Purification and partial characterization of a Paracoccidioides brasiliensis protein with capacity to bind to extracellular matrix proteins

Microorganisms adhere to extracellular matrix proteins by means of their own surface molecules. Paracoccidioides brasiliensis conidia have been shown to be capable of interacting with extracellular matrix proteins. We aimed at determining the presence of fungal proteins that could interact with extracellular matrix protein and, if found, attempt their purification and characterization. Various extracts were prepared from P. brasiliensis mycelial and yeast cultures (total homogenates, beta-mercaptoethanol, and sodium dodecyl sulfate [SDS] extracts) and analyzed by ligand affinity assays with fibronectin, fibrinogen and laminin. Two polypeptides were detected in both fungal forms. SDS extracts that interacted with all the extracellular matrix protein were tested; their molecular masses were 19 and 32 kDa. Analysis of the N-terminal amino acid sequence of the purified 32-kDa mycelial protein showed substantial homology with P. brasiliensis, Histoplasma capsulatum, and Neurospora crassa hypothetical proteins. Additionally, a monoclonal antibody (MAb) produced against this protein recognized the 32-kDa protein in the SDS extracts of both fungal forms for immunoblot. Immunofluorescence analysis revealed that this MAb reacted not only with mycelia and yeast cells, but also with conidia, indicating that this protein was shared by the three fungal propagules. By immunoelectron microscopy, this protein was detected in the cell walls and in the cytoplasm. Both the 32-kDa purified protein and MAb inhibited the adherence of conidia to the three extracellular matrix proteins in a dose-dependent manner. These findings demonstrate the presence of two polypeptides capable of interacting with extracellular matrix proteins on the surface of P. brasiliensis propagules, indicating that there may be common receptors for laminin, fibronectin, and fibrinogen. These proteins would be crucial for initial conidial adherence and perhaps also in dissemination of paracoccidioidomycosis.     

Human papillomavirus typing with a polymerase chain reaction-based genotyping array compared with type-specific PCR

BACKGROUND: Type-specific persistence of human papillomavirus (HPV) infection can cause invasive cervical cancer. OBJECTIVES: To evaluate the efficacy of HPV detection and typing with a general polymerase chain reaction (PCR)-based genotyping array and to compare it with a type-specific PCR assay. STUDY DESIGN: Four hundred and thirty-three cervical samples were tested with a modified MY11/GP6+ PCR-based reverse-blot assay (EasyChip HPV Blot; King Car, Taiwan [hereafter HPV Blot]) and with 20 genotypes of L1-type-specific PCR (HPV-6, -11, -16, -18, -31, -33, -35, -39, -45, -51, -52, -53, -56, -58, -59, -62, -66, -68, -70, and -71 [CP8061]). RESULTS: The concordance of the two tests in determining HPV positivity was 96.8% (419/433), with a Cohen's kappa=0.93 (95% CI: 0.90-0.97) and McNemar's test of P=1.0, which indicates excellent agreement. The overall concordance of the two tests in the identification of type-specific HPV was 91.0% (394/433). Sensitivity (90-100%), specificity (99.2-100%), and accuracy (98.6-100%) rates of HPV Blot against the gold standard were satisfactory for HPV-16, -18, -58, -33, -52, -39, -45, -31, -51, -70 while HPV-71 (63.6%) had suboptimal sensitivity. Though the kappa values between the two tests for many individual genotypes could not be reliably calculated because of low positivity, the kappa values for HPV-16, -52, and -58 were excellent (0.93, 0.96, and 0.95, respectively). CONCLUSION: The modified MY11/GP6+ PCR-based HPV Blot assay is accurate and sensitive for detection and genotyping of HPV in cervical swab samples.     

Expansion and clonal deletion of peripheral T cells induced by bacterial superantigen is independent of the interleukin-2 pathway

Injection of the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) into mice provokes a rapid expansion and subsequent contraction of the pool of SEB-reactive T cells bearing T cell receptor (TcR) V_β8 gene products. Given that interleukin 2 (IL-2) stimulates proliferation, abolishes anergy, and counteracts apoptotic cell death in T cells in vitro, we tested whether the IL-2 synthesis inhibitor cyclosporin A (CsA) or a vaccinia virus recombinant releasing high amounts of human IL-2 modulate SEB responses in vivo. Surprisingly, neither IL-2 nor CsA were able to change the in vivo kinetics and magnitude of SEB-induced expansion, unresponsiveness to SEB, and peripheral clonal deletion of T cells expressing products of the SEB-reactive TcR V_β8 gene family. In accord with these in vivo observations, IL-2 is incapable of reversing “anergy” and apoptotic cell death of V_β8⁺ SEB-reactive T cells isolated from SEB-primed mice in vitro. Accordingly, upon SEB injection V_β8⁺ T cells expand rapidly, without expressing IL-2 receptor (IL-2R)α chains in vivo, although SEB induces IL-2R α in vitro. Altogether, these results indicate that the IL-2/IL-2R-mediated pathway is not involved in T cell repertoire modulation by bacterial superantigens. Moreover, the data suggest that unresponsiveness of V_β8⁺ T cells from SEB-primed mice is not a reversible process, but involves an unreversible commitment to programmed cell death. Absence or presence of IL-2 responsiveness could be a hallmark to distinguish truly reversible anergy and peripheral clonal deletion.     

Identification of 26 new constitutional RB1 gene mutations in Spanish, Colombian, and Cuban retinoblastoma patients

Constitutional mutations in the RB1 gene predispose to retinoblastoma development. Hence genetic screening of retinoblastoma patients and relatives is important for genetic counseling purposes. In addition, RB1 gene mutation studies may help decipher the molecular mechanisms leading to tumors with different degrees of penetrance or expressivity. In the course of genetically screening of 107 hereditary and non-hereditary retinoblastoma patients (11 familiar bilateral, 4 familiar unilateral, 49 sporadic bilateral and 43 sporadic unilateral) and kindred from Spain, Colombia and Cuba, using direct PCR sequencing, we observed 45 distinct mutations and four RB1 deletions in 53 patients (9 familiar bilateral, 2 familiar unilateral, 31 sporadic bilateral and 11 sporadic unilateral). Most of these mutations (26/45, 57%) have not been reported before. In 32 patients, the predisposing mutations correspond to nonsense (mainly CpG transitions) and small insertions or deletions whose expected outcome is a truncated Rb protein that lacks the functional pockets and tail. Five single aminoacid replacements and seventeen mutations affecting splicing sites were also observed in retinoblastoma patients. Two of these sixteen mutations are of unclear pathogenic nature.     

ICAM-3, the third LFA-1 counterreceptor,is a co-stimulatory molecule for both resting and activated T lymphocytes

Optimal activation of human T cells mediated by ligation of CD3/T cell receptor (TcR) complex requires co-stimulatory signals. These can be provided by the adhesive interaction between receptor molecules on T cells and their counter-receptors on antigen-presenting cells. Soluble ICAM-3, anti-ICAM-3 and anti-CD3 mAb were utilized to address the role of the ICAM-3/LFA-1 pathway in TcR/CD3-dependent or -independent T cell activation. Immunoaffinity-purified ICAM-3 co-immobilized with suboptimal concentrations of anti-CD3 monoclonal antibody (mAb) stimulated T lymphocytes as monitored by the expression of the lymphocyte activation antigens CD25 and CD69. The mechanism underlaying this activation appear to involve the interaction of ICAM-3 with a β2 integrin, likely to be LFA-1, since mAb to the CD18 chain completely inhibited T cell activation. Similar experiments demonstrated that anti-ICAM-3 mAb were able to co-stimulate both resting (cord blood) and activated (T cell clones) T lymphocytes. On the contrary, anti-ICAM-1 mAb were only co-stimulatory for CD25 expression on activated but not on resting T cells. In addition, we have found that some γδ T cell clones bearing the Vδ1 segment were activated by direct mAb engagement of ICAM-3 in the absence of TcR/CD3 occupancy. Furthermore, immobilized anti-ICAM-3 mAb also induced development of dentritic processes. In conclusion, our data suggest that ICAM-3 on the surface of both T cells and antigen-presenting cells plays an essential role in the initiation of the immune response.     

Nutritional Importance of Selected Fresh Fishes,Shrimps and Mollusks to Meet Compliance with Nutritional Guidelines of n-3 LC-PUFA Intake in Spain

Fishery products are the main source of dietary n-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA). Following the European Commission’s request to address the risks and benefits of seafood consumption, and taking into account the great variability of nutrient and contaminant levels in fishery products, the present work aims to estimate the n-3 LC-PUFA provided per serving of selected fishes, shrimps and mollusks that are commonly consumed in Spain. This would enable the establishment of a risk–benefit analysis of fish consumption and provide recommendations for fish intake to comply with nutritional guidelines of n-3 LC-PUFA intake. We confirmed high variation in the pattern and contents of fatty acids for different species. n-6 PUFA were minor fatty acids, whereas palmitic (C16:0), oleic (C18:1 n-9), and mainly eicosapentaenoic (C20:5 n-3) and docosahexaenoic (C22:6 n-3) acids were the major fatty acids in the sample. Therefore, consumption of 2–3 servings per week of a variety of fishery products may contribute to compliance with the recommended daily n-3 LC-PUFA intake while maintaining an adequate balance to avoid contaminant-derived potential risks (metals and others). Taking the fatty acid content of fishery products described in this study into consideration, it is advisable to include one serving of fatty fish per week in order to meet recommended n-3 LC-PUFA levels.     

A Randomized Study of Nutritional Supplementation in Patients with Unilateral Wet Age-Related Macular Degeneration

The purpose of this study is evaluate the efficacy and safety of medicinal products containing the original Age-Related Eye Disease group (AREDS) formulation at doses approved in Europe (EU, control group; n = 59) with a product that adds DHA, lutein, zeaxanthin, resveratrol and hydroxytyrosol to the formula (intervention group; n = 50). This was a multicenter, randomized, observer-blinded trial conducted in patients aged 50 years or older diagnosed with unilateral exudative Age related Macular Degeneration AMD. At month 12, the intervention did not have a significant differential effect on visual acuity compared with the control group, with an estimated treatment difference in Early Treatment Diabetic Retinopathy Study (ETDRS) of −1.63 (95% CI −0.83 to 4.09; p = 0.192). The intervention exhibited a significant and, in most cases, relevant effect in terms of a reduction in some inflammatory cytokines and a greater improvement in the fatty acid profile and serum lutein and zeaxantin concentration. In patients with unilateral wet AMD, the addition of lutein, zeaxanthin, resveratrol, hydroxytyrosol and DHA to the AREDS EU recommended doses in the short-term did not have a differential effect on visual acuity compared to a standard AREDS EU formula but, in addition to improving the fatty acid profile and increasing carotenoid serum levels, may provide a beneficial effect in improving the proinflammatory and proangiogenic profile of patients with AMD.     

Human Milk Virome Analysis: Changing Pattern Regarding Mode of Delivery,Birth Weight,and Lactational Stage

The human milk (HM) microbiota is a significant source of microbes that colonize the infant gut early in life. The aim of this study was to compare transient and mature HM virome compositions, and also possible changes related to the mode of delivery, gestational age, and weight for gestational age. Overall, in the 81 samples analyzed in this study, reads matching bacteriophages accounted for 79.5% (mainly Podoviridae, Myoviridae, and Siphoviridae) of the reads, far more abundant than those classified as eukaryotic viruses (20.5%, mainly Herpesviridae). In the whole study group of transient human milk, the most abundant families were Podoviridae and Myoviridae. In mature human milk, Podoviridae decreased, and Siphoviridae became the most abundant family. Bacteriophages were predominant in transient HM samples (98.4% in the normal spontaneous vaginal delivery group, 92.1% in the premature group, 89.9% in the C-section group, and 68.3% in the large for gestational age group), except in the small for gestational age group (only ~45% bacteriophages in transient HM samples). Bacteriophages were also predominant in mature HM; however, they were lower in mature HM than in transient HM (71.7% in the normal spontaneous vaginal delivery group, 60.8% in the C-section group, 56% in the premature group, and 80.6% in the large for gestational age group). Bacteriophages still represented 45% of mature HM in the small for gestational age group. In the transient HM of the normal spontaneous vaginal delivery group, the most abundant family was Podoviridae; however, in mature HM, Podoviridae became less prominent than Siphoviridae. Myoviridae was predominant in both transient and mature HM in the premature group (all C-section), and Podoviridae was predominant in transient HM, while Siphoviridae and Herpesviridae were predominant in mature HM. In the small for gestational age group, the most abundant taxa in transient HM were the family Herpesviridae and a species of the genus Roseolovirus. Bacteriophages constituted the major component of the HM virome, and we showed changes regarding the lactation period, preterm birth, delivery mode, and birth weight. Early in life, the HM virome may influence the composition of an infant’s gut microbiome, which could have short- and long-term health implications. Further longitudinal mother–newborn pair studies are required to understand the effects of these variations on the composition of the HM and the infant gut virome.     

Pointwise‐constrained optimal control of a semiactive vehicle suspension

This article presents an optimal control strategy (OCS) for semiactive vehicle suspensions with road profile sensors. The suspension is modeled as a quarter‐car model with a magnetorheological (MR) damper. The OCS main objective is to minimize the fourth‐power acceleration of the sprung mass. In addition, three pointwise constraints of the model are taken into account when the optimal control problem is solved: suspension travel limits (upper and lower) and tyre vertical force. In order to deal with a large number of constraints, we implement the gradient optimization method based on the method of moving asymptotes routine, which shows very good performance reaching optimal controls while satisfying the constrains. The solution has been compared with two passive MR damper configurations (low and high damping) as well as Skyhook and Balance control strategies for three different road inputs. Results show that OCS fulfills the constraints and reduces the sprung mass acceleration peak and the root‐mean‐quad acceleration up to 59% , in comparison to passive strategies.