High frequency of alpha7-HPV in Colombian Caribbean coast women: cervical cancer screening analysis.

BACKGROUND: Cervical cancer (CC) is a significant global public health concern, particularly in developing countries such as Colombia. The main risk factor involves high-risk HPV types (HR-HPV) infection, coupled with population-specific variables. The Caribbean region in Colombia lacks research on HR-HPV-type frequencies. Therefore, this study aims to establish the prevalence of type-specific HR-HPV and its association with sociodemographic factors among women undergoing cervical cytology screening. METHODS: A cross-sectional study involving voluntary women who provided informed consent and completed a questionnaire capturing sociodemographic, clinical, and sexual behavior information was conducted. All participants underwent cervical cytology and molecular analysis. Generic HPV detection employed three simultaneous PCRs (GP5+/6+, MY09/11, and PU1R/2 M), and positive samples were genotyped using the Optiplex HPV Genotyping kit. The analysis encompassed the 12 types of high-risk HPV (HR-HPV-16,-18,-31,-33,-35,-39,-45,-51,-52,-56,-58, and - 59). Frequencies were reported based on geographic subregions within the Córdoba department, and disparities were made between single and multiple infections. Sociodemographic and clinical variables were subjected to ordinal logistic regression, with statistical significance at a p-value < 0.05. The statistical analyses utilized STATA 14® and R-Core Team-software. RESULTS: We included 450 women, mean age 40 (SD±11.44). PCR analysis revealed 43% HPV-positive (n=192). GP5+/6+ detected the most positives at 26% (n=119), followed by PU1R/2 M at 22% (n = 100) and MY09/11 at 15% (n=69). Multiple infections occurred in 87.3% (n=142), primarily 2 to 4 types (47.37%, n=90). Dominant types were HPV-18 (15.6%, n=61), HPV-16 (14.9%, n=58), HPV-31 (13.0%, n = 51), and HPV-45 (11.5%, n=45). Logistic regression identified age above 60 as a risk for concurrent multiple types (OR=6.10; 95% CI 1.18-31.63). Menopause was protective (OR=0.31; 95% CI 0.11-0.89). CONCLUSIONS: Our study reveals a notable prevalence of multiple (2-4) high-risk HPV infections among adult women engaged in CC detection initiatives. Predominantly, α7 species constitute the prevalent HR-viral types, with the Medio Sinú subregion showing elevated prevalence. Menopausal status confers protection against diverse HR-HPV infections. Nevertheless, advancing age, particularly beyond 60 years, is linked to an increased susceptibility to simultaneous infections by multiple HPV-types.

Development and Optimization of a Multiplex Real-Time RT-PCR to Detect SARS-CoV-2 in Human Samples.

PCR and its variants (RT-PCR and qRT-PCR) are valuable and innovative molecular techniques for studying nucleic acids. qPCR has proven to be highly sensitive, efficient, and reproducible, generating reliable results that are easy to analyze. During the COVID-19 pandemic, qPCR became the gold standard technique for detecting the SARS-CoV-2 virus that allowed to confirm the infection event, and those asymptomatic ones, and thus save millions of lives. In-house multiplex qPCR tests were developed worldwide to detect different viral targets and ensure results, follow the infections, and favor the containment of a pandemic. Here, we present the detailed fundamentals of the qPCR technique based on fluorogenic probes and processes to develop and optimize a successful multiplex RT-qPCR test for detecting SARS-CoV-2 that could be used to diagnose COVID-19 accurately.

A Rapid and Simple Method for Purification of Nucleic Acids on Porous Membranes: Simulation vs. Experiment.

Paper-based microfluidic systems have emerged as one of the most promising technologies for developing point-of-care diagnostic platforms (POCT) for detecting and monitoring various diseases. Saliva is a non-invasive biofluid easily collected, transported, and stored. Due to its accessibility and connection to systemic diseases, saliva is one of the best candidates for medical advancement at the point of care, where people can easily monitor their health. However, saliva is a complex mixture of DNA, RNA, proteins, exosomes, and electrolytes. Thus, nucleic acid separation from the salivary components is essential for PCR applications. Paper membranes are a highly porous and foldable structure capable of transporting fluids without pumps and sophisticated systems. The current work presents an insight into simulations for nucleic acid extraction on three types of porous paper membranes for use in point-of-care devices. The flow fluid model is solved on a COMSOL Multiphysics 5.3 free version platform, and the results are compared with experimental assays. The results show that pore uniformity, wet strength, porosity, and functional groups of MF1™ and Fusion 5™ paper membranes are vital parameters affecting nucleic acid extraction and PCR amplification efficiency.

Antiproliferative and Pro-Apoptotic Effects of a Phenolic-Rich Extract from Lycium barbarum Fruits on Human Papillomavirus (HPV) 16-Positive Head Cancer Cell Lines.

The in vitro antiproliferative activity of a phenolic-rich extract from Lycium barbarum fruits against head and neck HPV16 squamous cell carcinoma (OSCC) has been demonstrated, indicating for the first time that L. barbarum extract inhibits human papillomavirus (HPV) type 16 cell lines. Ethanol extract of L. barbarum was used for cell viability evaluation on SCC090, CAL27, and HGnF cell lines. After 24 and 48 h, the cell cycle effect of L. barbarum extract (at 1.0, 10, and 100 µg/mL) was measured via flow cytometry. In addition, the mRNA expression on E6/E7 and p53 via RT-PCR and the expression of p16, p53, Ki-67, and Bcl-2 via immunohistochemistry were also determined. Untreated cells, 20 µM cisplatin, and a Camellia sinensis-derived extract were used as negative and positive controls, respectively. We demonstrated that the studied L. barbarum extract resulted in G0/G1 arrest and S phase accumulation in SCC090 at 1.0 and 10 µg/mL. A reduction in mRNA levels of E6/E7 oncogenes (p < 0.05) with p53 overexpression was also observed through PCR, while immunohistochemical analyses indicated p16 overexpression (p > 0.05) and a decrease in p53 overexpression. The observed effects were associated with anticancer and immunomodulatory phenolics, such as flavonols/flavan-3-ols and tyramine-conjugated hydroxycinnamic acid amides, identified in the studied extract. These findings revealed that the phenolic-rich extract of L. barbarum fruits has promising properties to be considered further for developing new therapies against oral and oropharyngeal HPV lesions.

Exploring complement-dependent cytotoxicity by rituximab isotypes in 2D and 3D-cultured B-cell lymphoma.

BACKGROUND: The therapeutic IgG1 anti-CD20 antibody, rituximab (RTX), has greatly improved prognosis of many B-cell malignancies. Despite its success, resistance has been reported and detailed knowledge of RTX mechanisms are lacking. Complement-dependent cytotoxicity (CDC) is one important mode of action of RTX. The aim of this study was to systematically evaluate factors influencing complement-mediated tumor cell killing by RTX. METHODS: Different RTX isotypes, IgG1, IgG3, IgA1 and IgA2 were evaluated and administered on four human CD20+ B-cell lymphoma cell lines, displaying diverse expression of CD20 and complement-regulatory protein CD59. Complement activation was assessed on lymphoma cells grown in 2 and 3-dimensional (3D) culture systems by trypan blue exclusion. CDC in 3D spheroids was additionally analyzed by Annexin V and propidium iodide staining by flow cytometry, and confocal imaging. Anti-CD59 antibody was used to evaluate influence of CD59 in RTX-mediated CDC responses. Statistical differences were determined by one-way ANOVA and Tukey post hoc test. RESULTS: We found that 3 out of 4 lymphomas were sensitive to RTX-mediated CDC when cultured in 2D, while 2 out of 4 when grown in 3D. RTX-IgG3 had the greatest CDC potential, followed by clinical standard RTX-IgG1 and RTX-IgA2, whereas RTX-IgA1 displayed no complement activation. Although the pattern of different RTX isotypes to induce CDC were similar in the sensitive lymphomas, the degree of cell killing differed. A greater CDC activity was seen in lymphoma cells with a higher CD20/CD59 expression ratio. These lymphomas were also sensitive to RTX when grown in 3D spheroids, although the CDC activity was substantially reduced compared to 2D cultures. Analysis of RTX-treated spheroids demonstrated apoptosis and necrosis essentially in the outer cell-layers. Neutralization of CD59 overcame resistance to RTX-mediated CDC in 2D-cultured lymphoma cells, but not in spheroids. CONCLUSIONS: The results demonstrate that CDC outcome in CD20+ B-cell lymphoma is synergistically influenced by choice of RTX isotype, antigen density, tumor structure, and degree of CD59 expression. Assessment of tumor signatures, such as CD20/CD59 ratio, can be advantageous to predict CDC efficiency of RTX in vivo and may help to develop rational mAbs to raise response rates in patients.

Quantitative Analysis of the Transcriptome of Two Commonly Used Human Monocytic Cell Lines-THP-1 and Mono Mac 6-Reveals Their Arrest during Early Monocyte/Neutrophil Differentiation.

Cell lines of monocyte/macrophage origin are often used as model systems to study monocyte/macrophage biology. A relevant question is how similar these cell lines are to their in vivo counterparts? To address this issue, we performed a detailed analysis of the transcriptome of two commonly used human monocyte/macrophage cell lines, Mono Mac 6 and THP-1. Both of these cell lines originate from leukemic cells with myelo-monocytic characteristics. We found that both Mono Mac 6 and THP-1 represent cells of very immature origin. Their transcriptomes show more similarities to immature neutrophils than cells of the monocyte/macrophage lineage. They express significant levels of N-elastase, proteinase 3, cathepsin G, and azurocidin but very low levels of CD14, ficolin, and complement factor P. All major MHC class II genes are also expressed at low levels. They show high levels of lysozyme and low levels of one of the immunoglobulin Fc receptors, FCGRIIA, which is characteristic of both neutrophils and monocytes. THP-1, but not Mono Mac 6, also expresses the high-affinity receptor for IgG, FCGRIA. Both cell lines lack the expression of the connective tissue components fibronectin, proteoglycan 4, and syndecan 3, which are characteristics of tissue macrophages but are absent in blood monocytes, indicating that they originate from bone marrow precursors and not yolk sac-derived hematopoietic cells. Both of these cell lines seem, therefore, to represent cells arrested during early myelo-monocytic development, at a branch point between neutrophil and monocyte differentiation. Their very immature phenotype indicates that great care should be taken when using these cell lines as models for normal monocyte/macrophage biology.

Structural Characterization of a Thorarchaeota Profilin Indicates Eukaryotic-Like Features but with an Extended N-Terminus.

The emergence of the first eukaryotic cell is preceded by evolutionary events, which are still highly debatable. Clues of the exact sequence of events are beginning to emerge. Recent metagenomics analyses has uncovered the Asgard super-phylum as the closest yet known archaea host of eukaryotes. Some of these have been tested and confirmed experimentally. However, the bulk of eukaryotic signature proteins predicted to be encoded by the Asgard super-phylum have not been studied, and their true functions, at least in the context of a eukaryotic cell, are still elusive. For example, there are several different variants of the profilin within each Asgardian Achaea, and there are some conflicting results of their actual roles. Here, the 3D structure of profilin from Thorarchaeota is determined by nuclear magnetic resonance spectroscopy and shows that this profilin has a eukaryotic-like profilin with a rigid core and an extended N-terminus previously implicated in polyproline binding. In addition, it is also shown that Thorarchaeota Profilin co-localizes with eukaryotic actin in cultured HeLa cells. This finding reaffirms the notion that Asgardian encoded proteins possess eukaryotic-like characteristics and strengthen the likely existence of a complex cytoskeleton already in a last eukaryotic common ancestor.

The Human Monocyte-A Circulating Sensor of Infection and a Potent and Rapid Inducer of Inflammation.

Monocytes were previously thought to be the precursors of all tissue macrophages but have recently been found to represent a unique population of cells, distinct from the majority of tissue macrophages. Monocytes and intestinal macrophages seem now to be the only monocyte/macrophage populations that originate primarily from adult bone marrow. To obtain a better view of the biological function of monocytes and how they differ from tissue macrophages, we have performed a quantitative analysis of its transcriptome in vivo and after in vitro stimulation with E. coli LPS. The monocytes rapidly responded to LPS by producing extremely high amounts of mRNA for the classical inflammatory cytokines, IL-1α, IL-1ß, IL-6 and TNF-α, but almost undetectable amounts of other cytokines. IL-6 was upregulated 58,000 times, from almost undetectable levels at baseline to become one of the major transcripts already after a few hours of cultivation. The cells also showed very strong upregulation of a number of chemokines, primarily IL-8, Ccl2, Ccl3, Ccl3L3, Ccl20, Cxcl2, Cxcl3 and Cxcl4. IL-8 became the most highly expressed transcript in the monocytes already after four hours of in vitro culture in the presence of LPS. A high baseline level of MHC class II chains and marked upregulation of super oxide dismutase (SOD2), complement factor B, complement factor C3 and coagulation factor 3 (F3; tissue factor) at four hours of in vitro culture were also observed. This indicates a rapid protective response to high production of oxygen radicals, to increase complement activation and possibly also be an inducer of local coagulation. Overall, these findings give strong support for monocytes acting primarily as potent mobile sensors of infection and rapid activators of a strong inflammatory response.

Consideraciones actuales sobre los teratógenos y sus efectos durante el embarazo / Current considerations on the teratogens and their effects during pregnancy

Introducción: El desarrollo alcanzado por la teratología como ciencia y las observaciones clínicas efectuadas por los científicos, permitieron establecer sus principios, representados por una serie de factores que determinan la capacidad de un agente de provocar trastornos congénitos, los cuales resultan actualmente de gran utilidad e importancia en la formación del profesional de la salud y repercuten en el Programa de Atención Materno Infantil. Objetivo: Describir los agentes teratógenos asociados a la aparición de defectos congénitos. Métodos: Se realizó una revisión bibliográfica acerca de los agentes teratógenos y sus posibles efectos teratogénicos en las bases de datos LILACS, SciELO, Clinicalkey y Google Académico, así como en la plataforma Biblioteca Virtual en Salud. Resultados: Las consecuencias de la exposición al teratógeno dependen, entre otras cosas, del momento en que se encuentre el proceso de gestación. Existen múltiples agentes teratógenos y algunos factores de riesgo que frecuentemente concomitan con ellos, por lo que se deben considerar en la prevención primaria para disminuir alteraciones y defectos congénitos por estas noxas ambientales. Consideraciones finales: El conocimiento de los efectos de los agentes teratógenos permite valorar el posible daño al feto.

Introduction: The development reached by teratology as science and the clinical observations made by the scientists, allowed to establish their principles, represented by a series of factors that determine the capacity of an agent to cause congenital dysfunctions, which are at the moment of great utility and importance in the training of health professional and rebound in the Infantile Maternal Care Program. Objective: To describe the teratogen agents associated with the appearance of congenital defects. Methods: A literatura review was carried out about the teratogen agents and their possible teratogenic effects in the LILACS, SciELO, Clinicalkey databases and Academic Google, as well as in the platform Virtual Library in Health. Results: The consequences of the exhibition to the teratogen depend, among other things, of the moment in which is the gestation process. There are multiple teratogen agents and some risk factors that frequently concomitan with them, for what should be considered in the primary prevention to diminish alterations and congenital defects for these environmental noxas. Final considerations: The knowledge of the effects of the agents teratogen allows to value the possible damage to the fetus.

Human recombinant cementum protein 1, dental pulp stem cells, and PLGA/hydroxyapatite scaffold as substitute biomaterial in critical size osseous defect repair in vivo.

OBJECTIVE: To evaluate the healing response of critical defects in rat calvaria with recombinant cementum protein 1 (hrCEMP-1) combined with human dental pulp stem cells (hDPSC) and polylactide-co-glycolide/hydroxyapatite (PLGA/HA) scaffold. METHODS: The effect of hrCEMP-1 on proliferation and differentiation of human dental stem cells (hDPSCs) toward a mineralizing-like phenotype was evaluated in monolayer and PLGA/HA scaffold by qPCR. 5 mm calvarial defects were created in Wistar rats and filled with: 1) PLGA/HA scaffold; 2) hDPSCs-PLGA/HA scaffold; 3) hrCEMP-1-hDPSc-PLGA/HA scaffold; 4) control (without scaffold). Bone formation was evaluated by histological-histomorphometric analysis, scanning electron microscopy (SEM) and radiographic evaluation. Comparisons between groups were made with a one-way analysis of variance ANOVA and Bonferroni post-hoc test. RESULTS: In vitro results showed that the PLGA/HA scaffold loaded with hrCEMP-1 improved the proliferation and differentiation of hDPSCs towards a mineralization phenotype by inducing mRNA expression of ALP, OSX, RUNX2, OP, and COL-I genes. The hrCEMP-1/hDPSCs/-PLGA/HA scaffold resulted only in connective tissue formed after ten weeks of healing, larger central radiolucency, and a low peripheral density. We showed superior bone growth and repair with a PLGA/HA matrix scaffold alone and containing hDPSCs compared to the hrCEMP/cells group. CONCLUSIONS: PLGA/HA scaffold with hrCEMP-1 induces hDPSC commitment to mineralizing phenotype in vitro, but does not promote critical size osseous defect repair in vivo when it is included in a substitute biomaterial with hDPSc-PLGA/HA scaffold.

Quantitative In-Depth Transcriptome Analysis Implicates Peritoneal Macrophages as Important Players in the Complement and Coagulation Systems.

To obtain a more detailed picture of macrophage (MΦ) biology, in the current study, we analyzed the transcriptome of mouse peritoneal MΦs by RNA-seq and PCR-based transcriptomics. The results show that peritoneal MΦs, based on mRNA content, under non-inflammatory conditions produce large amounts of a number of antimicrobial proteins such as lysozyme and several complement components. They were also found to be potent producers of several chemokines, including platelet factor 4 (PF4), Ccl6, Ccl9, Cxcl13, and Ccl24, and to express high levels of both TGF-ß1 and TGF-ß2. The liver is considered to be the main producer of most complement and coagulation components. However, we can now show that MΦs are also important sources of such compounds including C1qA, C1qB, C1qC, properdin, C4a, factor H, ficolin, and coagulation factor FV. In addition, FX, FVII, and complement factor B were expressed by the MΦs, altogether indicating that MΦs are important local players in both the complement and coagulation systems. For comparison, we analyzed human peripheral blood monocytes. We show that the human monocytes shared many characteristics with the mouse peritoneal MΦs but that there were also many major differences. Similar to the mouse peritoneal MΦs, the most highly expressed transcript in the monocytes was lysozyme, and high levels of both properdin and ficolin were observed. However, with regard to connective tissue components, such as fibronectin, lubricin, syndecan 3, and extracellular matrix protein 1, which were highly expressed by the peritoneal MΦs, the monocytes almost totally lacked transcripts. In contrast, monocytes expressed high levels of MHC Class II, whereas the peritoneal MΦs showed very low levels of these antigen-presenting molecules. Altogether, the present study provides a novel view of the phenotype of the major MΦ subpopulation in the mouse peritoneum and the large peritoneal MΦs and places the transcriptome profile of the peritoneal MΦs in a broader context, including a comparison of the peritoneal MΦ transcriptome with that of human peripheral blood monocytes and the liver.

A Nanoscale Shape-Discovery Framework Supporting Systematic Investigations of Shape-Dependent Biological Effects and Immunomodulation.

Since it is now possible to make, in a controlled fashion, an almost unlimited variety of nanostructure shapes, it is of increasing interest to understand the forms of biological control that nanoscale shape allows. However, a priori rational investigation of such a vast universe of shapes appears to present intractable fundamental and practical challenges. This has limited the useful systematic investigation of their biological interactions and the development of innovative nanoscale shape-dependent therapies. Here, we introduce a concept of biologically relevant inductive nanoscale shape discovery and evaluation that is ideally suited to, and will ultimately become, a vehicle for machine learning discovery. Combining the reproducibility and tunability of microfluidic flow nanochemistry syntheses, quantitative computational shape analysis, and iterative feedback from biological responses in vitro and in vivo, we show that these challenges can be mastered, allowing shape biology to be explored within accepted scientific and biomedical research paradigms. Early applications identify significant forms of shape-induced biological and adjuvant-like immunological control.

Glassy carbon microelectrode arrays enable voltage-peak separated simultaneous detection of dopamine and serotonin using fast scan cyclic voltammetry.

Progress in real-time, simultaneous in vivo detection of multiple neurotransmitters will help accelerate advances in neuroscience research. The need for development of probes capable of stable electrochemical detection of rapid neurotransmitter fluctuations with high sensitivity and selectivity and sub-second temporal resolution has, therefore, become compelling. Additionally, a higher spatial resolution multi-channel capability is required to capture the complex neurotransmission dynamics across different brain regions. These research needs have inspired the introduction of glassy carbon (GC) microelectrode arrays on flexible polymer substrates through carbon MEMS (C-MEMS) microfabrication process followed by a novel pattern transfer technique. These implantable GC microelectrodes provide unique advantages in electrochemical detection of electroactive neurotransmitters through the presence of active carboxyl, carbonyl, and hydroxyl functional groups. In addition, they offer fast electron transfer kinetics, capacitive electrochemical behavior, and wide electrochemical window. Here, we combine the use of these GC microelectrodes with the fast scan cyclic voltammetry (FSCV) technique to optimize the co-detection of dopamine (DA) and serotonin (5-HT) in vitro and in vivo. We demonstrate that using optimized FSCV triangular waveform at scan rates ≤700 V s-1 and holding and switching at potentials of 0.4 and 1 V respectively, it is possible to discriminate voltage reduction and oxidation peaks of DA and 5-HT, with 5-HT contributing distinct multiple oxidation peaks. Taken together, our results present a compelling case for a carbon-based MEA platform rich with active functional groups that allows for repeatable and stable detection of electroactive multiple neurotransmitters at concentrations as low as 1.1 nM.

Importance of antibody isotypes in antitumor immunity by monocytes and complement using human-immune tumor models.

Monoclonal antibodies (mAbs) have revolutionized clinical medicine, especially in the field of cancer immunotherapy. The challenge now is to improve the response rates, as immunotherapy still fails for many patients. Strategies to enhance tumor cell death is a fundamental aim, but relevant model systems for human tumor immunology are lacking. Herein, we have developed a preclinical human immune - three-dimensional (3D) tumor model (spheroids) to map the efficiency of tumor-specific isotypes for improved tumor cell killing. Different anti-CD20 Rituximab (RTX) isotypes alone or in combination, were evaluated for mediating complement-dependent cytotoxicity and antibody-dependent phagocytosis by human monocytic cells in 3D spheroids, in parallel with monolayer cultures, of human CD20+ B-cell lymphomas. We demonstrate that the IgG3 variant of RTX has the greatest tumoricidal effect over other isotypes, and when combined with apoptosis-inducing RTX-IgG2 isotype the therapeutic effect can be substantially enhanced. The results show further that the treatment outcome by RTX isotypes is influenced by tumor morphology and expression of the complement inhibitor CD59. Hence, the human immune-3D tumor model is a clinical relevant and attractive ex vivo system to predict mAbs for best efficacy in cancer immunotherapy.

Human papilloma virus genotypes in dysplasia and epithelial hyperplasia of oral cavity using the luminex xmap technology. A multicenter study

BACKGROUND: Oral cancer associated with high risk (HPV-HR) human papilloma virus (HPV) has been increasing. HPV-HR has been associated with epithelial dysplasia, however, little information exists on its frequency in epithelial hyperplasia lesions. The aim of this study is to compare HPV genotypes in dysplastic and hyperplastic lesions of oral cavity. MATERIAL AND METHODS: Two hundred and fifty oral lesions: 131 dysplasia and 119 hyperplasia from two regions of Colombia were evaluated. One hundred seventy-four coming from urban area and 104 from a high risk population to oral cancer from a rural area. HPV was identified by qPCR and Twenty-four HPVs genotypes were evaluated by Luminex(R) technology. Logistic regressions were performed to establish the associations between HPV infections with oral dysplasia. RESULTS: Twenty-eight percent (70/250) of the samples were positives for any HPV and HPV-HRs were more frequently than low risk HPVs. HPV-16 was the most detected genotype (16%) followed by HPV-31, 53, 18 and 45. HPV, HPV-HRs and HPV-16 were only associated with dysplasia in urban area; OR 3.28 (CI 95% 1.49-7.17), OR 7.94 (CI 95% 2.97-21.2) and OR 5.90 (CI 95% 2.05-17). Individuals in rural area showed more HPV and HPV-HRs infection in hyperplasic lesions than urban population. The majority of HPV+ lesions had multi-type of HPV (52/70) and the urban individuals showed more genotypes than rural population. CONCLUSIONS: HPV-.HRs are frequently found in hyperplastic and dysplastic epithelial lesions. HPV-HRs and HPV-16 were associated with dysplasia in urban population. Rural high risk population and urban population differ in the frequency and variety of HPV genotypes

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Acetyl-CoA carboxylase 1-dependent lipogenesis promotes autophagy downstream of AMPK.

Autophagy, a membrane-dependent catabolic process, ensures survival of aging cells and depends on the cellular energetic status. Acetyl-CoA carboxylase 1 (Acc1) connects central energy metabolism to lipid biosynthesis and is rate-limiting for the de novo synthesis of lipids. However, it is unclear how de novo lipogenesis and its metabolic consequences affect autophagic activity. Here, we show that in aging yeast, autophagy levels highly depend on the activity of Acc1. Constitutively active Acc1 (acc1S/A ) or a deletion of the Acc1 negative regulator, Snf1 (yeast AMPK), shows elevated autophagy levels, which can be reversed by the Acc1 inhibitor soraphen A. Vice versa, pharmacological inhibition of Acc1 drastically reduces cell survival and results in the accumulation of Atg8-positive structures at the vacuolar membrane, suggesting late defects in the autophagic cascade. As expected, acc1S/A cells exhibit a reduction in acetate/acetyl-CoA availability along with elevated cellular lipid content. However, concomitant administration of acetate fails to fully revert the increase in autophagy exerted by acc1S/A Instead, administration of oleate, while mimicking constitutively active Acc1 in WT cells, alleviates the vacuolar fusion defects induced by Acc1 inhibition. Our results argue for a largely lipid-dependent process of autophagy regulation downstream of Acc1. We present a versatile genetic model to investigate the complex relationship between acetate metabolism, lipid homeostasis, and autophagy and propose Acc1-dependent lipogenesis as a fundamental metabolic path downstream of Snf1 to maintain autophagy and survival during cellular aging.

Analysis of In Vitro Osteoblast Culture on Scaffolds for Future Bone Regeneration Purposes in Dentistry.

One of the main focuses of tissue engineering is to search for tridimensional scaffold materials, complying with nature's properties for tissue regeneration. Determining material biocompatibility is a fundamental step in considering its use. Therefore, the purpose of this study was to analyze osteoblast cell adhesion and viability on different materials to determine which was more compatible for future bone regeneration. Tridimensional structures were fabricated with hydroxyapatite, collagen, and porous silica. The bovine bone was used as material control. Biocompatibility was determined by seeding primary osteoblasts on each tridimensional structure. Cellular morphology was assessed by SEM and viability through confocal microscopy. Osteoblast colonization was observed on all evaluated materials' surface, revealing they did not elicit osteoblast cytotoxicity. Analyses of four different materials studied with diverse compositions and characteristics showed that adhesiveness was best seen for HA and viability for collagen. In general, the results of this investigation suggest these materials can be used in combination, as scaffolds intended for bone regeneration in dental and medical fields.

Applications of Nanomaterials for Immunosensing.

In biomedical science among several other growing fields, the detection of specific biological agents or biomolecular markers, from biological samples is crucial for early diagnosis and decision-making in terms of appropriate treatment, influencing survival rates. In this regard, immunosensors are based on specific antibody-antigen interactions, forming a stable immune complex. The antigen-specific detection antibodies (i.e., biomolecular recognition element) are generally immobilized on the nanomaterial surfaces and their interaction with the biomolecular markers or antigens produces a physico-chemical response that modulates the signal readout. Lowering the detection limits for particular biomolecules is one of the key parameters when designing immunosensors. Thus, their design by combining the specificity and versatility of antibodies with the intrinsic properties of nanomaterials offers a plethora of opportunities for clinical diagnosis. In this review, we show a comprehensive set of recent developments in the field of nanoimmunosensors and how they are progressing the detection and validation for a wide range of different biomarkers in multiple diseases and what are some drawbacks and considerations of the uses of such devices and their expansion.

In vitro response of dental pulp stem cells in 3D scaffolds: A regenerative bone material.

Three-dimensional-porous scaffolds of bone graft substitutes play a critical role in both cell targeting and transplantation strategies. These scaffolds provide surfaces that facilitate the response of stem cells related to attachment, survival, migration, proliferation, and differentiation. OBJECTIVE: The aim of this study was to evaluate the in vitro behavior of human dental pulp mesenchymal stem cells cultured on scaffolds of polylactic/polyglycolic acid with and without hydroxyapatite. METHOD: We performed an in vitro experimental study using dental pulp stem cells obtained from samples of premolars, molars. The cells were cultured on scaffolds with osteogenic differentiation medium. Cell proliferation, adhesion and cell differentiation to an osteoblastic linage in the biomaterial were evaluated at three different time points: 7, 15 and 30 days. Each experiment was performed in triplicate. Analysis of the data was performed with the Split Plot block and MANOVA model. RESULTS: The differentiation capability of hDPSCs towards the osteoblast lineage was better in the scaffold of PLGA/HA at 7, 15 and 30 days, as indicated by the high expression of osteogenic markers RUNX2, ALP, OPN and COL-I, compared with differentiation in the PLGA scaffold. No statistically significant differences were found in cell adhesion between the two types of scaffolds. CONCLUSION: The PLGA/HA scaffold provided better physical and chemical signals, as judged by the ability of dental pulp stem cells to adhere, proliferate and differentiate toward the osteogenic lineage.

Differential Recognition of Nanoparticle Protein Corona and Modified Low-Density Lipoprotein by Macrophage Receptor with Collagenous Structure.

Key practical challenges such as understanding the immunological processes at the nanoscale and controlling the targeting and accumulation of nano-objects in vivo now further stimulate efforts to underpin phenomenological knowledge of the nanoscale with more mechanistic and molecular insight. Thus, the question as to what constitutes nanoscale biological identity continues to evolve. Certainly nanoparticles in contact with a complex biological milieu develop a biological identity, differing from the original nanomaterial, now referred to as the "biomolecular corona". However, this surface-adsorbed layer of biomolecules may in some circumstance lead to different forms of receptor-particle interactions not evident only from the identity of the surface-adsorbed biomolecules and hard to predict or detect by current physicochemical methods. Here we show that scavenger receptors may recognize complex as yet unidentified biomolecular surface layer motifs, even when no current physicochemical analysis is capable of doing so. For instance, fluorescently labeled SiO2 nanoparticles in a biological milieu are strongly recognized by the macrophage receptor with collagenous structure (MARCO) in even dense biological media (human serum) apparently using a form of binding with which most of the MARCO's known ligands ( e. g., LPS, modified LDL) fail to compete. Such observations may suggest the need for a much stronger emphasis on nanoscale receptor-corona and other biomolecular interaction studies if one wishes to unravel how biomolecular recognition drives outcomes in the nanoscale biological domain.