Controlled silver electrodeposition on gold nanoparticle antibody tags for ultrasensitive prostate specific antigen sensing using electrochemical and optical smartphone detection.

One of the current challenges in medicine is to achieve a rapid and unequivocal detection and quantification of extremely low levels of disease biomarkers in complex biological samples. Here, we present the development and analytical evaluation of a low-cost smartphone-based system designed for ultrasensitive detection of the prostate-specific antigen (PSA) using two detection alternatives: electrochemical or optical, by coupling the smartphone with a portable potentiostat or magnifying lenses. An antibody tagged with gold nanoparticles (AuNPs), and indium tin oxide coated polyethylene terephthalate platform (ITO-PET) have been used to develop a sandwich-type immunoassay. Then, a controlled silver electrodeposition on the AuNPs surface is carried out, enhancing their size greatly. Due to such strong nanoparticle-size amplification (from nm to µm), the final detection can be dual, by measuring current intensity or the number of silver-enlarged microstructures generated. The proposed strategies exhibited limit detections (LOD) of 102 and 37 fg/mL for electrochemical and optical detection respectively. The developed immunosensor reaches excellent selectivity and performance characteristics to quantify biomarkers at clinically relevant values without any pretreatment. These proposed procedures could be useful to check and verify possible recurrence after clinical treatment of tumors or even report levels of disease serum biomarkers in early stages.

Relative and Transport Efficiency-Independent Approach for the Determination of Nanoparticle Size Using Single-Particle ICP-MS.

Herein, we introduce the first relative single-particle inductively coupled plasma mass spectrometry (spICP-MS) approach where size calibration is carried out using the target NP itself measured under different instrumental conditions without external dependence on the complex and prone-to-error determination of transport efficiency or mass flux calibrations, in contrast to most spICP-MS approaches. The simple approach proposed allows determining gold nanoparticle (AuNP) sizes, with errors ranging from 0.3 to 3.1% (corroborated by HR-TEM). It has been demonstrated that the changes observed in the single-particle histograms obtained for a suspension of AuNPs under different sensitivity conditions (n = 5) are directly and exclusively related to the mass (size) of the target AuNP itself. Interestingly, the relative nature of the approach shows that once the ICP-MS system has been calibrated with a generic NP standard, it is no longer necessary to repeat the calibration for the size determination of different unimetallic NPs carried out along time (at least 8 months), independently of their size (16-73 nm) and even nature (AuNP or AgNP). Additionally, neither the NP surface functionalization with biomolecules nor protein corona formation led to significant changes (relative errors slightly increased 1.3- to 1.5-fold, up to 7%) in the NP size determination, in contrast to conventional spICP-MS approaches where relative errors increased 2- to 8-fold, up to 32%. This feature could be especially valuable for the analysis of NPs in real samples without the need of matrix-matched calibration.

Correlations between the NMR Lipoprotein Profile, APOE Genotype, and Cholesterol Efflux Capacity of Fasting Plasma from Cognitively Healthy Elderly Adults.

Cholesterol efflux capacity (CEC) is of interest given its potential relationship with several important clinical conditions including Alzheimer's disease. The inactivation of the APOE locus in mouse models supports the idea that it is involved in determining the CEC. With that in mind, we examine the impact of the plasma metabolome profile and the APOE genotype on the CEC in cognitively healthy elderly subjects. The study subjects were 144 unrelated healthy individuals. The plasma CEC was determined by exposing cultured mouse macrophages treated with BODIPY-cholesterol to human plasma. The metabolome profile was determined using NMR techniques. Multiple regression was performed to identify the most important predictors of CEC, as well as the NMR features most strongly associated with the APOE genotype. Plasma 3-hydroxybutyrate was the variable most strongly correlated with the CEC (r = 0.365; p = 7.3 × 10-6). Male sex was associated with a stronger CEC (r = -0.326, p = 6.8 × 10-5). Most of the NMR particles associated with the CEC did not correlate with the APOE genotype. The NMR metabolomics results confirmed the APOE genotype to have a huge effect on the concentration of plasma lipoprotein particles as well as those of other molecules including omega-3 fatty acids. In conclusion, the CEC of human plasma was associated with ketone body concentration, sex, and (to a lesser extent) the other features of the plasma lipoprotein profile. The APOE genotype exerted only a weak effect on the CEC via the modulation of the lipoprotein profile. The APOE locus was associated with omega-3 fatty acid levels independent of the plasma cholesterol level.

Neurological Complications in Systemic Inflammatory Diseases.

Systemic inflammatory diseases could produce neurologic complications, and they are frequently incorporated in the differential diagnosis of neurological symptoms. There are wellestablished criteria to meet the diagnosis of neurologic manifestations of these systemic diseases. Methods: However, the range of clinical presentations varies in each condition, and the prevalence of these complications differs between studies. Hence, in many cases, an etiological relationship is not clearly defined. Results and Conclusion: For these reasons, it is challenging to make an accurate diagnosis. We analyzed the spectrum of neurological manifestations in a cohort of patients with systemic lupus erythematosus, rheumatoid arthritis, Behçet disease and sarcoidosis in order to improve our current knowledge of these complications.

Micro-Topographies Induce Epigenetic Reprogramming and Quiescence in Human Mesenchymal Stem Cells.

Biomaterials can control cell and nuclear morphology. Since the shape of the nucleus influences chromatin architecture, gene expression and cell identity, surface topography can control cell phenotype. This study provides fundamental insights into how surface topography influences nuclear morphology, histone modifications, and expression of histone-associated proteins through advanced histone mass spectrometry and microarray analysis. The authors find that nuclear confinement is associated with a loss of histone acetylation and nucleoli abundance, while pathway analysis reveals a substantial reduction in gene expression associated with chromosome organization. In light of previous observations where the authors found a decrease in proliferation and metabolism induced by micro-topographies, they connect these findings with a quiescent phenotype in mesenchymal stem cells, as further shown by a reduction of ribosomal proteins and the maintenance of multipotency on micro-topographies after long-term culture conditions. Also, this influence of micro-topographies on nuclear morphology and proliferation is reversible, as shown by a return of proliferation when re-cultured on a flat surface. The findings provide novel insights into how biophysical signaling influences the epigenetic landscape and subsequent cellular phenotype.

Comparison Study of an Optimized Ultrasound-Based Method versus an Optimized Conventional Method for Agar Extraction, and Protein Co-Extraction, from Gelidium sesquipedale.

Agar is a hydrocolloid found in red seaweeds, which has been of industrial interest over the last century due to its multiple applications in the food, cosmetic, and medical fields. This polysaccharide, extracted by boiling for several hours, is released from the cell wall of red seaweeds. However, the environmental impact coming from the long processing time and the energy required to reach the targeted processing temperature needs to be reduced. In this study, a response surface methodology was employed to optimize both conventional extraction and ultrasound-assisted extractions. Two different models were successfully obtained (R2 = 0.8773 and R2 = 0.7436, respectively). Additionally, a further re-extraction confirmed that more agar could be extracted. Protein was also successfully co-extracted in the seaweed residues. Optimized conditions were obtained for both the extractions and the re-extraction of the two methods (CE: 6 h, 100 °C; and UAE: 1 h, 100% power). Finally, FT-IR characterization demonstrated that the extracts had a similar spectrum to the commercial agar. Compared to commercial samples, the low gel strength of the agar extracts shows that these extracts might have novel and different potential applications.

Advances in quantum dots as diagnostic tools.

Quantum dots (QDs) are crystalline inorganic semiconductor nanoparticles a few nanometers in size that possess unique optical electronic properties vs those of larger materials. For example, QDs usually exhibit a strong and long-lived photoluminescence emission, a feature dependent on size, shape and composition. These special optoelectronic properties make them a promising alternative to conventional luminescent dyes as optical labels in biomedical applications including biomarker quantification, biomolecule targeting and molecular imaging. A key parameter for use of QDs is to functionalize their surface with suitable (bio)molecules to provide stability in aqueous solutions and efficient and selective tagging biomolecules of interest. Researchers have successfully developed biocompatible QDs and have linked them to various biomolecule recognition elements, i.e., antibodies, proteins, DNA, etc. In this chapter, QD synthesis and characterization strategies are reviewed as well as the development of nanoplatforms for luminescent biosensing and imaging-guided targeting. Relevant biomedical applications are highlighted with a particular focus on recent progress in ultrasensitive detection of clinical biomarkers. Finally, key future research goals to functionalize QDs as diagnostic tools are explored.

Absolute quantification of proteins using element mass spectrometry and generic standards.

Elemental mass spectrometry is a powerful analytical technique widely established in inorganic analysis. However, despite its quantitative capabilities, it is not yet fully integrated or considered in Life Sciences fields like proteomics. Whereas it is true that ICP-MS has suffered from several instrumental and analytical limitations that have hindered its applicability in protein analysis, significant developments during the last decades have turned ICP-MS into an interesting and, in our opinion, a powerful tool to consider for accurate protein quantification without recourse to specific protein standards. Herein we will try to discuss how these traditional limitations in ICP-MS have been overcome, what further improvements are yet necessary (some of which are shared with MS-based proteomics platforms) and enlighten some of the already existing and potential applications of ICP-MS in absolute quantitative proteomics. SIGNIFICANCE: ICP-MS has the potential to become a complementary tool to help molecular mass spectrometry cope with existing limitations, especially those related to standardization and accuracy, in the absolute proteomics field. It can provide absolute quantification of diverse proteoforms using a single generic compound containing sulfur and/or another target element (e.g., phosphorous). Moreover, its applications in quantitative proteomics are no longer limited to protein standards certification or quantification of simple or purified mixtures. Interestingly, absolute quantification of proteins using ICP-MS is favored when carried out at the intact level, making it very compatible with top-down proteomics approaches. Recent instrumental and methodological advances enable synergic combination of ICP-MS with stablished LC-MS proteomics methodologies, setting the basis for its implementation in quantitative proteomics workflows.

Prognostic Factor Utility of BAP1 Immunohistochemistry in Uveal Melanoma: A Single Center Study in Spain.

Even today, the mortality rate for uveal melanoma (UM) remains very high. In our research, we sought to determine which pathological and clinical features were correlated with the prognosis of UM. BAP1 (BRCA1-Associated Protein 1) gene mutation has been analyzed as one of the strongest predictors for metastasis in UM. The BAP1 gene codifies the BAP1 protein which has a tumor suppressor function. The presence of this protein can be determined by BAP1 immunohistochemical staining. Eighty-four uveal melanoma patients and forty enucleated eyeballs were examined. Metastasis was present in 24 patients. Nuclear BAP1 staining was low in 23 patients. The presence of a higher large basal diameter tumor (p < 0.001), tumor infiltrating lymphocytes (p = 0.020), and a lack of nuclear BAP1 immunostaining (p = 0.001) ocurred significantly more often in the metastatic group. Metastasis-free survival was lower in patients with low nuclear BAP1 staining (p = 0.003). In conclusion, to the best of our knowledge, this is the first time that BAP1 staining has been studied in uveal melanoma in a Spanish community. We believe that this technique should become routine in the pathological examination of uveal melanoma in order to allow adequate classification of patients and to establish an individual follow-up plan.

Bilateral Drug-Induced Uveitis and Epiretinal Membrane during the Treatment of a Metastatic Cutaneous Melanoma.

Purpose: To report the case of a drug-induced uveitis during the treatment of a metastatic cutaneous melanoma.Case report: A 75-year-old female treated with Dabrafenib and Trametinib due to a cutaneous melanoma stage IV presented with blurriness in both eyes. The examination revealed bilateral intraocular signs of inflammation, and fundoscopy showed bilateral changes at the posterior pole, such as chorioretinal folds and Neurosensory Retinal Detachment (NRD). Due to a worsening of Visual Acuity (VA) and persistence of intraocular inflammation in spite of topical prednisolone acetate treatment, the therapy with Dabrafenib + Trametinib was interrupted, after having been administered for 4 months, and replaced by Nivolumab. Fundus abnormalities and intraocular inflammation improved, but VA remained low due to the presence of an epiretinal membrane in the right eye. Then, a decreasing course of prednisolone eye drops was introduced for one more month and finally interrupted without the cessation of Nivolumab.Conclusion: Drug-induced uveitis has been increasing in the last few years due to the development of new biological agents for treatment of different types of tumours. The management of these adverse events should be handled in collaboration with oncologists and ophthalmologists and must be individualised and based on the risk-benefit balance. A case report of an uveitis and subsequent development of an epiretinal membrane during the treatment with Dabrafenib, Trametinib and subsequent Nivolumab for a metastatic cutaneous melanoma is reported here, in order to note the importance of an adequate follow-up of patients treated with these drugs.

Haemophilus Influenzae Endogenous Endophthalmitis in an Immunocompetent Host.

Purpose: To report a case of endogenous endophthalmitis caused by Haemophilus influenzae in an immunocompetent host.Case report: A 13-year-old male presented with pain, blurriness, and decreased Visual Acuity (VA) of Hand Motion in his right eye. Slit-lamp examination revealed hypopyon. Fundoscopy showed vitritis, vasculitis, and retinal infiltrates. Echography revealed vitreous condensations. Empirical treatment with intravitreal Ganciclovir, oral Valacyclovir, and Dexamethasone was initiated with no improvement. Vitreous culture revealed Haemophilus influenzae growth. Then, intravenous Ciprofloxacin and Cefotaxime and intravitreal Ceftazidime were administered with gradual improvement. Lensectomy with Pars Plana Vitrectomy and intraocular tamponade was performed. Nevertheless, the visual outcome was poor.Conclusion: Pediatric endogenous bacterial endophthalmitis is a rare but potentially devastating infection which is often misdiagnosed. Moreover, we want to highlight the importance of an adequate clinical suspicion in cases of H. influenzae to prevent the serious complications seen in this report.

The histone demethylase KDM6B fine-tunes the host response to Streptococcus pneumoniae.

Streptococcus pneumoniae is a natural colonizer of the human respiratory tract and an opportunistic pathogen. Although epithelial cells are among the first to encounter pneumococci, the cellular processes and contribution of epithelial cells to the host response are poorly understood. Here, we show that a S. pneumoniae serotype 6B ST90 strain, which does not cause disease in a murine infection model, induces a unique NF-κB signature response distinct from an invasive-disease-causing isolate of serotype 4 (TIGR4). This signature is characterized by activation of p65 and requires a histone demethylase KDM6B. We show, molecularly, that the interaction of the 6B strain with epithelial cells leads to chromatin remodelling within the IL-11 promoter in a KDM6B-dependent manner, where KDM6B specifically demethylates histone H3 lysine 27 dimethyl. Remodelling of the IL-11 locus facilitates p65 access to three NF-κB sites that are otherwise inaccessible when stimulated by IL-1ß or TIGR4. Finally, we demonstrate through chemical inhibition of KDM6B with GSK-J4 inhibitor and through exogenous addition of IL-11 that the host responses to the 6B ST90 and TIGR4 strains can be interchanged both in vitro and in a murine model of infection in vivo. Our studies therefore reveal how a chromatin modifier governs cellular responses during infection.

Epithelial conjunctival neoplasias - the importance of an early diagnosis and optimal treatment.

Objective: To emphasize the importance of an early diagnosis and an adequate treatment in conjunctival tumors. Methods: We present two clinical cases and compare the course of each case: one of conjunctival intraepithelial neoplasia (CIN) which took a positive course, and a fatal case of squamous cell carcinoma (SCC) with intraocular and orbital extension. Results: Epithelial conjunctival malignancies are one of the most prevalent ocular surface tumors. Among these, CIN are the most common. CIN have an excellent prognosis, given adequate treatment. However, when the diagnosis of CIN is late, the epithelial basement membrane will be affected, resulting in SCC. SCC may have poorer results due to its capacity to infiltrate near tissues and create distant metastasis. Conclusion: It is not common today to treat patients with orbital extension of SCC; however, it is crucial to note the importance of an early diagnosis of conjunctival malignancies. An early diagnosis is essential to prevent the transformation to other life-threatening types.

Catalytic Gold Deposition for Ultrasensitive Optical Immunosensing of Prostate Specific Antigen.

A major challenge in the development of bioanalytical methods is to achieve a rapid and robust quantification of disease biomarkers present at very low concentration levels in complex biological samples. An immunoassay platform is presented herein for ultrasensitive and fast detection of the prostate-specific antigen (PSA), a well-recognized cancer biomarker. A sandwich type immunosensor has been developed employing a detection antibody labeled with inorganic nanoparticles acting as tags for further indirect quantification of the analyte. The required high sensitivity is then achieved through a controlled gold deposition on the nanoparticle surface, carried out after completing the recognition step of the immunoassay, thus effectively amplifying the size of the nanoparticles from nm to µm range. Due to such an amplification procedure, quantification of the biomolecule could be carried out directly on the immunoassay plates using confocal microscopy for measurement of the reflected light produced by gold-enlarged nanostructures. The high specificity of the immunoassay was demonstrated with the addition of a major abundant protein in serum (albumin) at much higher concentrations. An extremely low detection limit for PSA quantification (LOD of 1.1 fg·mL-1 PSA) has been achieved. Such excellent LOD is 2-3 orders of magnitude lower than the clinically relevant PSA levels present in biological samples (4-10 ng·mL-1) and even to monitor eventual recurrence after clinical treatment of a prostate tumor (0.1 ng·mL-1). In fact, the broad dynamic range obtained (4 orders of magnitude) would allow the PSA quantification of diverse samples at very different relevant levels.

Assessment of the Potential and Limitations of Elemental Mass Spectrometry in Life Sciences for Absolute Quantification of Biomolecules Using Generic Standards.

Inductively coupled plasma-mass spectrometry (ICP-MS) has been widely used in Life Sciences for the absolute quantification of biomolecules without specific standards, assuming the same response for generic compounds including complex biomolecules. However, contradictory results have been published on this regard. We present the first critical statistical comparison of the ICP-MS response factors obtained for 14 different relevant S-containing biomolecules (three peptides, four proteins, one amino acid, two cofactors, three polyethylene glycol (PEG) derivatives, and sulfate standard), covering a wide range of hydrophobicities and molecular sizes. Two regular flow nebulizers and a total consumption nebulizer (TCN) were tested. ICP-MS response factors were determined though calibration curves, and isotope dilution analysis was used to normalize the results. No statistical differences have been found for low-molecular-weight biocompounds, PEGs, and nonhydrophobic peptides using any of the nebulizers tested. Interestingly, while statistical differences were still found negligible (96-104%) for the proteins and hydrophobic peptide using the TCN, significantly lower response factors (87-40%) were obtained using regular flow nebulizers. Such differential behavior seems to be related mostly to hydrophobicity and partially to the molecular weight. Findings were validated using IDA in intact and digested bovine serum albumin solutions using the TCN (98 and 100%, respectively) and the concentric nebulizer (73 and 97%, respectively). Additionally, in the case of a phosphoprotein, results were corroborated using the P trace in parallel to the S trace used along the manuscript. This work seems to suggest that ICP-MS operated with regular nebulizers can offer absolute quantification using generic standards for most biomolecules except proteins and hydrophobic peptides.

Actin Assembly around the Shigella-Containing Vacuole Promotes Successful Infection.

The enteroinvasive bacterium Shigella flexneri forces its uptake into non-phagocytic host cells through the translocation of T3SS effectors that subvert the actin cytoskeleton. Here, we report de novo actin polymerization after cellular entry around the bacterium-containing vacuole (BCV) leading to the formation of a dynamic actin cocoon. This cocoon is thicker than any described cellular actin structure and functions as a gatekeeper for the cytosolic access of the pathogen. Host CDC42, TOCA-1, N-WASP, WIP, the Arp2/3 complex, cortactin, coronin, and cofilin are recruited to the actin cocoon. They are subverted by T3SS effectors, such as IpgD, IpgB1, and IcsB. IcsB immobilizes components of the actin polymerization machinery at the BCV dependent on its fatty acyltransferase activity. This represents a unique microbial subversion strategy through localized entrapment of host actin regulators causing massive actin assembly. We propose that the cocoon promotes subsequent invasion steps for successful Shigella infection.

Itching eyes after itching around the head.

Objective: To report the occurrence and management of an eyelashes infestation by Phthirus pubis. Methods: A 27-year-old female presented with itching in her right eye and head after she had been traveling in southern Italy five days earlier. Visual acuity (VA) was 20/20 in both eyes. Slit-lamp examination showed bilateral blepharitis. Moreover, mobile insects and eggs attached to the eyelashes were observed. A microbiological study was performed with a Phthirus pubis result. The patient was treated with mechanical dislodging. Results: The resolution of the infection was carried out removing every insect and egg. A vaseline application twice daily for 7 days was necessary to stifle any nits that could remain. Conclusions: The eyelashes pediculosis is frequently caused by Phthirus pubis. Only a minor percentage of the cases are due to Phthirus capitis, but the differential diagnosis is essential: there are Phthirus pubis pediculosis cases due to sexual abuse. A sexual history and screening for other sexually transmitted diseases is warranted.

B Cell Development and T-Dependent Antibody Response Are Regulated by p38γ and p38δ.

p38MAP kinase (MAPK) signal transduction pathways are important regulators of inflammation and the immune response; their involvement in immune cell development and function is still largely unknown. Here we analysed the role of the p38 MAPK isoforms p38γ and p38δ in B cell differentiation in bone marrow (BM) and spleen, using mice lacking p38γ and p38δ, or conditional knockout mice that lack both p38γ and p38δ specifically in the B cell compartment. We found that the B cell differentiation programme in the BM was not affected in p38γ/δ-deficient mice. Moreover, these mice had reduced numbers of peripheral B cells as well as altered marginal zone B cell differentiation in the spleen. Expression of co-stimulatory proteins and activation markers in p38γ/δ-deficient B cells are diminished in response to B cell receptor (BCR) and CD40 stimulation; p38γ and p38δ were necessary for B cell proliferation induced by BCR and CD40 but not by TLR4 signaling. Furthermore, p38γ/δ-null mice produced significantly lower antibody responses to T-dependent antigens. Our results identify unreported functions for p38γ and p38δ in B cells and in the T-dependent humoral response; and show that the combined activity of these kinases is needed for peripheral B cell differentiation and function.