 ppm of gluten). Reference immunoassay for detecting the same target has a limit of detection of 3 ppm, 6 times less sensitive than this method. Importantly, it also displays high specificity, detecting the other three prolamins toxic for celiac patients and not showing cross-reactivity to nontoxic proteins such as maize, soya, and rice. These features make the proposed method a valuable tool for gluten detection in foods.

Usefulness of lung-to-head ratio and intrapulmonary arterial Doppler in predicting neonatal morbidity in fetuses with congenital diaphragmatic hernia treated with fetoscopic tracheal occlusion.

OBJECTIVE: To explore the potential value of intrapulmonary artery Doppler velocimetry in predicting neonatal morbidity in fetuses with left-sided congenital diaphragmatic hernia (CDH) treated with fetoscopic tracheal occlusion (FETO). METHODS: Observed/expected lung-to-head ratio (O/E-LHR), and intrapulmonary Doppler pulsatility index and peak early-diastolic reversed flow were evaluated within 24 h before FETO in a consecutive cohort of 51 fetuses with left-sided CDH at between 24 and 33 weeks' gestation. Lung Doppler parameters were converted into Z-scores and defined as abnormal if the pulsatility index had a Z-score of > 1.0 or the peak early-diastolic reversed flow had a Z-score of > 3.5. The association of O/E-LHR and Doppler velocimetry with neonatal outcome was assessed using multiple linear or logistic regression analysis adjusted for gestational age at birth. RESULTS: Among the 26 fetuses that survived, 18 (69.2%) had normal and eight (30.8%) had abnormal Doppler values. O/E-LHR was not associated with neonatal morbidity in surviving fetuses. Compared with the group with normal Doppler parameters, cases with abnormal intrapulmonary Doppler were associated with a significant increase in the duration of mechanical ventilation (average increase of 21.2 (95% CI, 9.99-32.5) days; P < 0.01), conventional ventilation (15.2 (95% CI, 7.43-23.0) days; P < 0.01), high-frequency ventilation (6.34 (95% CI, 0.69-11.99) days; P < 0.05), nitric oxide therapy (5.73 (95% CI, 0.60-10.9) days; P < 0.05), oxygen support (36.5 (95% CI, 16.3-56.7) days; P < 0.01), parenteral nutrition (19.1 (95% CI, 7.53-30.7) days; P < 0.01) and stay in neonatal intensive care unit (42.7 (95% CI, 22.9-62.6) days; P < 0.001), and with significantly higher rates of high-frequency ventilation (87.5 vs. 44.4%;P < 0.05), oxygen requirement at 28 days of age (75.0 vs. 11.1%; P < 0.01), gastroesophageal reflux (62.5 vs. 22.2%; P < 0.05) and tube feeding at discharge (37.5 vs. 5.56%; P < 0.05). CONCLUSION: As previously reported, O/E-LHR did not predict neonatal morbidity. In contrast, intrapulmonary artery Doppler evaluation was predictive of neonatal morbidity in CDH fetuses treated with FETO.

Predictors of neonatal morbidity in fetuses with severe isolated congenital diaphragmatic hernia undergoing fetoscopic tracheal occlusion.

OBJECTIVES: To investigate neonatal morbidity in fetuses with severe congenital diaphragmatic hernia (CDH) treated with fetoscopic endoluminal tracheal occlusion (FETO) and compare it with historical controls with less severe forms of CDH that were managed expectantly. METHODS: This was a prospective, multicenter study on neonatal outcomes and prenatal predictors in 90 FETO survivors (78 left-sided, 12 right) and 41 controls from the antenatal CDH registry with either severe or moderate hypoplasia who were managed expectantly. We also investigated early neonatal morbidity indicators, including the need for patch repair, duration of mechanical ventilation and supplemental oxygen, age at full enteral feeding and incidence of pulmonary hypertension. RESULTS: Gestational age at delivery was predictive of duration of assisted ventilation (P = 0.046), days on supplemental oxygen (P = 0.019) and age at full enteral feeding (P = 0.020). When delivery took place after 34 weeks' gestation, neonatal morbidity of FETO cases was comparable with that of expectantly managed cases with moderate hypoplasia. CONCLUSIONS: Fetal intervention for severe CDH is associated with neonatal morbidity that is comparable with that of an expectantly managed group with less severe disease.

Decompression through tracheobronchial endoscopy of bronchial atresia presenting as massive pulmonary tumor: a new indication for fetoscopic surgery.

This is the first report of fetoscopy to successfully treat a case of bilobar bronchial atresia presenting in the form of a massive right lung tumor causing severe thoracic compression with mediastinal shift, pulmonary hypoplasia and eventually fetal hydrops. The presence of an echolucent tubular structure in the pulmonary hilum was identified as the intrapulmonary continuation of the interrupted main bronchus, and led to the diagnosis of main bronchial atresia. After extensive discussion and counseling, a fetoscopic operation was designed and indicated. The procedure consisted of a fetal tracheoscopy and access to the right main bronchus, which was perforated with a diode laser until a communication with the dead end of the intrapulmonary main bronchus was achieved. After the operation a significant reduction in size was observed, hydrops and mediastinal shift disappeared and O/E LHR increased to 85%. The fetus was born at term and lobectomy of two dysplastic pulmonary lobes was performed. The infant is developing well at 14 months of age. This report demonstrates that fetoscopic airway decompression can achieve fetal survival in the rare event of main bronchus atresia.

Evaluation of fetal vocal cords to select candidates for successful fetoscopic treatment of congenital high airway obstruction syndrome: preliminary case series.

OBJECTIVE: Congenital high airway obstruction syndrome (CHAOS) normally has a dire prognosis due to laryngeal atresia and association with other anomalies. However, rare cases with an isolated tracheal membrane have been described. We assessed systematically the larynx and vocal cords in fetuses with CHAOS, with the goal of identifying such cases and attempting fetoscopic deobstruction. METHODS: Between 2008 and 2012, 7 cases of CHAOS were referred to our institution. The ultrasonographic aspect of the larynx and trachea was assessed. We report the ultrasound findings, necropsy findings of 6 cases and perinatal outcome in 1 case undergoing therapy. RESULTS: In 6 cases, laryngeal atresia was presumed by ultrasound and confirmed by necropsy after termination of pregnancy. In 1 case, normal appearance of the vocal cords led to suspicion of a possible isolated tracheal web completely occluding the trachea. Fetoscopy confirmed the finding, thus the membrane was perforated. Lung size normalized and hydrops resolved. A normal fetus was delivered at term, requiring no respiratory support. At 9 months of age, the infant showed a normal laryngoscopy and was developing well. CONCLUSIONS: Systematic ultrasound assessment of the vocal cords can be performed in fetuses with CHAOS. We report the first case of long-term intact survival after fetoscopic airway deobstruction.

Outcome of fetuses with congenital diaphragmatic hernia and associated intrafetal fluid effusions managed in the era of fetal surgery.

OBJECTIVE: Fetuses with congenital diaphragmatic hernia (CDH) and for whom additional ultrasound findings are abnormal typically are considered to have a dismal prognosis. Our aim was to assess the outcome of fetuses with CDH and associated intrafetal fluid effusions. METHODS: This was a retrospective bicentric cohort study on the perinatal management of fetuses with CDH and intrafetal fluid effusions. RESULTS: The incidence of effusions was 5.2% (n = 14) in 269 consecutive cases of left-sided CDH and 29.2% (n = 14) in 48 cases of right-sided CDH. Hydrothorax (n = 19 (68%)) and ascites (n = 16 (57%)) were the most common effusions. A combination of both was present in 11 (39%) fetuses. Of 20 ongoing pregnancies with CDH and fluid effusions, without other anomalies, five with moderate or mild pulmonary hypoplasia were managed without fetoscopic endoluminal tracheal occlusion (FETO). The 15 other cases underwent FETO because of severe pulmonary hypoplasia. Neonatal survival rate was similar in both groups (n = 2/5 and n = 6/15, respectively (P = 1.0)). Survival among those who underwent FETO was similar to previously published results concerning isolated cases undergoing FETO. CONCLUSIONS: Our observations do not support the view that intrafetal effusions are an adverse prognostic factor in fetuses with CDH. In CDH fetuses with effusions and severe pulmonary hypoplasia treated with FETO, neonatal survival is similar to that in isolated cases undergoing the intervention. Whether pleural effusions should be addressed by thoracic drainage procedures remains unproven.

A competitive assay for the detection of a 16-mer peptide from α1 chain of human collagen XI.

Characterization of extracellular matrix (ECM) is becoming more and more important to decipher cancer progression. Constant remodeling results in ECM components degradation or unusual ECM accumulation that releases short fragments to the body fluids. These fragments might be potential cancer biomarkers but to detect them specific receptors are needed. In response to this demand, we present the first electrochemical aptamer-based competitive assay for the minor collagen XI, dysregulated in several carcinomas. It was performed on magnetic beads using enzymatic labeling. First, we selected the most appropriate tag for the aptamer (biotin or 6-carboxyfluorescein). The former yielded higher currents by chronoamperometry and it was used for the competitive assay. The collagen fragment, a 16mer peptide used as the target, was detected from 52 to 1000 nM with an RSD of about 5%. The LOD of the assay was estimated as 24 nM (44 ng/mL). The performance of the assay in serum diluted 1:2 was equivalent to the assay in PBS. The detection of α1 chain of human collagen XI was also possible in cell lysates and confirmed by aptacytofluorescence, which is promising as a new tool to validate this fragment as a cancer biomarker.

Contribution of intrapulmonary artery Doppler to improve prediction of survival in fetuses with congenital diaphragmatic hernia treated with fetal endoscopic tracheal occlusion.

OBJECTIVE: To evaluate the contribution of intrapulmonary artery Doppler in predicting the survival of fetuses with congenital diaphragmatic hernia (CDH) treated with fetoscopic tracheal occlusion (FETO). METHODS: A cohort of 41 fetuses (between 24 and 28 weeks of gestation) with CDH was treated with FETO. The observed/expected lung-to-head ratio (O/E-LHR), pulmonary artery pulsatility index (PI), peak early diastolic reversed flow (PEDRF) and peak systolic velocity (PSV) were evaluated before FETO, and their isolated and combined value to predict survival using multiple logistic regression and decision-tree analysis was assessed. RESULTS: O/E-LHR and intrapulmonary artery PI and PEDRF were significantly associated with the probability of survival (O/E-LHR > or = 26%, OR 14.2; PI < 1 Z-score, OR 8.4; and PEDRF < 3.5 Z-scores, OR 5.7). Decision-tree analysis showed that O/E-LHR was the best initial predictor of prognosis (O/E-LHR > or = 26%, 90% survival; O/E-LHR < 26%, 45% survival). For fetuses with an O/E-LHR of < 26%, Doppler parameters allowed discrimination of cases with moderate (66-71% survival) and very poor (0% survival) prognosis. CONCLUSION: Intrapulmonary artery Doppler evaluation helps to refine the prediction of survival after FETO in fetuses with severe CDH.

[Thoracoscopic lobectomy: initial experience]. / Lobectomía toracoscópica: experiencia inicial.

AIM: Analizing our preliminary experience in the treatment of pulmonary malformations with thoracoscopic lobectomy. MATERIAL AND METHODS: We have reviewed our first cases of thoracoscopic lobectomy during last year (2008-09). We have performed 6 thoracoscopic lobectomies in 6 male patients with a mean age of 16 months (6 months-4 years). They were 3 lower lobectomies (2 right and one left), 2 right upper lobectomies and one lingular sparing left upper lobectomy. Lung lesion was a congenital cystic adenomatoid malformation in 5 (2 of them asociated with intralobar sequestration) and one lobar emphysema due to bronquial atresia. RESULTS: Selective bronchial intubation was used in 4 cases and and tracheal intubation with bronchial blocker in 2. There were no conversions to open thoracotomy. Mean operative time was 202' (155-250'). There were no intraoperative nor postoperative complications. None of these patients needed blood transfusion. Mean drainage time was 3.5 days (3-4 d) and mean discharge time was 4.8 days (4-7 d). With a mean follow-up time of 5.3 months (2 m-12 m), all of the patients have no symptoms and X-ray are normal. CONCLUSIONS: Thoracoscopic lobectomy is a safe and efective approach to treat congenital pulmonary pathology. The keys to perform it safely are good anesthetic management, adequate instruments for children size, and careful disection of broncovascular structures. Advantages of the minimal access surgery and implementation and miniaturization of the instruments and sealing devices will favor its progressive use.

[Utility of the ECMO in patients with congenital diaphragmatic hernia]. / Utilidad de la ECMO en pacientes con hernia diafragmática congénita.

At the moment the extracorporeal membrane oxygenation (ECMO) constitutes the last link in the therapeutic one of the handling of the respiratory failure in patients with Congenital Diaphragmatic Hernia (HDC). We presented our experience. From January 2001 we arrange the ECMO in neonative UCI. 76 HDC, 13 (3 rights and 10 lefts) they have needed ECMO (one in two occasions; altogether 14 procedures). Criteria of inclusion: refractory hypoxaemia, oxigenaction index > 40 and weight > 2 kg. 5 girls and 8 boys with gestacional age between 35 and 41 weeks (average: 38) and weight when being born between 2,300 and 3,500 grams (average 2,817). In 6 cases (5 transferred from other centers) the diagnosis was posnatal. Of the 7 with prenatal diagnosis, in 4 cases fetal therapy by means of traqueal occlusion had been made. Veno-venous in 8 and veno-arterial procedure in 5. Rank of duration: 68-606 hours, average of 228.35. The surgery has been made before the ECMO in 9 cases, 2 during and 1 later. In an occasion there was no surgery. The complications have been of hemorrágico type in one patient and infectious in three cases with sudden sepsis in one. Precocious mortality has been of 6 patients and delayed the 2 (total 61%). Although this procedure has the high morbi-mortality, it is necessary to consider that is patients very badly prognosis without another alternative (with mortality of the 100%). Multicentric studies are needed to establish indicators prognoses pre and postbirthdays.

Aptamers targeting protein-specific glycosylation in tumor biomarkers: general selection, characterization and structural modeling.

Detecting specific protein glycoforms is attracting particular attention due to its potential to improve the performance of current cancer biomarkers. Although natural receptors such as lectins and antibodies have served as powerful tools for the detection of protein-bound glycans, the development of effective receptors able to integrate in the recognition both the glycan and peptide moieties is still challenging. Here we report a method for selecting aptamers toward the glycosylation site of a protein. It allows identification of an aptamer that binds with nM affinity to prostate-specific antigen, discriminating it from proteins with a similar glycosylation pattern. We also computationally predict the structure of the selected aptamer and characterize its complex with the glycoprotein by docking and molecular dynamics calculations, further supporting the binary recognition event. This study opens a new route for the identification of aptamers for the binary recognition of glycoproteins, useful for diagnostic and therapeutic applications.

The Barcelona-Asymptomatic Intracranial Atherosclerosis (AsIA) study: Subclinical cervico-cerebral stenosis and middle cerebral artery pulsatility index as predictors of long-term incident cognitive impairment.

BACKGROUND AND AIMS: We aimed to study subclinical non-invasive vascular markers as predictors of incident long-term cognitive impairment in a longitudinal population-based study. METHODS: The Barcelona-Asymptomatic Intracranial Atherosclerosis (AsIA) study is a population-based study that included a random sample of 933 Caucasian subjects (mean age 66 years, 64% male) with a moderate-high vascular risk and without history of stroke or dementia. Subclinical carotid and intracranial stenosis was assessed at baseline visit by cervical and transcranial color-coded duplex (TCCD) and confirmed by magnetic resonance angiography. Cervico-cerebral stenosis (CCS) was defined as the presence of extra and/or intracranial stenosis >50%. Baseline middle cerebral artery pulsatility index (MCA-PI) was measured bilaterally by TCCD, and mean PI of both sides was considered for analyses. Subjects were followed-up to determine incident long-term cognitive impairment (mild cognitive impairment or dementia). RESULTS: After a median of 7.16 [6.91-7.75] years of follow-up, 91 subjects (9.7%) developed cognitive impairment, 27 of them mild cognitive impairment, and 64 dementia. Incidence of cognitive impairment was significantly higher among subjects with subclinical CCS (21.4% versus 9% in those without CCS) and among those with mean MCA-PI>1 (13.5% versus 7.4% in those with MCA-PI<1). In multivariate Cox regression analyses, both CCS and MCA-PI>1 were independently associated with incident cognitive impairment with HR of 2.07 [1.11-3.88] and 1.58 [1.02-2.46], respectively. CONCLUSIONS: Subclinical cervico-cerebral stenosis and higher MCA-PI are non-invasive neurosonological markers of incident long-term cognitive impairment in our population.

Expression of plectin mutant cDNA in cultured cells indicates a role of COOH-terminal domain in intermediate filament association.

Plectin is an intermediate filament (IF) binding protein of exceptionally large size. Its molecular structure, revealed by EM and predicted by its sequence, indicates an NH2-terminal globular domain, a long rodlike central domain, and a globular COOH-terminal domain containing six highly homologous repeat regions. To examine the role of the various domains in mediating plectin's interaction with IFs, we have constructed rat cDNAs encoding truncated plectin mutants under the control of the SV-40 promoter. Mutant proteins expressed in mammalian COS and PtK2 cells could be distinguished from endogenous wild type plectin by virtue of a short carboxy-terminal antigenic peptide (P tag). As shown by conventional and confocal immunofluorescence microscopy, the transient expression of plectin mutants containing all six or the last four of the repeat regions of the COOH-terminus, or the COOH-terminus and the rod, associated with IF networks of both the vimentin and the cytokeratin type and eventually caused their collapse into perinuclear aggregates. Similar effects were observed upon expression of a protein encoded by a full length cDNA construct. Microtubules and microfilaments were unaffected. Unexpectedly, mutants containing the rod without any of the COOH-terminal repeats, accumulated almost exclusively within the nuclei of cells. When the rod was extended by the first one and a half of the COOH-terminal repeats, mutant proteins showed a partial cytoplasmic distribution, although association with intermediate filaments was not observed. Nuclear and diffuse cytoplasmic distribution was also observed upon expression of the NH2-terminal domain without rod. These results indicate that sequences located roughly within the last two thirds of the globular COOH-terminus are indispensable for association of plectin with intermediate filaments in living cells.

Cloning and sequencing of rat plectin indicates a 466-kD polypeptide chain with a three-domain structure based on a central alpha-helical coiled coil.

We have determined the complete cDNA sequence of rat plectin from a number of well-characterized overlapping lambda gt11 clones. The 4,140-residue predicted amino acid sequence (466,481 D) is consistent with a three-domain structural model in which a long central rod domain, having mainly an alpha-helical coiled coil conformation, is flanked by globular NH2- and COOH-terminal domains. The plectin sequence has a number of repeating motifs. The rod domain has five subregions approximately 200-residues long in which there is a strong repeat in the charged amino acids at 10.4 residues that may be involved in association between plectin molecules. The globular COOH-terminal domain has a prominent six-fold tandem repeat, with each repeat having a strongly conserved central region based on nine tandem repeats of a 19-residue motif. The plectin sequence has several marked similarities to that of desmoplakin (Green, K. J., D. A. D. Parry, P. M. Steinert, M. L. A. Virata, R. M. Wagner, B. D. Angst, and L.A. Nilles. 1990. J. Biol. Chem. 265:2,603-2,612), which has a shorter coiled-coil rod domain with a similar 10.4 residue charge periodicity and a COOH-terminal globular domain with three tandem repeats homologous to the six found in plectin. The plectin sequence also has homologies to that of the bullous pemphigoid antigen. Northern blot analysis indicated that there is a significant degree of conservation of plectin genes between rat, human, and chicken and that, as shown previously at the protein level, plectin has a wide tissue distribution. There appeared to be a single rat plectin gene that gave rise to a 15-kb message. Expression of polypeptides encoded by defined fragments of plectin cDNA in E. coli has also been used to localize the epitopes of a range of monoclonal and serum antibodies. This enabled us to tentatively map a sequence involved in plectin-vimentin and plectin-lamin B interactions to a restricted region of the rod domain.

Polymorphisms in CCL5 promoter are associated with pulmonary tuberculosis in northern Spain.

OBJECTIVE: To study whether two functional single nucleotide polymorphisms of the CC chemokine ligand 5 (CCL5) gene could affect susceptibility to pulmonary tuberculosis (TB) in a human immunodeficiency virus negative genetically homogeneous population, containing newly diagnosed patients with active disease. DESIGN: Seventy-six patients with active pulmonary TB (PTB) and 157 healthy control subjects from Cantabria, northern Spain, were genotyped for the CCL5 -403G/A and -28C/G polymorphisms. RESULTS: The frequency of the CCL5-403G/A and -28C/G promoter polymorphisms were significantly different between patients with active TB and control subjects. Three of the four possible haplotypes were also significantly different. The G/G-C/C diplotype was much more frequent in the healthy control group and the G/G-G/G and A/A-C/C diplotypes were more frequent in patients with PTB. CONCLUSION: Our findings indicate that CCL5 may play a role in conferring susceptibility to active PTB. Thus, the -403G and -28C alleles, either separately or combined in the G-C haplotype and the GG/CC diplotype, may be related to protection against PTB. By contrast, the -403A and -28G alleles, the G-G or A-C haplotypes and the G/G-G/G and A/A-C/C diplotypes may confer susceptibility to PTB.