Behaviour Hallmarks in Alzheimer's Disease 5xFAD Mouse Model.

The 5xFAD transgenic mouse model widely used in Alzheimer's disease (AD) research recapitulates many AD-related phenotypes with a relatively early onset and aggressive age-dependent progression. Besides developing amyloid peptide deposits alongside neuroinflammation by the age of 2 months, as well as exhibiting neuronal decline by the age of 4 months that intensifies by the age of 9 months, these mice manifest a broad spectrum of behavioural impairments. In this review, we present the extensive repertoire of behavioural dysfunctions in 5xFAD mice, organised into four categories: motor skills, sensory function, learning and memory abilities, and neuropsychiatric-like symptoms. The motor problems, associated with agility and reflex movements, as well as balance and coordination, and skeletal muscle function, typically arise by the time mice reach 9 months of age. The sensory function (such as taste, smell, hearing, and vision) starts to deteriorate when amyloid peptide buildups and neuroinflammation spread into related anatomical structures. The cognitive functions, encompassing learning and memory abilities, such as visual recognition, associative, spatial working, reference learning, and memory show signs of decline from 4 to 6 months of age. Concerning neuropsychiatric-like symptoms, comprising apathy, anxiety and depression, and the willingness for exploratory behaviour, it is believed that motivational changes emerge by approximately 6 months of age. Unfortunately, numerous studies from different laboratories are often contradictory on the conclusions drawn and the identification of onset age, making preclinical studies in rodent models not easily translatable to humans. This variability is likely due to a range of factors associated with animals themselves, housing and husbandry conditions, and experimental settings. In the forthcoming studies, greater clarity in experimental details when conducting behavioural testing in 5xFAD transgenic mice could minimise the inconsistencies and could ensure the reliability and the reproducibility of the results.

Insights on the Use of Transgenic Mice Models in Alzheimer's Disease Research.

Alzheimer's disease (AD), the leading cause of dementia, presents a significant global health challenge with no known cure to date. Central to our understanding of AD pathogenesis is the ß-amyloid cascade hypothesis, which underlies drug research and discovery efforts. Despite extensive studies, no animal models of AD have completely validated this hypothesis. Effective AD models are essential for accurately replicating key pathological features of the disease, notably the formation of ß-amyloid plaques and neurofibrillary tangles. These pathological markers are primarily driven by mutations in the amyloid precursor protein (APP) and presenilin 1 (PS1) genes in familial AD (FAD) and by tau protein mutations for the tangle pathology. Transgenic mice models have been instrumental in AD research, heavily relying on the overexpression of mutated APP genes to simulate disease conditions. However, these models do not entirely replicate the human condition of AD. This review aims to provide a comprehensive evaluation of the historical and ongoing research efforts in AD, particularly through the use of transgenic mice models. It is focused on the benefits gathered from these transgenic mice models in understanding ß-amyloid toxicity and the broader biological underpinnings of AD. Additionally, the review critically assesses the application of these models in the preclinical testing of new therapeutic interventions, highlighting the gap between animal models and human clinical realities. This analysis underscores the need for refinement in AD research methodologies to bridge this gap and enhance the translational value of preclinical studies.

Multifunctional Poly(2-ethyl-2-oxazoline) Copolymers Containing Dithiolane and Pentafluorophenyl Esters as Effective Reactive Linkers for Gold Surface Coatings.

Surface functionalization with biological macromolecules is an important task for the development of sensor materials, whereby the interaction with other biological materials should be suppressed. In this work, we developed a novel multifunctional poly(2-ethyl-2-oxazoline)-dithiolane conjugate as a versatile linker for gold surface immobilization of amine-containing biomolecules, containing poly(2-ethyl-2-oxazoline) as antifouling polymer, dithiolane for surface immobilization, and activated esters for protein conjugation. First, a well-defined carboxylic acid containing copoly(2-ethyl-2-oxazoline) was synthesized by cationic ring-opening copolymerization of 2-ethyl-2-oxazoline with a methyl ester-containing 2-oxazoline monomer, followed by postpolymerization modifications. The side-chain carboxylic groups were then converted to amine-reactive pentafluorophenyl (PFP) ester groups. Part of the PFP groups was used for the attachment of the dithiolane moiety, which can efficiently bind to gold surfaces. The final copolymer contained 1.4 mol% of dithiolane groups and 4.5 mol% of PFP groups. The copolymer structure was confirmed by several analytical techniques, including NMR spectroscopy and size-exclusion chromatography. The kinetics of the PFP ester aminolysis and hydrolysis demonstrated significantly faster amidation compared to hydrolysis, which is essential for subsequent protein conjugation. Successful coating of gold surfaces with the polymer was confirmed by spectroscopic ellipsometry, showing a polymer brush thickness of 4.77 nm. Subsequent modification of the coated surfaces was achieved using bovine serum albumin as a model protein. This study introduces a novel reactive polymer linker for gold surface functionalization and offers a versatile polymer platform for various applications including biosensing and surface functionalization.

A three-dimensionally printed otological model for cholesteatoma mastoidectomy training.

PURPOSE: To relate the creation and expert validation (face and content validity) of an affordable three-dimensional (3-D) printed model of temporal bones with chronic otitis media with cholesteatoma (COMC) as a simulator for mastoidectomy. METHODS: We performed computed tomography (CT) of the temporal bones of a patient with COMC followed at the University of São Paulo (USP) Hospital with 3-D Slicer to create a 3-D model of the affected bone using light-curing resin and silicone (cholesteatoma). The final 3-D printed images were scored by 10 otologists using a customized version of the Michigan Standard Simulation Scale Experience (MiSSES). Internal consistency and inter-rater reliability were assessed using Cronbach's α and intraclass correlations. RESULTS: Otologists consistently scored the model positively for fidelity, educational value, reactions, and the overall model quality. Nine otologists agreed that the model was a good educational device for surgical training of COMC. All experts deemed the model ready-or nearly ready-for use. The final cost of the model, including raw materials and manufacturing, was 120 USD. CONCLUSIONS: Using 3-D printing technology, we created the first anatomically accurate, low-cost, disease-reproducing 3-D model of temporal bones for mastoidectomy training for cholesteatoma.

Combined effects of dietary Laminaria digitata with alginate lyase on plasma metabolites and hepatic lipid, pigment and mineral composition of broilers.

BACKGROUND: The Laminaria digitata is an abundant macroalga and a sustainable feedstock for poultry nutrition. L. digitata is a good source of essential amino acids, carbohydrates and vitamins, including A, D, E, and K, as well as triacylglycerols and minerals, in particular iron and calcium. However, the few studies available in the literature with broilers document the application of this macroalga as a dietary supplement rather than a feed ingredient. No study has addressed up until now the effects of a high-level incorporation (> 2% in the diet) of L. digitata on plasma biochemical markers and hepatic lipid composition, as well as minerals and pigments profile in the liver of broilers. Our experimental design included one hundred and twenty Ross 308 male birds contained in 40 wired-floor cages and distributed to the following diets at 22 days of age (n = 10) for 15 days: 1) a corn-soybean basal diet (Control); 2) the basal diet plus 15% of L. digitata (LA); 3) the basal diet plus 15% of L. digitata with 0.005% of Rovabio® Excel AP (LAR); and 4) the basal diet plus 15% of L. digitata with 0.01% of the recombinant CAZyme, alginate lyase (LAE). RESULTS: L. digitata compromised birds' growth performance by causing a reduction in final body weight. It was found an increase in hepatic n-3 and n-6 fatty acids, in particular C18:2n-6, C18:3n-6, C20:4n-6, C20:5n-3, C22:5n-3 and C22:6n-3 with the addition of the macroalga, with or without feed enzymes, to the broiler diets. Also, the beneficial C18:3n-3 fatty acid was increased by combining L. digitata and commercial Rovabio® Excel AP compared to the control diet. The sum of SFA, MUFA and the n-6/n-3 PUFA ratio were decreased by L. digitata, regardless the addition of exogenous enzymes. ß-carotene was enhanced by L. digitata, individually or combined with CAZymes, being also responsible for a positive increase in total pigments. Macrominerals, in particular phosphorous and sulphur, were increased in the liver of broilers fed L. digitata individually relative to the control. For microminerals, copper, iron and the correspondent sum were consistently elevated in the liver of broilers fed L. digitata, individually or combined with exogenous CAZymes. The powerful discriminant analysis tool based on the hepatic characterization revealed a good separation between the control group and L. digitata diets but failed to discriminate the addition of feed enzymes. CONCLUSIONS: Overall, this study highlights the value of L. digitata as a feed ingredient for the poultry industry. Moreover, we can conclude that the effect of L. digitata overpowers the effect of feed enzymes, both the Rovabio® Excel AP and the alginate lyase. Having in mind the negative effects observed on birds' performance, our main recommendation at this stage is to restraint L. digitata incorporation level in forthcoming nutritional studies.

Three-dimensional printing in otolaryngology education: a systematic review.

PURPOSE: The progressive expansion of the technology that facilitates the development of three-dimensional (3D) printing within the field of otorhinolaryngology has opened up a new study front in medicine. The objective of this study is to systematically review scientific publications describing the development of 3D models having applications in otorhinolaryngology, with emphasis on subareas with a large number of publications, as well as the countries in which the publications are concentrated. METHODS: In this literature review, specific criteria were used to search for publications on 3D models. The review considered articles published in English on the development of 3D models to teach otorhinolaryngology. The studies with presurgical purposes or without validation of the task by surgeons were excluded from this review. RESULTS: This review considered 39 articles published in 10 countries between 2012 and 2021. The works published prior to 2012 were not considered as per the inclusion criteria for the research. Among the 39 simulators selected for review, otology models comprised a total of 15 publications (38%); they were followed by rhinology, with 12 (31%); laryngology, with 8 (21%); and head and neck surgery, with 4 publications (10%). CONCLUSION: The use of 3D technology and printing is well established in the context of surgical education and simulation models. The importance of developing new technological tools to enhance 3D printing and the current limitations in obtaining appropriate animal and cadaver models signify the necessity of investing more in 3D models.

"Benefits of the pedicled osteoplastic flap as a surgical approach of mastoidectomy in cochlear implant surgery".

OBJECTIVE: To evaluate the esthetic and functional results of an osteoplastic flap for mastoid cavity closure in cochlear implant surgery. STUDY DESIGN: Double-blind, prospective, randomized clinical trial. SETTING: tertiary referral center. INTERVENTION(S): On hundred and twenty-six patients were randomized in 2 groups for cochlear implant surgery. Cases (n: 63) underwent simple mastoidectomy using an anteriorly pedicled osteoplastic flap for mastoid closure. In controls (n: 63), a traditional periosteal flap was used. Evaluation with the POSAS questionnaire was performed 1 year after surgery to assess surgical wound esthetics. Sixteen patients from each group had postoperative CT-scans and wideband tympanometry to assess mastoid aeration and middle ear absorbance. Gender and time after surgery were correlated. MAIN OUTCOME MEASURE(S): Evaluation of the quality of the surgical wound with the application of a questionnaire validated in the medical literature and translated into Portuguese language called POSAS, considering the perception of the blinded patient and doctor regarding the surgical technique proceeded. A lower POSAS score suggests better esthetics of the surgical wound. Secondary outcomes are volumetric measurement of aeration inside mastoid cavity using 3D computer tomography exam, which aims to analyze the influence of fibrocicatricial retraction in the surgical wound into the mastoid and the interference of its aeration volume in the absorption of sound in the middle ear, using the wideband tympanometry exam. RESULTS: The POSAS questionnaire in the Case group showed a lower level of local pain and itchiness, a skin color and thickness more similar to the surrounding skin and less irregularity and stiffness, with no influence from time after surgery and gender compared to the Control group. The median tomographic volume was 6.37 cc in the cases and 4.60 cc in controls. Wideband tympanometry showed general smaller sound absorbance in the Case group results, specially, at 1000 Hz frequency. No intraoperative or postoperative complications were observed with the osteoplastic flap. CONCLUSIONS: This technique is an effective and safe alternative to alleviate common problems of mastoid surgery for cochlear implantation. In addition to esthetic benefits, it has less interference in middle ear physiology of sound absorbance and less fibrous tissue into the mastoid cavity during the follow-up of more than 1 year.

Plasma Metabolites and Liver Composition of Broilers in Response to Dietary Ulva lactuca with Ulvan Lyase or a Commercial Enzyme Mixture.

The effect of a high incorporation level of Ulva lactuca, individually and supplemented with a Carbohydrate-Active enZyme (CAZyme) on broilers' plasma parameters and liver composition is assessed here. Twenty one-day-old Ross 308 male broilers were randomly assigned to one of four treatments (n = 10): corn/soybean meal based-diet (Control); based-diet with 15% U. lactuca (UL); UL diet with 0.005% of commercial carbohydrase mixture; and UL diet with 0.01% of recombinant ulvan lyase. Supplementing U. lactuca with the recombinant CAZyme slightly compromised broilers' growth by negatively affecting final body weight and average daily gain. The combination of U. lactuca with ulvan lyase also increased systemic lipemia through an increase in total lipids, triacylglycerols and VLDL-cholesterol (p < 0.001). Moreover, U. lactuca, regardless of the CAZyme supplementation, enhanced hepatic n-3 PUFA (mostly 20:5n-3) with positive decrease in n-6/n-3 ratio. However, broilers fed with U. lactuca with ulvan lyase reduced hepatic α- and γ-tocopherol concentrations relative to the control. Conversely, the high amount of pigments in macroalga diets led to an increase in hepatic ß-carotene, chlorophylls and total carotenoids. Furthermore, U. lactuca, alone or combined with CAZymes, enhanced hepatic total microminerals, including iron and manganese. Overall, plasma metabolites and liver composition changed favorably in broilers that were fed 15% of U. lactuca, regardless of enzyme supplementation.

Dietary Arthrospira platensis improves systemic antioxidant potential and changes plasma lipids without affecting related hepatic metabolic pathways in post-weaned piglets.

BACKGROUND: The ability of a high level of dietary Arthrospira platensis, individually or in combination with two exogenous carbohydrate-degrading enzymes (lysozyme and Rovabio®), to improve systemic antioxidant potential and hepatic lipid metabolism was tested in piglets. Forty male post-weaned piglets, sons of Large White × Landrace sows crossed with Pietrain boars, were allocated into 4 groups (n = 10) and fed during 28 days one of the following diets: 1) a control basal diet (cereal and soybean meal); 2) a basal diet with 10% of A. platensis (AP); 3) the AP diet supplemented with 0.005% of Rovabio® (AP + R); 4) the AP diet supplemented with 0.01% of lysozyme (AP + L). RESULTS: Arthrospira platensis decreased BW gain of piglets, regardless the addition of feed enzymes. The majority of plasma metabolites were affected by diets. A. platensis increased total lipids, total cholesterol and LDL-cholesterol, without changing hepatic fatty acid content or modulating, in an expressive manner, the transcriptional profile of lipid sensitive mediators. The antioxidant potential in general, and total carotenoids in particular, were improved by the microalga, regardless lysozyme or Rovabio®. CONCLUSIONS: Summing up, A. platensis, individually and combined with feed enzymes, impacts negatively on piglets' growth but improves the systemic antioxidant potential and changes plasma lipids with a minor modulation on related hepatic metabolic pathways.

Can Age Influence Functional Tests Differently to Predict Falls in Nursing Home and Community-Dwelling Older Adults?

Aim: The aim of this cross-sectional study was to determine which functional tests are able to predict falls in nursing home residents (NHR) and community-dwelling (CDW) older adults grouped by age. Methods: Two hundred and fifty-two older adults were assessed in the following tests, Mini-Mental State Examination, anthropometric characteristics, fall history and functional tests. The participants were assigned to one of the following groups: Young Older Adults (YOA; 60-69 years, n = 102; 27.5% NHR), Old Older Adults (OOA; 70-79 years; n = 100; 23.5% NHR) and Very Older Adults (VOA, ≥80 years; n = 50; 52% NHR). Results: The five times sit-to-stand showed 1.41 odds to experience a fall in the NHR of the VOA. In the CDW, the handgrip strength score decreased the odds to fall occurrence (0.92 odds) in the YOA, while the low score in timed up and go test of the OOA showed 1.21 odds to experience a fall. Conclusions: Therefore, the five times sit-to-stand test can predict falls in NHR of VOA groups, while handgrip strength and timed up and go tests can predict falls in CDW of YOA and OOA groups, respectively.

Future Perspectives in Detecting EGFR and ALK Gene Alterations in Liquid Biopsies of Patients with NSCLC.

Non-small-cell lung cancer (NSCLC) is a major cause of death worldwide. Alterations in such genes as EGFR and ALK are considered important biomarkers in NSCLC due to the existence of targeted therapies with specific tyrosine kinase inhibitors (TKIs). However, specific resistance-related mutations can occur during TKI treatment, which often result in therapy inefficacy. Liquid biopsies arise as a reliable tool for the early detection of these types of alterations, allowing a non-invasive follow-up of the patients. Furthermore, they can be essential for cancer screening, initial diagnosis and to check surgery success. Despite the great advantages of liquid biopsies in NSCLC and the high input that next-generation sequencing (NGS) approaches can provide in this field, its use in oncology is still limited. With improvement of assay sensitivity and the establishment of clinical guidelines for liquid biopsy analysis, it is expected that they will be used in routine procedures. This review focuses on the usefulness of liquid biopsies of NSCLC patients as a means to detect alterations in EGFR and ALK genes and in disease management, highlighting the impact of NGS methods.

Proteomic research and diagnosis in bladder cancer: state of the art review.

PURPOSE: Proteomic biomarkers have been emerging as alternative methods to the gold standard procedures of cystoscopy and urine cytology in the diagnosis and surveillance of bladder cancer (BC). This review aims to update the state of the art of proteomics research and diagnosis in BC. MATERIALS AND METHODS: We reviewed the current literature related to BC research on urinary, tissue, blood and cell line proteomics, using the Pubmed database. FINDINGS: Two urinary protein biomarkers are FDA-approved (NMP22® and BTA® tests), only if performed along with cystoscopy for surveillance after initial diagnosis, but not in the primary diagnostic setting due to high false-positive rates in case of infections, stones and hematuria. There are a great number of non-FDA approved proteins being studied, with good preliminary results; panels of proteins seem valuable tools to be refined in ongoing trials. Blood proteins are a bigger challenge, because of the complexity of the serum protein profile and the scarcity of blood proteomic studies in BC. Previous studies with the BC tissue proteome do not correlate well with the urinary proteome, likely due to the tumor heterogeneity. Cell line proteomic research helps in the understanding of basic mechanisms that drive BC development and progression; the main difficulty is culturing low-grade tumors in vitro, which represents the majority of BC tumors in clinical practice. CONCLUSION: Protein biomarkers have promising value in the diagnosis, surveillance and prognostic of BC. Urine is the most appropriate body fluid for biomarker research in BC due to its easiness of sampling, stability and enrichment of shed and secreted tumor-specific proteins. Panels of biomarkers may exhibit higher sensitivity than single proteins in the diagnosis of BC at larger populations due to clinical and tumor heterogeneity. Prospective clinical trials are warranted to validate the relevance of proteomic data in the clinical management of BC.

Effect of dietary inclusion of Spirulina on production performance, nutrient digestibility and meat quality traits in post-weaning piglets.

The effect of Spirulina (Arthrospira platensis), individually or in combination with two commercial carbohydrases, in piglet diets was assessed on growth performance, nutrient digestibility and meat quality traits. Forty post-weaned male piglets from Large White × Landrace sows crossed with Pietrain boars with an initial live weight of 12.0 ± 0.89 kg were used. Piglets were assigned to one of four dietary treatments (n = 10): cereal and soya bean meal base diet (control), base diet with 10% Spirulina (SP), SP diet supplemented with 0.005% Rovabio® Excel AP (SP + R) and SP diet supplemented with 0.01% lysozyme (SP + L). Animals were slaughtered after a 4-week experimental period. Growth performance was negatively affected by the incorporation of Spirulina in the diets, with an average decrease of 9.1% on final weight, in comparison with control animals. Total tract apparent digestibility (TTAD) of crude protein was higher (p < .05) in the control group than in other groups. In addition, lysozyme increased TTAD of crude fat and acid detergent fibre, relative to the SP and control groups, respectively. In addition, the incorporation of Spirulina, individually and supplemented with enzymes, did not impair meat quality traits. Surprisingly, no protective effect against lipid oxidation was observed with the inclusion of Spirulina in pork after 7 days of storage. This study indicates that growth performance of post-weaning piglets was impaired by the incorporation of 10% Spirulina in the diets, which is mediated by an increase in digesta viscosity and a lower protein digestibility, as a consequence of the resistance of microalga proteins to the action of endogenous peptidases. In addition, it also indicates that lysozyme, in contrast to Rovabio® Excel AP, is efficient in the degradation of Spirulina cell wall in piglet's intestine. However, the digestion of proteins liberated by Spirulina cell wall disruption is still a challenge.

Digital imaging-assisted quantification of H3K27me3 immunoexpression in luminal A/B-like, HER2-negative, invasive breast cancer predicts patient survival and risk of recurrence.

BACKGROUND: Breast cancer (BC) is a major health concern and better understanding of its biology might improve treatment decisions and patient outcomes. Histone3 Lysine27 tri-methylation (H3K27me3) is a post-translational histone modification frequently associated with altered gene expression. In BC patients, lower H3K27me3 expression has been associated with worse prognosis. We assessed H3K27me3 immunoexpression with digital imaging software assistance, in a cohort of luminal-like BC patients with long-term follow-up time and evaluated its association with clinically relevant endpoints and its clinical usefulness. METHODS: H3K27me3 immunoexpression was assessed, by means of digital-imaging system, in archival tissue samples of 160 luminal A/B-like HER2-negative invasive BC, stages I-III. Survival analysis was performed using Kaplan-Meier and Cox regression. Cases were categorized as 'low' or 'high' expression based on cut-off defined by receiver operating characteristic (ROC) curve analysis. RESULTS: The patient cohort showed a median age of 61-years, with a median follow-up time of 11.7 years. Low H3K27me3 expression (below 85% cut-off) was significantly associated with recurrence, both in univariable (HR = 1.99, 95%CI 1.066-3.724) and multivariable analysis when adjusting for grade and age (HR = 1.89, 95%CI 1.004-3.559). A trend for higher risk of death in low H3K27me3 expression BC was observed (p = 0.069), reaching statistical significance in younger patients (p = 0.021). CONCLUSIONS: H3K27me3 immunoexpression assessed by digital imaging scoring software is an independent prognosis biomarker in luminal-like BC patients and may assist in more individualized adjuvant treatment decisions, thus potentially reducing recurrences after curative-intent treatment, while sparing unnecessary toxicity.

Practicability of clinical application of bladder cancer molecular classification and additional value of epithelial-to-mesenchymal transition: prognostic value of vimentin expression.

BACKGROUND: Bladder cancer (BlCa) taxonomy has proved its impact in patient outcome and selection for targeted therapies, but such transcriptomic-based classification has not yet translated to routine practice. Moreover, epithelial-to-mesenchymal transition (EMT) has shown relevance in acquisition of more aggressive BlCa phenotype. We aimed to test the usefulness of the molecular classification, as defined by immunohistochemistry (a routinely performed and easy-to-implement technique), in a well-defined BlCa cohort of both non-muscle invasive (NMIBC) and muscle invasive (MIBC) disease. Also, we aimed to assess the additional prognostic value of the mesenchymal marker vimentin to the stratification strategy. METHODS: A total of 186 samples were available. Immunohistochemistry/RT-qPCR for luminal markers GATA3/FOXA1, basal markers KRT5/KRT6A and vimentin were performed. RESULTS: mRNA expression levels of the markers positively correlated with immunoexpression scores. We found substantial overlapping in immunoexpression of luminal and basal markers, evidencing tumor heterogeneity. In MIBC, basal tumors developed recurrence more frequently. NMIBC patients with higher vimentin immunoexpression endured poorer disease-free survival, and increased expression was observed from normal bladder-NMIBC-MIBC-metastases. CONCLUSIONS: The classification has the potential to be implemented in routine, but further adjustments in practical scoring should be defined; focusing on additional markers, including those related to EMT, may further refine BlCa molecular taxonomy.

Circulating miR-99a-5p Expression in Plasma: A Potential Biomarker for Early Diagnosis of Breast Cancer.

MicroRNAs have emerged as new diagnostic and therapeutic biomarkers for breast cancer. Herein, we analysed miR-99a-5p expression levels in primary tumours and plasma of breast cancer patients to evaluate its usefulness as a minimally invasive diagnostic biomarker. MiR-99a-5p expression levels were determined by quantitative real-time PCR in three independent cohorts of patients: (I) Discovery cohort: breast cancer tissues (n = 103) and healthy breast tissues (n = 26); (II) Testing cohort: plasma samples from 105 patients and 98 healthy donors; (III) Validation cohort: plasma samples from 89 patients and 85 healthy donors. Our results demonstrated that miR-99a-5p was significantly downregulated in breast cancer tissues compared to healthy breast tissues. Conversely, miR-99a-5p levels were significantly higher in breast cancer patients than in healthy controls in plasma samples from both testing and validation cohorts, and ROC curve analysis revealed that miR-99a-5p has good diagnostic potential even to detect early breast cancer. In conclusion, miR-99a-5p's deregulated expression distinguished healthy patients from breast cancer patients in two different types of samples (tissues and plasma). Interestingly, expression levels in plasma were significantly lower in healthy controls than in early-stage breast cancer patients. Our findings suggest circulating miR-99a-5p as a novel promising non-invasive biomarker for breast cancer detection.

The nonsense mutation MSH2 c.2152C>T shows a founder effect in Portuguese Lynch syndrome families.

The mutational spectrum of the MMR genes is highly heterogeneous, but specific mutations are observed at high frequencies in well-defined populations or ethnic groups, due to founder effects. The MSH2 mutation c.2152C>T, p.(Gln718*), has occasionally been described in Lynch families worldwide, including in Portuguese Lynch syndrome families. During genetic testing for Lynch syndrome at the Portuguese Oncology Institutes of Porto and Lisbon, this mutation was identified in 28 seemingly unrelated families. In order to evaluate if this alteration is a founder mutation, haplotype analysis using microsatellite and SNP markers flanking the MSH2 gene was performed in the 28 probands and 87 family members. Additionally, the geographic origin of these families was evaluated and the age of the mutation estimated. Twelve different haplotypes were phased for 13 out of the 28 families and shared a conserved region of ∼3.6 Mb. Based on the mutation and recombination events observed in the microsatellite haplotypes and assuming a generation time of 25 years, the age estimate for the MSH2 mutation was 273 ± 64 years. The geographic origins of these families were mostly from the Northern region of Portugal. Concluding, these results suggest that the MSH2 c.2152C>T alteration is a founder mutation in Portugal with a relatively recent origin. Furthermore, its high proportion indicates that screening for this mutation as a first step, together with the previously reported Portuguese founder mutations, may be cost-effective in genetic testing of Lynch syndrome suspects of Portuguese ancestry.

A two-enzyme constituted mixture to improve the degradation of Arthrospira platensis microalga cell wall for monogastric diets.

The main goal of this study was to test a rational combination of pre-selected carbohydrate-active enzymes (CAZymes) and sulphatases, individually or in combination, in order to evaluate its capacity to disrupt Arthrospira platensis cell wall, allowing the release of its valuable nutritional bioactive compounds. By the end, a two-enzyme constituted mixture (Mix), composed by a lysozyme and a α-amylase, was incubated with A. platensis suspension. The microalga cell wall disruption was evaluated through the amount of reducing sugars released from the cell wall complemented with the oligosaccharide profile by HPLC. An increase of the amount of reducing sugars up to 2.42 g/L in microalgae treated with the Mix relative to no treatment (p < .05), as well as a 7-fold increase of oligosaccharides amount (p < .001), were obtained. With resort of fluorescence microscopy, a 36% reduction of fluorescence intensity (p < .001) was observed using Calcofluor White staining. In the supernatant, the Mix caused a 1.34-fold increase in protein content (p = .018) relative to the control. Similarly, n-6 polyunsaturated fatty acids (PUFA) (p = .007), in particular 18:2n-6 (p = .016), monounsaturated fatty acids (MUFA) (p = .049) and chlorophyll a (p = .025) contents were higher in the supernatant of microalgae treated with the enzyme mixture in relation to the control. Taken together, these results point towards the disclosure of a novel two-enzyme mixture able to partial degrade A. platensis cell wall, improving its nutrients bioavailability for monogastric diets with the cost-effective advantage use of microalgae in animal feed industry.

E-cadherin clone 36 nuclear staining dictates adverse disease outcome in lobular breast cancer patients.

Breast cancer is a heterogeneous disease and additional biomarkers for individually predicting patient outcomes are needed. Aberrant membrane E-cadherin immunoexpression has been demonstrated in lobular breast cancer. Also, E-cadherin nuclear staining has been reported, associating with prognosis in various tumors. Here, we explore whether membrane or nuclear staining of E-cadherin has the potential to dictate prognosis of patients with lobular breast cancer. We selected a cohort of 285 consecutively diagnosed lobular breast cancer patients and performed immunohistochemistry for E-cadherin (clones 36, EP700Y, and NCH38) and P-cadherin (clone 56C1) in representative formalin-fixed paraffin-embedded blocks. All patients were female, HER2-negative and surgically treated in a single institution. Survival curves were computed by Kaplan-Meier analysis. Hazard ratios and respective 95% confidence intervals were estimated using Cox regression models. Statistical significance was set at p < 0.05. Nuclear staining for E-cadherin clone 36 was frequent (35%), contrarily to other antibodies tested. Negative correlation was found between nuclear and membrane E-cadherin clone 36 immunostaining (rs = -0.30, p < 0.001), whereas positive correlation was found between membrane immunoexpression of E-cadherin clone 36 and P-cadherin (rs = 0.31, p < 0.001). Patients with any evidence of E-cadherin clone 36 nuclear immunostaining disclosed significantly worse overall survival, disease-specific-survival and disease/progression-free survival (hazard ratio = 2.059, 95% confidence interval 1.313-3.230; hazard ratio = 1.980, 95% confidence interval 1.121-3.495; and hazard ratio = 2.341, 95% confidence interval 1.403-3.905, respectively). Differences in survival were more remarkable when considering nuclear E-cadherin immunoexpression in ≥50% tumor cells. Poorer survival was maintained in multivariable analysis, after adjusting for age, menopausal and PR status, treatment course, vascular invasion, tumor grade and stage. Our results support the use of antibodies against the cytoplasmic domain of E-cadherin, such as clone 36, which may reveal nuclear immunostaining and indicate more aggressive clinical course in patients with lobular breast cancer. We hypothesize that E-cadherin is cleaved and translocated to nucleus functioning as transcription factor.

m6A RNA modification and its writer/reader VIRMA/YTHDF3 in testicular germ cell tumors: a role in seminoma phenotype maintenance.

BACKGROUND: Covalent RNA modifications, such as N-6-methyladenosine (m6A), have been associated with various biological processes, but their role in cancer remains largely unexplored. m6A dynamics depends on specific enzymes whose deregulation may also impact in tumorigenesis. Herein, we assessed the differential abundance of m6A, its writer VIRMA and its reader YTHDF3, in testicular germ cell tumors (TGCTs), looking for clinicopathological correlates. METHODS: In silico analysis of TCGA data disclosed altered expression of VIRMA (52%) and YTHDF3 (48%), prompting subsequent validation. Formalin-fixed paraffin-embedded tissues from 122 TGCTs (2005-2016) were selected. RNA extraction, cDNA synthesis and real-time qPCR (Taqman assays) for VIRMA and YTHDF3 were performed, as well as immunohistochemistry for VIRMA, YTHDF3 and m6A, for staining intensity assessment. Associations between categorical variables were assessed using Chi square and Fisher's exact test. Distribution of continuous variables between groups was compared using the nonparametric Mann-Whitney and Kruskal-Wallis tests. Biomarker performance was assessed through receiver operating characteristics (ROC) curve construction and a cut-off was established by Youden's index method. Statistical significance was set at p < 0.05. RESULTS: In our cohort, VIRMA and YTHDF3 mRNA expression levels differed among TGCT subtypes, with Seminomas (SEs) depicting higher levels than Non-Seminomatous tumors (NSTs) (p < 0.01 for both). A positive correlation was found between VIRMA and YTHDF3 expression levels. VIRMA discriminated SEs from NSTs with AUC = 0.85 (Sensitivity 77.3%, Specificity 81.1%, PPV 71.6%, NPV 85.3%, Accuracy 79.7%). Immunohistochemistry paralleled transcript findings, as patients with strong m6A immunostaining intensity depicted significantly higher VIRMA mRNA expression levels and stronger VIRMA immunoexpression intensity (p < 0.001 and p < 0.01, respectively). CONCLUSION: Abundance of m6A and expression of VIRMA/YTHDF3 were different among TGCT subtypes, with higher levels in SEs, suggesting a contribution to SE phenotype maintenance. VIRMA and YTHDF3 might cooperate in m6A establishment in TGCTs, and their transcript levels accurately discriminate between SEs and NSTs, constituting novel candidate biomarkers for patient management.