The links of stress, substance use and socio-demographic factors with domestic violence during the Covid-19 pandemic in Portugal.

BACKGROUND: Lockdown, as a measure implemented to combat the coronavirus disease 2019 (COVID-19) pandemic, left many domestic violence (DV) victims trapped with their abusers. This study intends to explore the links between perceived stress, substance use and socio-demographic factors with DV experiences during COVID-19 pandemic in Portugal. METHODS: A cross-sectional study was carried out on a sample of 1062 participants over 16 years old, residing in Portugal. Data were collected through an online survey conducted between April and October 2020. The associations between potential factors and DV were investigated using bivariable analysis and multivariable logistic regression. RESULTS: The prevalence of DV reported was 13.75% (n = 146), disaggregated into psychological violence (13%, n = 138), sexual violence (1.0%, n = 11) and physical violence (0.9%, n = 10). Multivariable analyses confirmed that perceived financial difficulties (OR = 1.608; P = 0.019), use of medications to sleep or calm down (OR = 1.851; P = 0.002) and perceived stress (OR = 2.443; P = 0.003) were responsible for DV exposure during COVID-19 pandemic. Younger age (<25 years old) and consumption of alcohol were associated with a higher risk of DV victimization. CONCLUSIONS: Interventions aimed at preventing and confronting DV are necessary within the strategies to combat COVID-19 in Portugal, especially aimed at groups in vulnerable situations, during and after the pandemic.

Adolescent and young adult patients as co-researchers: A scoping review.

BACKGROUND: As part of a research project aimed at evaluating a hospital-based adolescent transition programme, we asked ourselves what is known about the ethical and methodological challenges of research involving adolescent patients as co-researchers. The aim of our review was to summarize empirical evidence and identify knowledge gaps about the involvement of young patients as co-researchers. METHODS: We conducted a scoping review through searches in MEDLINE, EMBASE, PsychINFO, AMED. RESULTS: We found reports of young patients being actively engaged as co-researchers in any stage of a research project, although commonly they were not involved in every stage. Including young patients as co-researchers is resource demanding and time-consuming. Involving young patients as co-researchers contributes to the fulfilment of their right to participation and may improve the relevance of research. Benefits for the young co-researcher include empowerment, skills building and raised self-esteem. Few authors go into detail about ethical considerations when involving young co-researchers. None of the included articles discuss legal considerations. DISCUSSION AND CONCLUSION: No lists of recommendations are given, but recommendations can be deduced from the articles. There is need for time, funding and flexibility when including young patients as co-researchers. Knowledge gaps concern legal and ethical dilemmas of including a vulnerable group as co-researchers. More reflection is needed about what meaningful participation is and what it entails in this context. PATIENT OR PUBLIC CONTRIBUTION: This review is part of a research project where the hospital youth council has been involved in discussions of focus area and methods.

Sequential dual site-selective protein labelling enabled by lysine modification.

Methods that allow for chemical site-selective dual protein modification are scarce. Here, we provide proof-of-concept for the orthogonality and compatibility of a method for regioselective lysine modification with strategies for protein modification at cysteine and genetically encoded ketone-tagged amino acids. This sequential, orthogonal approach was applied to albumin and a therapeutic antibody to create functional dual site-selectively labelled proteins.

Usability of an Intelligent Virtual Assistant for Promoting Behavior Change and Self-Care in Older People with Type 2 Diabetes.

In the context of the VASelfCare project, we developed an application prototype of an intelligent anthropomorphic virtual assistant. Designed as a relational agent, the virtual assistant has the role of supporting older people with Type 2 Diabetes Mellitus (T2D) in medication adherence and lifestyle changes. Our paper has two goals: describing the essentials of this prototype, and reporting on usability evaluation. We describe the general architecture of the prototype, including the graphical component, and focus on its main feature: the incorporation, in the way the dialogue flows, of Behavior Change Techniques, identified through a theoretical framework, the Behaviour Change Wheel. Usability was experimentally evaluated in field tests in a purposive sample of 20 participants (11 older adults with T2D and 9 experts). The Portuguese version of the System Usability Scale was employed, supplemented with qualitative data from open questions, diaries, digital notes and telephone follow-ups. The aggregated mean SUS score was 73,75 (SD 13,31), which corresponds to a borderline rating of excellent. Textual data were content analyzed and will be prioritized to further improve usability.

Tetrazine Carbon Nanotubes for Pretargeted In†Vivo "Click-to-Release" Bioorthogonal Tumour Imaging.

The bioorthogonal inverse-electron-demand Diels-Alder (IEDDA) cleavage reaction between tetrazine and trans-cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single-walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO-caged molecule was used to deliver active effector molecules. To optimize a turn-on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near-infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real-time, non-invasive tumour visualization with a high target-to-background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine-functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off-site activation of fluorophore/drug.

Structure-Based Design of Potent Tumor-Associated Antigens: Modulation of Peptide Presentation by Single-Atom O/S or O/Se Substitutions at the Glycosidic Linkage.

GalNAc-glycopeptides derived from mucin MUC1 are an important class of tumor-associated antigens. α- O-glycosylation forces the peptide to adopt an extended conformation in solution, which is far from the structure observed in complexes with a model anti-MUC1 antibody. Herein, we propose a new strategy for designing potent antigen mimics based on modulating peptide/carbohydrate interactions by means of O → S/Se replacement at the glycosidic linkage. These minimal chemical modifications bring about two key structural changes to the glycopeptide. They increase the carbohydrate-peptide distance and change the orientation and dynamics of the glycosidic linkage. As a result, the peptide acquires a preorganized and optimal structure suited for antibody binding. Accordingly, these new glycopeptides display improved binding toward a representative anti-MUC1 antibody relative to the native antigens. To prove the potential of these glycopeptides as tumor-associated MUC1 antigen mimics, the derivative bearing the S-glycosidic linkage was conjugated to gold nanoparticles and tested as an immunogenic formulation in mice without any adjuvant, which resulted in a significant humoral immune response. Importantly, the mice antisera recognize cancer cells in biopsies of breast cancer patients with high selectivity. This finding demonstrates that the antibodies elicited against the mimetic antigen indeed recognize the naturally occurring antigen in its physiological context. Clinically, the exploitation of tumor-associated antigen mimics may contribute to the development of cancer vaccines and to the improvement of cancer diagnosis based on anti-MUC1 antibodies. The methodology presented here is of general interest for applications because it may be extended to modulate the affinity of biologically relevant glycopeptides toward their receptors.

FGF2 induces breast cancer growth through ligand-independent activation and recruitment of ERα and PRBΔ4 isoform to MYC regulatory sequences.

Progression to hormone-independent growth leading to endocrine therapy resistance occurs in a high proportion of patients with estrogen receptor alpha (ERα) and progesterone receptors (PR) positive breast cancer. We and others have previously shown that estrogen- and progestin-induced tumor growth requires ERα and PR interaction at their target genes. Here, we show that fibroblast growth factor 2 (FGF2)-induces cell proliferation and tumor growth through hormone-independent ERα and PR activation and their interaction at the MYC enhancer and proximal promoter. MYC inhibitors, antiestrogens or antiprogestins reverted FGF2-induced effects. LC-MS/MS identified 700 canonical proteins recruited to MYC regulatory sequences after FGF2 stimulation, 397 of which required active ERα (ERα-dependent). We identified ERα-dependent proteins regulating transcription that, after FGF2 treatment, were recruited to the enhancer as well as proteins involved in transcription initiation that were recruited to the proximal promoter. Also, among the ERα-dependent and independent proteins detected at both sites, PR isoforms A and B as well as the novel protein product PRBΔ4 were found. PRBΔ4 lacks the hormone-binding domain and was able to induce reporter gene expression from estrogen-regulated elements and to increase cell proliferation when cells were stimulated with FGF2 but not by progestins. Analysis of the Cancer Genome Atlas data set revealed that PRBΔ4 expression is associated with worse overall survival in luminal breast cancer patients. This discovery provides a new mechanism by which growth factor signaling can engage nonclassical hormone receptor isoforms such as PRBΔ4, which interacts with growth-factor activated ERα and PR to stimulate MYC gene expression and hence progression to endocrine resistance.

Triaminopyrimidine derivatives as transmembrane HCl transporters.

Small synthetic molecules capable of inducing transmembrane anion transport have received a lot of attention as potential anti-cancer agents due to their ability to interfere with intracellular pH homeostasis. A series of triaminopyrimidine-based anion transporters have been synthesised, and they are found to diminish proton gradients across lipid bilayers at physiologically relevant pH. The compounds have pKa values (≈7.2) that allow protonation/deprotonation processes coupled with anion binding/unbinding events in physiologically relevant conditions. Synthetic vesicle transport experiments as well as solid state structures indicate synergistic binding of HCl. Cell assays show that the transporters induce apoptosis in various cancerous cell lines.

Quaternization of Vinyl/Alkynyl Pyridine Enables Ultrafast Cysteine-Selective Protein Modification and Charge Modulation.

Quaternized vinyl- and alkynyl-pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine-tagged proteins at near-stoichiometric quantities. We have demonstrated that this method can effectively create a homogenous antibody-drug conjugate that features a precise drug-to-antibody ratio of 2, which was stable in human plasma and retained its specificity towards Her2+ cells. Finally, the developed warhead introduces a +1 charge to the overall net charge of the protein, which enabled us to show that the electrophoretic mobility of the protein may be tuned through the simple attachment of a quaternized vinyl pyridinium reagent at the cysteine residues. We anticipate the generalized use of quaternized vinyl- and alkynyl-pyridine reagents not only for bioconjugation, but also as warheads for covalent inhibition and as tools to profile cysteine reactivity.

Chemo- and Regioselective Lysine Modification on Native Proteins.

Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest p Ka was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein-antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.

Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation.

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins.

Regulator LdhR and d-Lactate Dehydrogenase LdhA of Burkholderia multivorans Play Roles in Carbon Overflow and in Planktonic Cellular Aggregate Formation.

LysR-type transcriptional regulators (LTTRs) are the most commonly found regulators in Burkholderia cepacia complex, comprising opportunistic pathogens causing chronic respiratory infections in cystic fibrosis (CF) patients. Despite LTTRs being global regulators of pathogenicity in several types of bacteria, few have been characterized in Burkholderia Here, we show that gene ldhR of B. multivorans encoding an LTTR is cotranscribed with ldhA encoding a d-lactate dehydrogenase and evaluate their implication in virulence traits such as exopolysaccharide (EPS) synthesis and biofilm formation. A comparison of the wild type (WT) and its isogenic ΔldhR mutant grown in medium with 2% d-glucose revealed a negative impact on EPS biosynthesis and on cell viability in the presence of LdhR. The loss of viability in WT cells was caused by intracellular acidification as a consequence of the cumulative secretion of organic acids, including d-lactate, which was absent from the ΔldhR mutant supernatant. Furthermore, LdhR is implicated in the formation of planktonic cellular aggregates. WT cell aggregates reached 1,000 µm in size after 24 h in liquid cultures, in contrast to ΔldhR mutant aggregates that never grew more than 60 µm. The overexpression of d-lactate dehydrogenase LdhA in the ΔldhR mutant partially restored the formed aggregate size, suggesting a role for fermentation inside aggregates. Similar results were obtained for surface-attached biofilms, with WT cells producing more biofilm. A systematic evaluation of planktonic aggregates in Burkholderia CF clinical isolates showed aggregates in 40 of 74. As CF patients' lung environments are microaerophilic and bacteria are found as free aggregates/biofilms, LdhR and LdhA might have central roles in adapting to this environment.IMPORTANCE Cystic fibrosis patients often suffer from chronic respiratory infections caused by several types of microorganisms. Among them are the Burkholderia cepacia complex bacteria, which cause progressive deterioration of lung function that, in some patients, might develop into fatal necrotizing pneumoniae with bacteremia, known as "cepacia syndrome." Burkholderia pathogenesis is multifactorial as they express several virulence factors, form biofilms, and are highly resistant to antimicrobial compounds, making their eradication from the CF patients' airways very difficult. As Burkholderia is commonly found in CF lungs in the form of cell aggregates and biofilms, the need to investigate the mechanisms of cellular aggregation is obvious. In this study, we demonstrate the importance of a d-lactate dehydrogenase and a regulator in regulating carbon overflow, cellular aggregates, and surface-attached biofilm formation. This not only enhances our understanding of Burkholderia pathogenesis but can also lead to the development of drugs against these proteins to circumvent biofilm formation.

Vinyl Ether/Tetrazine Pair for the Traceless Release of Alcohols in Cells.

The cleavage of a protecting group from a protein or drug under bioorthogonal conditions enables accurate spatiotemporal control over protein or drug activity. Disclosed herein is that vinyl ethers serve as protecting groups for alcohol-containing molecules and as reagents for bioorthogonal bond-cleavage reactions. A vinyl ether moiety was installed in a range of molecules, including amino acids, a monosaccharide, a fluorophore, and an analogue of the cytotoxic drug duocarmycin. Tetrazine-mediated decaging proceeded under biocompatible conditions with good yields and reasonable kinetics. Importantly, the nontoxic, vinyl ether duocarmycin double prodrug was successfully decaged in live cells to reinstate cytotoxicity. This bioorthogonal reaction presents broad applicability and may be suitable for in vivo applications.

Synaptic Homeostasis and Restructuring across the Sleep-Wake Cycle.

Sleep is critical for hippocampus-dependent memory consolidation. However, the underlying mechanisms of synaptic plasticity are poorly understood. The central controversy is on whether long-term potentiation (LTP) takes a role during sleep and which would be its specific effect on memory. To address this question, we used immunohistochemistry to measure phosphorylation of Ca2+/calmodulin-dependent protein kinase II (pCaMKIIα) in the rat hippocampus immediately after specific sleep-wake states were interrupted. Control animals not exposed to novel objects during waking (WK) showed stable pCaMKIIα levels across the sleep-wake cycle, but animals exposed to novel objects showed a decrease during subsequent slow-wave sleep (SWS) followed by a rebound during rapid-eye-movement sleep (REM). The levels of pCaMKIIα during REM were proportional to cortical spindles near SWS/REM transitions. Based on these results, we modeled sleep-dependent LTP on a network of fully connected excitatory neurons fed with spikes recorded from the rat hippocampus across WK, SWS and REM. Sleep without LTP orderly rescaled synaptic weights to a narrow range of intermediate values. In contrast, LTP triggered near the SWS/REM transition led to marked swaps in synaptic weight ranking. To better understand the interaction between rescaling and restructuring during sleep, we implemented synaptic homeostasis and embossing in a detailed hippocampal-cortical model with both excitatory and inhibitory neurons. Synaptic homeostasis was implemented by weakening potentiation and strengthening depression, while synaptic embossing was simulated by evoking LTP on selected synapses. We observed that synaptic homeostasis facilitates controlled synaptic restructuring. The results imply a mechanism for a cognitive synergy between SWS and REM, and suggest that LTP at the SWS/REM transition critically influences the effect of sleep: Its lack determines synaptic homeostasis, its presence causes synaptic restructuring.

Daily rhythms in the cyanobacterium synechococcus elongatus probed by high-resolution mass spectrometry-based proteomics reveals a small defined set of cyclic proteins.

Circadian rhythms are self-sustained and adjustable cycles, typically entrained with light/dark and/or temperature cycles. These rhythms are present in animals, plants, fungi, and several bacteria. The central mechanism behind these "pacemakers" and the connection to the circadian regulated pathways are still poorly understood. The circadian rhythm of the cyanobacterium Synechococcus elongatus PCC 7942 (S. elongatus) is highly robust and controlled by only three proteins, KaiA, KaiB, and KaiC. This central clock system has been extensively studied functionally and structurally and can be reconstituted in vitro. These characteristics, together with a relatively small genome (2.7 Mbp), make S. elongatus an ideal model system for the study of circadian rhythms. Different approaches have been used to reveal the influence of the central S. elongatus clock on rhythmic gene expression, rhythmic mRNA abundance, rhythmic DNA topology changes, and cell division. However, a global analysis of its proteome dynamics has not been reported yet. To uncover the variation in protein abundances during 48 h under light and dark cycles (12:12 h), we used quantitative proteomics, with TMT 6-plex isobaric labeling. We queried the S. elongatus proteome at 10 different time points spanning a single 24-h period, leading to 20 time points over the full 48-h period. Employing multidimensional separation and high-resolution mass spectrometry, we were able to find evidence for a total of 82% of the S. elongatus proteome. Of the 1537 proteins quantified over the time course of the experiment, only 77 underwent significant cyclic variations. Interestingly, our data provide evidence for in- and out-of-phase correlation between mRNA and protein levels for a set of specific genes and proteins. As a range of cyclic proteins are functionally not well annotated, this work provides a resource for further studies to explore the role of these proteins in the cyanobacterial circadian rhythm.

A Minimal, Unstrained S-Allyl Handle for Pre-Targeting Diels-Alder Bioorthogonal Labeling in Live Cells.

The unstrained S-allyl cysteine amino acid was site-specifically installed on apoptosis protein biomarkers and was further used as a chemical handle and ligation partner for 1,2,4,5-tetrazines by means of an inverse-electron-demand Diels-Alder reaction. We demonstrate the utility of this minimal handle for the efficient labeling of apoptotic cells using a fluorogenic tetrazine dye in a pre-targeting approach. The small size, easy chemical installation, and selective reactivity of the S-allyl handle towards tetrazines should be readily extendable to other proteins and biomolecules, which could facilitate their labeling within live cells.

Automatic tracking of cells for video microscopy in patch clamp experiments.

BACKGROUND: Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. METHODS: Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). RESULTS: We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. CONCLUSION: The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices.

Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses.

Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy-entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen-antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development.

Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) is a UDP-dependent galactosyltransferase.

Glycosyltransferases (GTs) are enzymes that catalyze the formation of glycosidic bonds and hundreds of GTs have been identified so far in humans. Glycosyltransferase 8 domain-containing protein 1 (GLT8D1) has been associated with central nervous system diseases and cancer. However, evidence on its enzymatic properties, including its substrates, has been scarcely described. In this paper, we have produced and purified recombinant secretory GLT8D1. The enzyme was found to be N-glycosylated. Differential scanning fluorimetry was employed to analyze the stabilization of GLT8D1 by Mn2+ and nucleotides, revealing UDP as the most stabilizing nucleotide scaffold. GLT8D1 displayed glycosyltransferase activity from UDP-galactose onto N-acetylgalactosamine but with a low efficiency. Modeling of the structure revealed similarities with other GT-A fold enzymes in CAZy family GT8 and glycosyltransferases in other families with galactosyl-, glucosyl-, and xylosyltransferase activities, each with retaining catalytic mechanisms. Our study provides novel structural and functional insights into the properties of GLT8D1 with implications in pathological processes.

Fuzzy method for pre-diagnosis of breast cancer from the Fine Needle Aspirate analysis.

BACKGROUND: Across the globe, breast cancer is one of the leading causes of death among women and, currently, Fine Needle Aspirate (FNA) with visual interpretation is the easiest and fastest biopsy technique for the diagnosis of this deadly disease. Unfortunately, the ability of this method to diagnose cancer correctly when the disease is present varies greatly, from 65% to 98%. This article introduces a method to assist in the diagnosis and second opinion of breast cancer from the analysis of descriptors extracted from smears of breast mass obtained by FNA, with the use of computational intelligence resources--in this case, fuzzy logic. METHODS: For data acquisition of FNA, the Wisconsin Diagnostic Breast Cancer Data (WDBC), from the University of California at Irvine (UCI) Machine Learning Repository, available on the internet through the UCI domain was used. The knowledge acquisition process was carried out by the extraction and analysis of numerical data of the WDBC and by interviews and discussions with medical experts. The PDM-FNA-Fuzzy was developed in four steps: 1) Fuzzification Stage; 2) Rules Base; 3) Inference Stage; and 4) Defuzzification Stage. Performance cross-validation was used in the tests, with three databases with gold pattern clinical cases randomly extracted from the WDBC. The final validation was held by medical specialists in pathology, mastology and general practice, and with gold pattern clinical cases, i.e. with known and clinically confirmed diagnosis. RESULTS: The Fuzzy Method developed provides breast cancer pre-diagnosis with 98.59% sensitivity (correct pre-diagnosis of malignancies); and 85.43% specificity (correct pre-diagnosis of benign cases). Due to the high sensitivity presented, these results are considered satisfactory, both by the opinion of medical specialists in the aforementioned areas and by comparison with other studies involving breast cancer diagnosis using FNA. CONCLUSIONS: This paper presents an intelligent method to assist in the diagnosis and second opinion of breast cancer, using a fuzzy method capable of processing and sorting data extracted from smears of breast mass obtained by FNA, with satisfactory levels of sensitivity and specificity. The main contribution of the proposed method is the reduction of the variation hit of malignant cases when compared to visual interpretation currently applied in the diagnosis by FNA. While the MPD-FNA-Fuzzy features stable sensitivity at 98.59%, visual interpretation diagnosis provides a sensitivity variation from 65% to 98% (this track showing sensitivity levels below those considered satisfactory by medical specialists). Note that this method will be used in an Intelligent Virtual Environment to assist the decision-making (IVEMI), which amplifies its contribution.