Contributing to the management of viral infections through simple immunosensing of the arachidonic acid serum level.

A trendsetting direct competitive-based biosensing tool has been developed and implemented for the determination of the polyunsaturated fatty acid arachidonic acid (ARA), a highly significant biological regulator with decisive roles in viral infections. The designed methodology involves a competitive reaction between the target endogenous ARA and a biotin-ARA competitor for the recognition sites of anti-ARA antibodies covalently attached to the surface of carboxylic acid-coated magnetic microbeads (HOOC-MµBs), followed by the enzymatic label of the biotin-ARA residues with streptavidin-horseradish peroxidase (Strep-HRP) conjugate. The resulting bioconjugates were magnetically trapped onto the sensing surface of disposable screen-printed carbon transducers (SPCEs) to monitor the extent of the biorecognition reaction through amperometry. The operational functioning of the exhaustively optimized and characterized immunosensing bioplatform was highly convenient for the quantitative determination of ARA in serum samples from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2-) and respiratory syncytial virus (RSV)-infected individuals in a rapid, affordable, trustful, and sensitive manner.

Gold Nanoparticle-Decorated Catalytic Micromotor-Based Aptassay for Rapid Electrochemical Label-Free Amyloid-ß42 Oligomer Determination in Clinical Samples from Alzheimer's Patients.

Micromotor (MM) technology offers a valuable and smart on-the-move biosensing microscale approach in clinical settings where sample availability is scarce in the case of Alzheimer's disease (AD). Soluble amyloid-ß protein oligomers (AßO) (mainly AßO42) that circulate in biological fluids have been recognized as a molecular biomarker and therapeutic target of AD due to their high toxicity, and they are correlated much more strongly with AD compared to the insoluble Aß monomers. A graphene oxide (GO)-gold nanoparticles (AuNPs)/nickel (Ni)/platinum nanoparticles (PtNPs) micromotors (MMGO-AuNPs)-based electrochemical label-free aptassay is proposed for sensitive, accurate, and rapid determination of AßO42 in complex clinical samples such as brain tissue, cerebrospinal fluid (CSF), and plasma from AD patients. An approach that implies the in situ formation of AuNPs on the GO external layer of tubular MM in only one step during MM electrosynthesis was performed (MMGO-AuNPs). The AßO42 specific thiolated-aptamer (AptAßO42) was immobilized in the MMGO-AuNPs via Au-S interaction, allowing for the selective recognition of the AßO42 (MMGO-AuNPs-AptAßO42-AßO42). AuNPs were smartly used not only to covalently bind a specific thiolated-aptamer for the design of a label-free electrochemical aptassay but also to improve the final MM propulsion performance due to their catalytic activity (approximately 2.0× speed). This on-the-move bioplatform provided a fast (5 min), selective, precise (RSD < 8%), and accurate quantification of AßO42 (recoveries 94-102%) with excellent sensitivity (LOD = 0.10 pg mL-1) and wide linear range (0.5-500 pg mL-1) in ultralow volumes of the clinical sample of AD patients (5 µL), without any dilution. Remarkably, our MM-based bioplatform demonstrated the competitiveness for the determination of AßO42 in the target samples against the dot blot analysis, which requires more than 14 h to provide qualitative results only. It is also important to highlight its applicability to the potential analysis of liquid biopsies as plasma and CSF samples, improving the reliability of the diagnosis given the heterogeneity and temporal complexity of neurodegenerative diseases. The excellent results obtained demonstrate the analytical potency of our approach as a future tool for clinical/POCT (Point-of-care testing) routine scenarios.

Bringing to Light the Importance of the miRNA Methylome in Colorectal Cancer Prognosis Through Electrochemical Bioplatforms.

This work reports the first electrochemical bioplatforms developed for the determination of the total contents of either target miRNA or methylated target miRNA. The bioplatforms are based on the hybridization of the target miRNA with a synthetic biotinylated DNA probe, the capture of the formed DNA/miRNA heterohybrids on the surface of magnetic microcarriers, and their recognition with an antibody selective to these heterohybrids or to the N6-methyladenosine (m6A) epimark. The determination of the total or methylated target miRNA was accomplished by labeling such secondary antibodies with the horseradish peroxidase (HRP) enzyme. In both cases, amperometric transduction was performed on the surface of disposable electrodes after capturing the resulting HRP-tagged magnetic bioconjugates. Because of their increasing relevance in colorectal cancer (CRC) diagnosis and prognosis, miRNA let-7a and m6A methylation were selected. The proposed electrochemical bioplatforms showed attractive analytical and operational characteristics for the determination of the total and m6A-methylated target miRNA in less than 75 min. These bioplatforms, innovative in design and application, were applied to the analysis of total RNA samples extracted from cultured cancer cells with different metastatic profiles and from paired healthy and tumor tissues of patients diagnosed with CRC at different stages. The obtained results demonstrated, for the first time using electrochemical platforms, the potential of interrogating the target miRNA methylation level to discriminate the metastatic capacities of cancer cells and to identify tumor tissues and, in a pioneering way, the potential of the m6A methylation in miRNA let-7a to serve as a prognostic biomarker for CRC.

Micromotor-based electrochemical immunoassays for reliable determination of amyloid-ß (1-42) in Alzheimer's diagnosed clinical samples.

Alzheimer's disease (AD), in addition to being the most common cause of dementia, is very difficult to diagnose, with the 42-amino acid form of Aß (Aß-42) being one of the main biomarkers used for this purpose. Despite the enormous efforts made in recent years, the technologies available to determine Aß-42 in human samples require sophisticated instrumentation, present high complexity, are sample and time-consuming, and are costly, highlighting the urgent need not only to develop new tools to overcome these limitations but to provide an early detection and treatment window for AD, which is a top-challenge. In recent years, micromotor (MM) technology has proven to add a new dimension to clinical biosensing, enabling ultrasensitive detections in short times and microscale environments. To this end, here an electrochemical immunoassay based on polypyrrole (PPy)/nickel (Ni)/platinum nanoparticles (PtNPs) MM is proposed in a pioneering manner for the determination of Aß-42 in left prefrontal cortex brain tissue, cerebrospinal fluid, and plasma samples from patients with AD. MM combines the high binding capacity of their immunorecognition external layer with self-propulsion through the catalytic generation of oxygen bubbles in the internal layer due to decomposition of hydrogen peroxide as fuel, allowing rapid bio-detection (15 min) of Aß-42 with excellent selectivity and sensitivity (LOD = 0.06 ng/mL). The application of this disruptive technology to the analysis of just 25 µL of the three types of clinical samples provides values concordant with the clinical values reported, thus confirming the potential of the MM approach to assist in the reliable, simple, fast, and affordable diagnosis of AD by determining Aß-42.

A Non-Canonical Hippo Pathway Represses the Expression of ΔNp63.

The p63 transcription factor, a member of the p53 family, plays an oncogenic role in squamous cell carcinomas, while in breast cancers its expression is often repressed. In the canonical conserved Hippo pathway, known to play a complex role in regulating growth of cancer cells, protein kinases MST1/2 and LATS1/2 act sequentially to phosphorylate and inhibit the YAP/TAZ transcription factors. We found that in MCF10A mammary epithelial cells as well as in squamous and breast cancer cell lines, expression of ΔNp63 RNA and protein is strongly repressed by inhibition of the Hippo pathway protein kinases. While MST1/2 and LATS1 are required for p63 expression, the next step of the pathway, namely phosphorylation and degradation of the YAP/TAZ transcriptional activators is not required for p63 repression. This suggests that regulation of p63 expression occurs by a noncanonical version of the Hippo pathway. We identified similarly regulated genes, suggesting the broader importance of this pathway. Interestingly, lowering p63 expression lead to increased YAP protein levels, indicating crosstalk of the YAP/TAZ-independent and -dependent branches of the Hippo pathway. These results, which reveal the intersection of the Hippo and p63 pathways, may prove useful for the control of their activities in cancer cells.

Functional Proteomics Characterization of the Role of SPRYD7 in Colorectal Cancer Progression and Metastasis.

SPRY domain-containing protein 7 (SPRYD7) is a barely known protein identified via spatial proteomics as being upregulated in highly metastatic-to-liver KM12SM colorectal cancer (CRC) cells in comparison to its isogenic poorly metastatic KM12C CRC cells. Here, we aimed to analyze SPRYD7's role in CRC via functional proteomics. Through immunohistochemistry, the overexpression of SPRYD7 was observed to be associated with the poor survival of CRC patients and with an aggressive and metastatic phenotype. Stable SPRYD7 overexpression was performed in KM12C and SW480 poorly metastatic CRC cells and in their isogenic highly metastatic-to-liver-KM12SM-and-to-lymph-nodes SW620 CRC cells, respectively. Upon upregulation of SPRYD7, in vitro and in vivo functional assays confirmed a key role of SPRYD7 in the invasion and migration of CRC cells and in liver homing and tumor growth. Additionally, transient siRNA SPRYD7 silencing allowed us to confirm in vitro functional results. Furthermore, SPRYD7 was observed as an inductor of angiogenesis. In addition, the dysregulated SPRYD7-associated proteome and SPRYD7 interactors were elucidated via 10-plex TMT quantitative proteins, immunoproteomics, and bioinformatics. After WB validation, the biological pathways associated with the stable overexpression of SPRYD7 were visualized. In conclusion, it was demonstrated here that SPRYD7 is a novel protein associated with CRC progression and metastasis. Thus, SPRYD7 and its interactors might be of relevance in identifying novel therapeutic targets for advanced CRC.

Benefits of FAIMS to Improve the Proteome Coverage of Deteriorated and/or Cross-Linked TMT 10-Plex FFPE Tissue and Plasma-Derived Exosomes Samples.

The proteome characterization of complex, deteriorated, or cross-linked protein mixtures as paired clinical FFPE or exosome samples isolated from low plasma volumes (250 µL) might be a challenge. In this work, we aimed at investigating the benefits of FAIMS technology coupled to the Orbitrap Exploris 480 mass spectrometer for the TMT quantitative proteomics analyses of these complex samples in comparison to the analysis of protein extracts from cells, frozen tissue, and exosomes isolated from large volume plasma samples (3 mL). TMT experiments were performed using a two-hour gradient LC-MS/MS with or without FAIMS and two compensation voltages (CV = -45 and CV = -60). In the TMT experiments of cells, frozen tissue, or exosomes isolated from large plasma volumes (3 mL) with FAIMS, a limited increase in the number of identified and quantified proteins accompanied by a decrease in the number of peptides identified and quantified was observed. However, we demonstrated here a noticeable improvement (>100%) in the number of peptide and protein identifications and quantifications for the plasma exosomes isolated from low plasma volumes (250 µL) and FFPE tissue samples in TMT experiments with FAIMS in comparison to the LC-MS/MS analysis without FAIMS. Our results highlight the potential of mass spectrometry analyses with FAIMS to increase the depth into the proteome of complex samples derived from deteriorated, cross-linked samples and/or those where the material was scarce, such as FFPE and plasma-derived exosomes from low plasma volumes (250 µL), which might aid in the characterization of their proteome and proteoforms and in the identification of dysregulated proteins that could be used as biomarkers.

Brain and immune system-derived extracellular vesicles mediate regulation of complement system, extracellular matrix remodeling, brain repair and antigen tolerance in Multiple sclerosis.

BACKGROUND: Multiple sclerosis (MS) is an immune-mediated central nervous system disease whose course is unpredictable. Finding biomarkers that help to better comprehend the disease's pathogenesis is crucial for supporting clinical decision-making. Blood extracellular vesicles (EVs) are membrane-bound particles secreted by all cell types that contain information on the disease's pathological processes. PURPOSE: To identify the immune and nervous system-derived EV profile from blood that could have a specific role as biomarker in MS and assess its possible correlation with disease state. RESULTS: Higher levels of T cell-derived EVs and smaller size of neuron-derived EVs were associated with clinical relapse. The smaller size of the oligodendrocyte-derived EVs was related with motor and cognitive impairment. The proteomic analysis identified mannose-binding lectin serine protease 1 and complement factor H from immune system cell-derived EVs as autoimmune disease-associated proteins. We observed hepatocyte growth factor-like protein in EVs from T cells and inter-alpha-trypsin inhibitor heavy chain 2 from neurons as white matter injury-related proteins. In patients with MS, a specific protein profile was found in the EVs, higher levels of alpha-1-microglobulin and fibrinogen ß chain, lower levels of C1S and gelsolin in the immune system-released vesicles, and Talin-1 overexpression in oligodendrocyte EVs. These specific MS-associated proteins, as well as myelin basic protein in oligodendrocyte EVs, correlated with disease activity in the patients with MS. CONCLUSION: Neural-derived and immune-derived EVs found in blood appear to be good specific biomarkers in MS for reflecting the disease state.

Electrochemical biotool for the dual determination of epithelial mucins associated to prognosis and minimal residual disease in colorectal cancer.

This work reports a dual immunoplatform for the simultaneous detection of two epithelial glycoproteins of the mucin family, mucin 1 (MUC1) and mucin 16 (MUC16), whose expression is related to adverse prognosis and minimal residual disease (MRD) in colorectal cancer (CRC). The developed immunoplatform involves functionalised magnetic microparticles (MBs), a set of specific antibody pairs (a capture antibody, cAb, and a biotinylated detector antibody b-dAb labelled with a streptavidin-horseradish peroxidase, Strep-HRP, polymer) for each target protein and amperometric detection at dual screen-printed carbon electrodes (SPdCEs) using the hydroquinone (HQ)/horseradish peroxidase (HRP)/H2O2 system. This dual immunoplatform allows, under the optimised experimental conditions, to achieve LOD values of 50 and 1.81 pg mL-1 (or mU mL-1) for MUC1 and MUC16, respectively, and adequate selectivity for the determination of the two targets in the clinic. The developed immunoplatform was employed to analyse CRC cell protein extracts (1.0 µg/determination) with different metastatic potential providing results in agreement with those obtained by blotting technologies but using affordable and applicable point-of-care instruments. This new biotool also emerges competitive in state-of-the-art electrochemical immunoplatforms seeking a compromise among simplicity, reduction of test time and analytical characteristics.

Advanced penile lymphoma: Case report and review of the literature.

Primary penile lymphomas are extremely rare. They are aggressive neoplasms that can present as double-or triple-hit lymphomas, and because the associate with a high risk of central nervous system dissemination, treatment consists of high-dose chemotherapy regimens plus intrathecal prophylaxis. Pathology can be confused with squamous cell carcinoma of the penis, leading to inappropriate treatments and unnecessary amputations. We report the case of a patient diagnosed with clinical Stage IV penile non-Hodgkin lymphoma that was treated with a complete and durable response. In addition, we review the available literature on penile lymphoma.

Characteristics of patients with hypersensitivity reactions to chemotherapeutic and biological agents and desensitization behavior / Características de pacientes com reações de hipersensibilidade a agentes quimioterápicos e biológicos e comportamento de dessensibilização

Introduction: Hypersensitivity to chemotherapeutic and biological agents has increased in recent years due to their frequent use. Avoidance has been the first line of defense, leading to decreased treatment efficacy and increased adverse events. Objective: To characterize the sociodemographic and clinical aspects of patients with hypersensitivity reactions to chemotherapeutic agents who underwent desensitization and biological procedures in a Colombian city. Methods: This observational, descriptive, retrospective, multicenter study was conducted in patients with hypersensitivity reactions to chemotherapeutic and biological agents who underwent desensitization. Results: In the 14 included patients with a history of hypersensitivity reactions to chemotherapeutic and biological agents (57.1% women; median age 42.5 years), 45 desensitization procedures were performed. The most commonly prescribed drug was rituximab (57%). The skin was the most frequent reaction site (78.6%), and systemic corticosteroids were the most common treatment (78.6%). Breakthrough reactions occurred in 31.1% of the patients and only premedication with corticosteroids was associated with less severe reactions. All cases of desensitization were successful. Conclusions: Desensitization to chemotherapeutic and biological agents proved to be a useful and safe tool in a Colombian population.

Introdução: A hipersensibilidade aos agentes quimioterápicos e biológicos aumentou nos últimos anos devido ao seu uso frequente. Evitar tem sido a primeira linha de ação, levando à diminuição da eficácia do tratamento e ao aumento de eventos adversos. Objetivos: Caracterizar os aspectos sociodemográficos e clínicos de pacientes com reações de hipersensibilidade a agentes quimioterápicos submetidos a dessensibilização e procedimentos biológicos em uma cidade colombiana. Métodos: Foi realizado um estudo observacional, descritivo, retrospectivo e multicêntrico em pacientes com reações de hipersensibilidade a agentes quimioterápicos e biológicos submetidos à dessensibilização. Resultados: Foram incluídos 45 procedimentos de dessensibilização em 14 pacientes com histórico de reações de hipersensibilidade a agentes quimioterápicos e biológicos (57,1% mulheres, com mediana de idade de 42,5 anos). O medicamento mais relatado foi o rituximabe (57%). O envolvimento cutâneo foi o mais frequente (78,6%) e os corticosteroides sistêmicos foram o tratamento mais utilizado (78,6%). As reações ocorreram em 31,1% e apenas a pré-medicação com corticosteroides foi associada a uma menor gravidade destas. Todos os casos de dessensibilização foram bem-sucedidos. Conclusões: A dessensibilização a agentes quimioterápicos e biológicos provou ser uma ferramenta útil e segura em uma população colombiana.

Proteomics analysis of prefrontal cortex of Alzheimer's disease patients revealed dysregulated proteins in the disease and novel proteins associated with amyloid-ß pathology.

BACKGROUND: Alzheimer's disease (AD) is a progressive, chronic, and neurodegenerative disease, and the most common cause of dementia worldwide. Currently, the mechanisms underlying the disease are far from being elucidated. Thus, the study of proteins involved in its pathogenesis would allow getting further insights into the disease and identifying new markers for AD diagnosis. METHODS: We aimed here to analyze protein dysregulation in AD brain by quantitative proteomics to identify novel proteins associated with the disease. 10-plex TMT (tandem mass tags)-based quantitative proteomics experiments were performed using frozen tissue samples from the left prefrontal cortex of AD patients and healthy individuals and vascular dementia (VD) and frontotemporal dementia (FTD) patients as controls (CT). LC-MS/MS analyses were performed using a Q Exactive mass spectrometer. RESULTS: In total, 3281 proteins were identified and quantified using MaxQuant. Among them, after statistical analysis with Perseus (p value < 0.05), 16 and 155 proteins were defined as upregulated and downregulated, respectively, in AD compared to CT (Healthy, FTD and VD) with an expression ratio ≥ 1.5 (upregulated) or ≤ 0.67 (downregulated). After bioinformatics analysis, ten dysregulated proteins were selected as more prone to be associated with AD, and their dysregulation in the disease was verified by qPCR, WB, immunohistochemistry (IHC), immunofluorescence (IF), pull-down, and/or ELISA, using tissue and plasma samples of AD patients, patients with other dementias, and healthy individuals. CONCLUSIONS: We identified and validated novel AD-associated proteins in brain tissue that should be of further interest for the study of the disease. Remarkably, PMP2 and SCRN3 were found to bind to amyloid-ß (Aß) fibers in vitro, and PMP2 to associate with Aß plaques by IF, whereas HECTD1 and SLC12A5 were identified as new potential blood-based biomarkers of the disease.

Nonsteroidal Anti-inflammatory Drug (NSAID) Tolerance After Biological Therapy in Patients With NSAID-Exacerbated Respiratory Disease: A Randomized Comparative Trial.

BACKGROUND: There are no prospective studies comparing how biological therapies affect nonsteroidal anti-inflammatory drug (NSAID) tolerance in NSAID-exacerbated respiratory disease. OBJECTIVE: To study the induction of NSAID tolerance after biological therapy in patients with NSAID-exacerbated respiratory disease. METHODS: A prospective pilot study in a real-world clinic setting was conducted among subjects with severe asthma and type 2 inflammation. A random allocation of therapy was carried out: benralizumab, dupilumab, mepolizumab, or omalizumab. NSAID intolerance was confirmed by an oral challenge test (OCT) using acetyl-salicylic acid (ASA-OCT). The principal outcome was NSAID tolerance according to OCT before and after 6 months of each biological therapy (intragroup comparisons). As exploratory outcomes, we compared NSAID tolerance between biological therapies (intergroup comparisons). RESULTS: A total of 38 subjects were included; 9 received benralizumab, 10 dupilumab, 9 mepolizumab, and 10 omalizumab. There was an increase in the concentration needed to produce a reaction during ASA-OCT with omalizumab (P < .001) and dupilumab (P = .004) but not with mepolizumab and benralizumab. Omalizumab and dupilumab achieved the highest frequency of NSAID tolerance (omalizumab 60%, dupilumab 40%, mepolizumab 22%, and benralizumab 22%). CONCLUSIONS: Biological therapies for asthma are useful for inducing NSAID tolerance; however, in patients with type 2 inflammation and high levels of total IgE, atopy, and eosinophils, anti-IgE or anti-IL4/13 seem to be more effective than antieosinophilic therapies. Omalizumab and dupilumab increased ASA tolerance, whereas mepolizumab and benralizumab did not. Future trials will be able to clarify this finding.

PKD phosphorylation and COP9/Signalosome modulate intracellular Spry2 protein stability.

Spry2 is a molecular modulator of tyrosine kinase receptor signaling pathways that has cancer-type-specific effects. Mammalian Spry2 protein undergoes tyrosine and serine phosphorylation in response to growth factor stimulation. Spry2 expression is distinctly altered in various cancer types. Inhibition of the proteasome functionality results in reduced intracellular Spry2 degradation. Using in vitro and in vivo assays, we show that protein kinase D (PKD) phosphorylates Spry2 at serine 112 and interacts in vivo with the C-terminal half of this protein. Importantly, missense mutation of Ser112 decreases the rate of Spry2 intracellular protein degradation. Either knocking down the expression of all three mammalian PKD isoforms or blocking their kinase activity with a specific inhibitor contributes to the stabilization of Spry2 wild-type protein. Downregulation of CSN3, a component of the COP9/Signalosome that binds PKD, significantly increases the half-life of Spry2 wild-type protein but does not affect the stability of a Spry2 after mutating Ser112 to the non-phosphorylatable residue alanine. Our data demonstrate that both PKD and the COP9/Signalosome play a significant role in control of Spry2 intracellular stability and support the consideration of the PKD/COP9 complex as a potential therapeutic target in tumors where Spry2 expression is reduced.

p53 and p63 Proteoforms Derived from Alternative Splicing Possess Differential Seroreactivity in Colorectal Cancer with Distinct Diagnostic Ability from the Canonical Proteins.

Colorectal cancer (CRC) is the third most common cancer and the second most frequent cause of cancer-related death worldwide. The detection in plasma samples of autoantibodies against specific tumor-associated antigens has been demonstrated to be useful for the early diagnosis of CRC by liquid biopsy. However, new studies related to the humoral immune response in cancer are needed to enable blood-based diagnosis of the disease. Here, our aim was to characterize the humoral immune response associated with the different p53 and p63 proteoforms derived from alternative splicing and previously described as aberrantly expressed in CRC. Thus, here we investigated the diagnostic ability of the twelve p53 proteoforms and the eight p63 proteoforms described to date, and their specific N-terminal and C-terminal end peptides, by means of luminescence HaloTag beads immunoassays. Full-length proteoforms or specific peptides were cloned as HaloTag fusion proteins and their seroreactivity analyzed using plasma from CRC patients at stages I-IV (n = 31), individuals with premalignant lesions (n = 31), and healthy individuals (n = 48). p53γ, Δ40p53ß, Δ40p53γ, Δ133p53γ, Δ160p53γ, TAp63α, TAp63δ, ΔNp63α, and ΔNp63δ, together with the specific C-terminal end α and δ p63 peptides, were found to be more seroreactive against plasma from CRC patients and/or individuals with premalignant lesions than from healthy individuals. In addition, ROC (receiver operating characteristic) curves revealed a high diagnostic ability of those p53 and p63 proteoforms to detect CRC and premalignant individuals (AUC higher than 85%). Finally, electrochemical biosensing platforms were employed in POC-like devices to investigate their usefulness for CRC detection using selected p53 and p63 proteoforms. Our results demonstrate not only the potential of these biosensors for the simultaneous analysis of proteoforms' seroreactivity, but also their convenience and versatility for the clinical detection of CRC by liquid biopsy. In conclusion, we here show that p53 and p63 proteoforms possess differential seroreactivity in CRC patients in comparison to controls, distinctive from canonical proteins, which should improve the diagnostic panels for obtaining a blood-based biomarker signature for CRC detection.

DRESS syndrome: A literature review and treatment algorithm.

Drug reaction with eosinophilia and systemic symptoms, known by its acronym in English as DRESS (Drug Reaction with Eosinophilia and Systemic Symptoms), clinically manifests with fever, facial edema, lymphadenopathy, a morbilliform rash, and organ involvement. Laboratory results reveal leukocytosis, atypical lymphocytes, eosinophilia, and alterations of liver and kidney function tests. The actual incidence of DRESS is unknown, because it may vary depending on the type of medication and the immune status of each patient; also, because many cases remain undiagnosed or untreated. The drugs most associated with DRESS include antiepileptics, antibiotics, antituberculosis, and non-steroidal anti-inflammatory agents (NSAIDs). Its diagnosis is sometimes made late and can become a challenge. The diagnostic criteria proposed by the international Registry of Severe Cutaneous Adverse Reactions (RegiSCAR) help to establish the diagnosis through a score system based on clinical and laboratory findings. The first step to identify the culprit is a thorough clinical history that includes all suspects, emphasizing those most known to cause DRESS syndrome according to the context and the literature. A skin biopsy may also be helpful in the diagnostic process. Patch testing is the test of choice to search for the culprit in cases of DRESS. Regarding prognosis, the estimated mortality due to DRESS is 3.8%. The main causes of mortality include fulminant hepatitis and liver necrosis. Several indicators of poor prognosis have been identified and these include an eosinophil count above 6000 × 103/µL, thrombocytopenia, pancytopenia, leukocytosis and coagulopathy. This article aims to review the evidence available regarding the epidemiology, pathophysiology, clinical and laboratory findings, diagnosis, and treatment of DRESS.

Li-Fraumeni Syndrome-Associated Dimer-Forming Mutant p53 Promotes Transactivation-Independent Mitochondrial Cell Death.

Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li-Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein. SIGNIFICANCE: A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches. See related commentary by Stieg et al., p. 1046. See related article by Gencel-Augusto et al., p. 1230. This article is highlighted in the In This Issue feature, p. 1027.

Anti-double stranded DNA antibodies: Electrochemical isotyping in autoimmune and neurological diseases.

This work reports the first amperometric biosensor for the simultaneous determination of the single or total content of the most relevant human immunoglobulin isotypes (hIgs) of anti-dsDNA antibodies, dsDNA-hIgG, dsDNA-hIgM, dsDNA-hIgA and dsDNA-three hIgs, which are considered relevant biomarkers in prevalent autoimmune diseases such as systemic lupus erythematosus (SLE) as well as of interest in neurodegenerative diseases such as Alzheimer's disease (AD). The bioplatform involves the use of neutravidin-functionalized magnetic microparticles (NA-MBs) modified with a laboratory-prepared biotinylated human double-stranded DNA (b-dsDNA) for the efficient capture of specific autoantibodies that are enzymatically labeled with horseradish peroxidase (HRP) enzyme using specific secondary antibodies for each isotype or a mixture of secondary antibodies for the total content of the three isotypes. Transduction was performed by amperometry (-0.20 V vs. the Ag pseudo-reference electrode) using the H2O2/hydroquinone (HQ) system after trapping the resulting magnetic bioconjugates on each of the four working electrodes of a disposable quadruple transduction platform (SP4CEs). The bioplatform demonstrated attractive operational characteristics for clinical application and was employed to determine the individual or total hIgs classes in serum from healthy individuals and from patients diagnosed with SLE and AD. The target concentrations in AD patients are provided for the first time in this work. In addition, the results for SLE patients and control individuals agree with those obtained by applying ELISA tests as well as with the clinical ranges reported by other authors, using individual detection methodologies restricted to centralized settings or clinical laboratories.

In-depth quantitative proteomics analysis revealed C1GALT1 depletion in ECC-1 cells mimics an aggressive endometrial cancer phenotype observed in cancer patients with low C1GALT1 expression.

BACKGROUND: Endometrial cancer (EC) is the most common cancer of the female reproductive organs. Despite the good overall prognosis of most low-grade ECs, FIGO I and FIGO II patients might experience tumor recurrence and worse prognosis. The study of alterations related to EC pathogenesis might help to get insights into underlying mechanisms involved in EC development and progression. METHODS: Core tumoral samples were used to investigate the role of C1GALT1 in EC by immunohistochemistry (IHC). ECC-1 cells were used as endometrioid EC model to investigate the effect of C1GALT1 depletion using C1GALT1 specific shRNAs. SILAC quantitative proteomics analyses and cell-based assays, PCR, qPCR, WB, dot-blot and IHC analyses were used to identify, quantify and validate dysregulation of proteins. RESULTS: Low C1GALT1 protein expression levels associate to a more aggressive phenotype of EC. Out of 5208 proteins identified and quantified by LC-MS/MS, 100 proteins showed dysregulation (log2fold-change ≥ 0.58 or ≤-0.58) in the cell protein extracts and 144 in the secretome of C1GALT1 depleted ECC-1 cells. Nine dysregulated proteins were validated. Bioinformatics analyses pointed out to an increase in pathways associated with an aggressive phenotype. This finding was corroborated by loss-of-function cell-based assays demonstrating higher proliferation, invasion, migration, colony formation and angiogenesis capacity in C1GALT1 depleted cells. These effects were associated to the overexpression of ANXA1, as demonstrated by ANXA1 transient silencing cell-based assays, and thus, correlating C1GALT and ANXA1 protein expression and biological effects. Finally, the negative protein expression correlation found by proteomics between C1GALT1 and LGALS3 was confirmed by IHC. CONCLUSION: C1GALT1 stably depleted ECC-1 cells mimic an EC aggressive phenotype observed in patients and might be useful for the identification and validation of EC markers of progression.

A non-canonical Hippo pathway represses the expression of ΔNp63.

The p63 transcription factor, a member of the p53 family, plays an oncogenic role in squamous cancers, while in breast cancers its expression is often repressed. In the canonical conserved Hippo pathway, known to play a complex role in regulating growth of cancer cells, the protein kinases MST1/2 and LATS1/2 act sequentially to phosphorylate and inhibit the YAP/TAZ transcription factors. We found that in the MCF10A mammary epithelial cell line as well as in squamous and breast cancer cell lines, expression of ΔNp63 RNA and protein is strongly repressed by inhibition of the Hippo pathway protein kinases in a manner that is independent of p53. While MST1/2 and LATS1 are required for p63 expression, the next step of the pathway, namely phosphorylation and degradation of the YAP/TAZ transcriptional activators is not required for repression of p63. This suggests that regulation of p63 expression occurs by a non-canonical version of the Hippo pathway. We additionally identified additional genes that were similarly regulated suggesting the broader importance of this pathway. Interestingly, we observed that experimentally lowering p63 expression leads to increased YAP protein levels, thereby constituting a feedback loop. These results, which reveal the intersection of the Hippo and p63 pathways, may prove useful for the control of their activities in cancer cells. One Sentence Summary: Regulation of p63 expression occurs by a non-canonical version of the Hippo pathway in mammary epithelial, breast carcinoma and head and neck squamous carcinoma cells.