Microrheological comparison of melanoma cells by atomic force microscopy.

Melanoma is one of the most severe cancers due to its great potential to form metastasis. Recent studies showed the importance of mechanical property assessment in metastasis formation which depends on the cytoskeleton dynamics and cell migration. Although cells are considered purely elastic, they are viscoelastic entities. Microrheology atomic force microscopy (AFM) enables the assessment of elasticity and viscous properties, which are relevant to cell behavior regulation. The current work compares the mechanical properties of human neonatal primary melanocytes (HNPMs) with two melanoma cell lines (WM793B and 1205LU cells), using microrheology AFM. Immunocytochemistry of F-actin filaments and phosphorylated focal adhesion kinase (p-FAK) and cell migration assays were performed to understand the differences found in microrheology AFM regarding the tumor cell lines tested. AFM revealed that HNPMs and tumor cell lines had distinct mechanical properties. HNPMs were softer, less viscous, presenting a higher power-law than melanoma cells. Immunostaining showed that metastatic 1205LU cells expressed more p-FAK than WM793B cells. Melanoma cell migration assays showed that WM73B did not close the gap, in contrast to 1205LU cells, which closed the gap at the end of 23 h. These data seem to corroborate the high migratory behavior of 1205LU cells. Microrheology AFM applied to HNPMs and melanoma cells allowed the quantification of elasticity, viscous properties, glassy phase, and power-law properties, which have an impact in cell migration and metastasis formation. AFM study is important since it can be used as a biomarker of the different stages of the disease in melanoma.

Bone Metastases Detection in Patients with Breast Cancer: Does Bone Scintigraphy Add Information to PET/CT?

BACKGROUND: Positron emission tomography/computed tomography (PET/CT) has become in recent years a tool for breast cancer (BC) staging. However, its accuracy to detect bone metastases is classically considered inferior to bone scintigraphy (BS). The purpose of this work is to compare the effectiveness of bone metastases detection between PET/CT and BS. MATERIALS AND METHODS: Prospective study of 410 female patients treated in a Comprehensive Cancer Center between 2014 and 2020 that performed PET/CT and BS for staging purposes. The image analysis was performed by 2 senior nuclear medicine physicians. The comparison was performed based on accuracy, sensitivity, and specificity on a patient and anatomical region level and was assessed using McNemar's Test. An average ROC was calculated for the anatomical region analysis. RESULTS: PET/CT presented higher values of accuracy and sensitivity (98.0% and 93.83%), surpassing BS (95.61% and 81.48%) in detecting bone disease. There was a significant difference in favor of PET/CT (sensitivity 93.83% vs. 81.48%), however, there is no significant difference in eliminating false positives (specificity 99.09% vs. 99.09%). PET/CT presented the highest accuracy and sensitivity values for most of the bone segments, only surpassed by BS for the cranium. There was a significant difference in favor of PET/CT in the upper limb, spine, thorax (sternum) and lower limb (pelvis and sacrum), and in favor of BS in the cranium. The ROC showed that PET/CT has a higher sensitivity and consistency across the bone segments. CONCLUSION: With the correct imaging protocol, PET/CT does not require BS for patients with BC staging.

Familial neurohypophyseal diabetes insipidus: clinical, genetic and functional studies of novel mutations in the arginine vasopressin gene.

PURPOSE: Familial neurohypophyseal diabetes insipidus (FNDI) is a rare disorder characterized by childhood-onset progressive polyuria and polydipsia due to mutations in the arginine vasopressin (AVP) gene. The aim of the study was to describe the clinical and molecular characteristics of families with neurohypophyseal diabetes insipidus. METHODS: Five Portuguese families with autosomal dominant FNDI underwent sequencing of the AVP gene and the identified mutations were functionally characterized by in vitro studies. RESULTS: Three novel and two recurrent heterozygous mutations were identified in the AVP gene. These consisted of one initiation codon mutation in the signal peptide coding region (c.2T > C, p.Met1?), three missense mutations in the neurophysin II (NPII) coding region (c.154T > C, p.Cys52Arg; c.289C > G, p.Arg97Gly; and c.293G > C, p.Cys98Ser), and one nonsense mutation in the NPII coding region (c.343G > T, p.Glu115Ter). In vitro transfection of neuronal cells with expression vectors containing each mutation showed that the mutations resulted in intracellular retention of the vasopressin prohormone. Patients showed progressive symptoms of polyuria and polydipsia, but with wide variability in severity and age at onset. No clear genotype-phenotype correlation was observed. CONCLUSION: The intracellular accumulation of mutant vasopressin precursors supports the role of cellular toxicity of the mutant proteins in the etiology of the disorder and explains the progressive onset of the disorder. These findings further expand the AVP mutational spectrum in FNDI and contribute to the understanding of the molecular pathogenic mechanisms involved in FNDI.

New prospects in skin regeneration and repair using nanophased hydroxyapatite embedded in collagen nanofibers.

This study reflects an exploitation of a composite matrix produced by electrospinning of collagen and electrospraying of nanophased hydroxyapatite (nanoHA), for skin regeneration applications. The main goal was to evaluate the effect of nanoHA, as source of localized calcium delivery, on human dermal fibroblasts, keratinocytes, and human mesenchymal stem cells (hMSCs) growth, proliferation, differentiation, and extracellular matrix production. This study revealed that calcium ions provided by nanoHA significantly enhanced cellular growth and proliferation rates and prevented adhesion of pathogenic bacteria strains typically found in human skin flora. Moreover, hMSCs were able to differentiate in both osteogenic and adipogenic lineages. Rat subcutaneous implantation of the membranes also revealed that no adverse reaction occurred. Therefore, the mechanically fit composite membrane presents a great potential to be used either as cell transplantation scaffold for skin wound regeneration or as wound dressing material in plastic surgery, burns treatment or skin diseases.

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.

Fifteen years of Servitude et Grandeur to the application of a biophysical technique in medicine: The tale of AFMBioMed.

AFMBioMed is the founding name under which international conferences and summer schools are organized around the application of atomic force microscopy in life sciences and nanomedicine. From its inception at the Atomic Energy Commission in Marcoule near 2004 to its creation in 2007 and to its 10th anniversary conference in Krakow, a brief narrative history of its birth and rise will demonstrate how and what such an organization brings to laboratories and the AFM community. With the current planning of the next AFMBioMed conference in Münster in 2019, it will be 15 years of commitment to these events.

Overexpression of circulating MiR-30b-5p identifies advanced breast cancer.

BACKGROUND: Breast cancer (BrC) remains the leading cause of cancer-related death in women, mainly due to recurrent and/or metastatic events, entailing the need for biomarkers predictive of progression to advanced disease. MicroRNAs hold promise as noninvasive cancer biomarkers due to their inherent stability and resilience in tissues and bodily fluids. There is increasing evidence that specific microRNAs play a functional role at different steps of the metastatic cascade, behaving as signaling mediators to enable the colonization of a specific organ. Herein, we aimed to evaluate the biomarker performance of microRNAs previously reported as associated with prognosis for predicting BrC progression in liquid biopsies. METHODS: Selected microRNAs were assessed using a quantitative reverse transcription-polymerase chain reaction in a testing cohort of formalin-fixed paraffin-embedded primary (n = 16) and metastatic BrC tissues (n = 22). Then, miR-30b-5p and miR-200b-3p were assessed in a validation cohort #1 of formalin-fixed paraffin-embedded primary (n = 82) and metastatic BrC tissues (n = 93), whereas only miR-30b-5p was validated on a validation cohort #2 of liquid biopsies from BrC patients with localized (n = 20) and advanced (n = 25) disease. ROC curve was constructed to evaluate prognostic performance. RESULTS: MiR-30b-5p was differentially expressed in primary tumors and paired metastatic lesions, with bone metastases displaying significantly higher miR-30b-5p expression levels, paralleling the corresponding primary tumors. Interestingly, patients with advanced disease disclosed increased circulating miR-30b-5p expression compared to patients with localized BrC. CONCLUSIONS: MiR-30b-5p might identify BrC patients at higher risk of disease progression, thus, providing a useful clinical tool for patients' monitoring, entailing earlier and more effective treatment. Nonetheless, validation in larger multicentric cohorts is mandatory to confirm these findings.

The contribution of 7q33 copy number variations for intellectual disability.

Copy number variations (CNVs) at the 7q33 cytoband are very rarely described in the literature, and almost all of the cases comprise large deletions affecting more than just the q33 segment. We report seven patients (two families with two siblings and their affected mother and one unrelated patient) with neurodevelopmental delay associated with CNVs in 7q33 alone. All the patients presented mild to moderate intellectual disability (ID), dysmorphic features, and a behavioral phenotype characterized by aggressiveness and disinhibition. One family presents a small duplication in cis affecting CALD1 and AGBL3 genes, while the other four patients carry two larger deletions encompassing EXOC4, CALD1, AGBL3, and CNOT4. This work helps to refine the phenotype and narrow the minimal critical region involved in 7q33 CNVs. Comparison with similar cases and functional studies should help us clarify the relevance of the deleted genes for ID and behavioral alterations.

Measurement of adsorption constants of laccase on gold nanoparticles to evaluate the enhancement in enzyme activity of adsorbed laccase.

Adsorption of enzymes to nanoparticles and the mechanisms responsible for enzyme activity modulation of adsorbed enzymes are not well understood. In this work, gold nanoparticles were used for electrostatic adsorption of a plant-derived laccase. Adsorption constants were determined by four independent techniques: dynamic light scattering, electrophoretic light scattering, agarose gel electrophoresis and fluorescence quenching. Stable bionanoconjugates were formed with log K in the range 6.8-8.9. An increase in enzyme activity was detected, in particular at acidic and close to neutral pH values, a feature that expands the useful pH range of the enzyme. A model for the adsorption was developed, based on geometrical considerations and volume increase data from dynamic light scattering. This indicates that enzymes adsorbed to gold nanoparticles are ca. 9 times more active than the free enzyme.

A glycolytic phenotype is associated with prostate cancer progression and aggressiveness: a role for monocarboxylate transporters as metabolic targets for therapy.

Metabolic adaptation is considered an emerging hallmark of cancer, whereby cancer cells exhibit high rates of glucose consumption with consequent lactate production. To ensure rapid efflux of lactate, most cancer cells express high levels of monocarboxylate transporters (MCTs), which therefore may constitute suitable therapeutic targets. The impact of MCT inhibition, along with the clinical impact of altered cellular metabolism during prostate cancer (PCa) initiation and progression, has not been described. Using a large cohort of human prostate tissues of different grades, in silico data, in vitro and ex vivo studies, we demonstrate the metabolic heterogeneity of PCa and its clinical relevance. We show an increased glycolytic phenotype in advanced stages of PCa and its correlation with poor prognosis. Finally, we present evidence supporting MCTs as suitable targets in PCa, affecting not only cancer cell proliferation and survival but also the expression of a number of hypoxia-inducible factor target genes associated with poor prognosis. Herein, we suggest that patients with highly glycolytic tumours have poorer outcome, supporting the notion of targeting glycolytic tumour cells in prostate cancer through the use of MCT inhibitors.

Role of SPARC in bone remodeling and cancer-related bone metastasis.

There is a growing socioeconomic recognition that clinical bone diseases such as bone infections, bone tumors and osteoporotic bone loss mainly associated with ageing, are major issues in today's society. SPARC (secreted protein, acidic and rich in cysteine), a matricellular glycoprotein, may be a promising therapeutic target for preventing or treating bone-related diseases. In fact, SPARC is associated with tissue remodeling, repair, development, cell turnover, bone mineralization and may also participate in growth and progression of tumors, namely cancer-related bone metastasis. Yet, the function of SPARC in such biological processes is poorly understood and controversial. The main objective of this work is to review the current knowledge related to the activity of SPARC in bone remodeling, tumorigenesis, and bone metastasis. Progress in understanding SPARC biology may provide novel strategies for bone regeneration and the development of anti-angiogenic, anti-proliferative, or counter-adhesive treatments specifically against bone metastasis.

Serotyping, a challenging approach for Toxoplasma gondii typing.

Genotype analysis has revealed a high genetic diversity in strains of Toxoplasma gondii, isolated from a wide range of intermediate hosts and different geographic origins. Diversity is notably striking for parasites from wild hosts in South America, generally referred as non-archetypal genotypes. Those genotypes are implicated in the etiology of severe clinical disease, multivisceral toxoplasmosis, associated with high rate of mortality in immunocompetent individuals. Can we accept specific antibodies produced during T. gondii infection as biomarkers to identify infecting genotypes? Scientific evidence supports a positive response to this question; however, the genetic diversity of T. gondii genotypes organized into 16 haplogroups and collectively defined in 6 major clades, provides a reminder of the complexity and difficulty for the purpose. This review discusses serological approaches to genotyping T. gondii.

Local Antibiotic Delivery Ceramic Bone Substitutes for the Treatment of Infected Bone Cavities and Bone Regeneration: A Systematic Review on What We Have Learned from Animal Models.

AIMS: the focus of this study is to evaluate if the combination of an antibiotic with a ceramic biomaterial is effective in treating osteomyelitis in an infected animal model and to define which model and protocol are best suited for in vivo experiments of local bone infection treatment. METHODS: a systematic review was carried out based on PRISMA statement guidelines. A PubMed search was conducted to find original papers on animal models of bone infections using local antibiotic delivery systems with the characteristics of bone substitutes. Articles without a control group, differing from the experimental group only by the addition of antibiotics to the bone substitute, were excluded. RESULTS: a total of 1185 records were retrieved, and after a three-step selection, 34 papers were included. Six manuscripts studied the effect of antibiotic-loaded biomaterials on bone infection prevention. Five articles studied infection in the presence of foreign bodies. In all but one, the combination of an antibiotic with bioceramic bone substitutes tended to prevent or cure bone infection while promoting biomaterial osteointegration. CONCLUSIONS: this systematic review shows that the combination of antibiotics with bioceramic bone substitutes may be appropriate to treat bone infection when applied locally. The variability of the animal models, time to develop an infection, antibiotic used, way of carrying and releasing antibiotics, type of ceramic material, and endpoints limits the conclusions on the ideal therapy, enhancing the need for consistent models and guidelines to develop an adequate combination of material and antimicrobial agent leading to an effective human application.

Studying the Degradation of Three Polymers under Different Chlorine Concentrations and Exposure Times.

Due to chlorine's ability to kill bacteria and fungi through a chemical reaction, chlorine solutions are commonly used to clean and disinfect numerous public facilities, although these actions are also dependent to the equipment present in those facilities. Accordingly, the interest in studying its effect when in contact with different materials is obvious. This study was carried out through accelerated degradation tests and various analysis methods (optical microscope, scanning electron microscope, and tensile tests). The objective was to observe the wear presented by three polymeric materials, polyvinyl chloride (PVC), high-density polyethylene (HDPE), and polypropylene (PP), when exposed to chlorine's action in swimming pools and drinking water treatment plants. The resulting effect depends on the chlorine content and the type of contact between the chemical agent and the material. The aim was to select the material less likely to be affected by chlorine through tests and analyses, allowing a longer component life. The use of certain more resistant polymeric materials can drastically reduce maintenance, reducing fundamental factors such as costs, the downtime of municipal facilities, and also the risk to public health. It was concluded that PVC has the most stable behaviour overall when in contact with chlorine solutions.

Upper airway assessment in obstructive sleep apnea patients: can computed tomography with lateral cephalometry replace drug-induced sleep endoscopy (DISE)?

OBJECTIVE: To evaluate the association between results from drug-induced sleep endoscopy (DISE) and computed tomography with lateral cephalometry (CTLC) of the pharynx in obstructive sleep apnea (OSA) patients, regarding the same anatomic level, in order to understand if CTLC could replace DISE in selected patients. STUDY DESIGN: Cross-sectional. SETTING: Tertiary hospital. METHODS: A total of 71 patients who attended the Sleep Medicine Consultation in the Otorhinolaryngology Department of Hospital CUF Tejo between 1.6.2019 and 30.9.2021, performed a polysomnographic sleep study and were elected to undergo DISE and CTLC of the pharynx for diagnostic purposes were selected. Obstructions at the same anatomic levels - tongue base, epiglottis and velum - were compared in both exams. RESULTS: Patients with reduction of epiglottis-pharynx space on CTLC had also a complete obstruction at epiglottis level on the VOTE classification of DISE (p = 0,027). Reduction of velum-pharynx space or tongue base-pharynx space were not related to complete obstruction of the velum (P = 0,623) or the tongue base (p = 0,594) found in DISE. Those with two or more space reductions had a tendency to multilevel obstruction observed in DISE (p = 0.089). CONCLUSION: When evaluating the obstruction level(s) of an OSA patient, efforts should be made to perform DISE, since CTLC measures, though regarding at the same structures, don´t correlate completely with obstructions observed in DISE.

Study of the Chlorine Influence on the Corrosion of Three Steels to Be Used in Water Treatment Municipal Facilities.

Many municipal facilities, such as pools and drinking water treatment facilities, are subject to ongoing maintenance due to the corrosion of their metallic materials caused by chlorine, leading to high costs and a possible risk to public health. A proper study of the employed product's effect could lead to the use of better materials, which significantly increase the lifetime of metallic equipment more attacked by corrosion, through studies evaluating their cost-effectiveness. This paper was carried out with the objective of studying the degradation of some metallic materials (AISI 316L, AISI 321 and Duplex 14462) used in the referred facilities in order to select the one that possessed a better behavior. It was observed that the introduction of some more adequate materials can drastically reduce maintenance operations, with Duplex 14462 showing the best results, ideal for greater chlorine concentrations, followed by AISI 321, which may be employed for components in less contact with chlorine, since it is more easily affordable.

The Impact of the Deterioration on Wood by Chlorine: An Experimental Study.

The use of disinfection and cleaning chemicals in several municipal facilities, such as swimming pools and drinking water treatment plants, causes the degradation of various types of wood, which leads to failures in equipment and the corresponding need for maintenance. This degradation creates added costs for municipalities, as well as the closure of certain facilities due to curative or preventive maintenance and, in many cases, public health issues, due to the water being contaminated with deteriorating products. Through a thorough study of the degradation effect on the products, more resistant materials can be found which are able to withstand these adversities and increase the lifespan of wood in regular contact with chemical agents. This is achievable by the determination of the cost-effectiveness of the substitute material to replace these components with alternative ones, with properties that better resist the deterioration effects promoted by aggressive environments. No studies have been found so far strictly focused on this matter. The objective of this study is to evaluate the degradation presented by two types of wood, beech and oak, which are exposed to the action of chlorine in municipal facilities. This degradation varies according to the chlorine content and the materials' time of contact with the chemical agent, allowing the selection of new materials which will provide an extended lifetime of the components, reducing maintenance drastically, as well as costs for the facilities and the risk to public health. The performed experimental tests have shown that the oak wood has the best results regarding chlorine degradation resistance.

Vancomycin-Loaded, Nanohydroxyapatite-Based Scaffold for Osteomyelitis Treatment: In Vivo Rabbit Toxicological Tests and In Vivo Efficacy Tests in a Sheep Model.

The treatment for osteomyelitis consists of surgical debridement, filling of the dead space, soft tissue coverage, and intravenous administration of antimicrobial (AM) agents for long periods. Biomaterials for local delivery of AM agents, while providing controllable antibiotic release rates and simultaneously acting as a bone scaffold, may be a valuable alternative; thus, avoiding systemic AM side effects. V-HEPHAPC is a heparinized nanohydroxyapatite (nHA)/collagen biocomposite loaded with vancomycin that has been previously studied and tested in vitro. It enables a vancomycin-releasing profile with an intense initial burst, followed by a sustained release with concentrations above the Minimum Inhibitory Concentration (MIC) for MRSA. In vitro results have also shown that cellular viability is not compromised, suggesting that V-HEPHAPC granules may be a promising alternative device for the treatment of osteomyelitis. In the present study, V-HEPHAPC (HEPHAPC with vancomycin) granules were used as a vancomycin carrier to treat MRSA osteomyelitis. First, in vivo Good Laboratory Practice (GLP) toxicological tests were performed in a rabbit model, assuring that HEPHAPC and V-HEPHAPC have no relevant side effects. Second, V-HEPHAPC proved to be an efficient drug carrier and bone substitute to control MRSA infection and simultaneously reconstruct the bone cavity in a sheep model.

360 Health Analysis (H360)-A Comparison of Key Performance Indicators in Breast Cancer Management across Health Institution Settings in Portugal.

BACKGROUND: The increased focus on quality indicators (QIs) and the use of clinical registries in real-world cancer studies have increased compliance with therapeutic standards and patient survival. The European Society of Breast Cancer Specialists (EUSOMA) established QIs to assess compliance with current standards in breast cancer care. METHODS: This retrospective study is part of H360 Health Analysis and aims to describe compliance with EUSOMA QIs in breast cancer management in different hospital settings (public vs. private; general hospitals vs. oncology centers). A set of key performance indicators (KPIs) was selected based on EUSOMA and previously identified QIs. Secondary data were retrieved from patients' clinical records. Compliance with target KPIs in different disease stages was compared with minimum and target EUSOMA standards. RESULTS: A total of 259 patient records were assessed. In stages I, II, and III, 18 KPIs met target EUSOMA standards, 5 met minimum standards, and 8 failed to meet minimum standards. Compliance with KPIs varied according to the type of hospital (particularly regarding diagnosis) and disease stage. Although small differences were found in KPI compliance among institutions, several statistical differences were found among treatment KPIs according to disease stage, particularly in stage III. CONCLUSIONS: This study represents the first assessment of the quality of breast cancer care in different hospital settings in Portugal and shows that, although most QIs meet EUSOMA standards, there is room for improvement. Differences have been found across institutions, particularly between oncology centers and general hospitals, in diagnosis and compliance with KPIs among disease stages. Stage III showed the greatest variability in compliance with treatment KPIs, probably related to the lower specificity of the guidelines in this disease stage.