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Cancer is associated with increased fracture risk, due either to metastasis or associated osteoporosis. After a fracture, blood clots form. Because proteins of the coagulation cascade and activated platelets promote cancer development, a fracture in patients with cancer often raises the question whether it is a pathologic fracture or whether the fracture itself might promote the formation of metastatic lesions. We therefore examined whether blood clot formation results in increased metastasis in a murine model of experimental breast cancer metastasis. For this purpose, a clot was surgically induced in the bone marrow of the left tibia of immundeficient mice. Either one minute prior to or five minutes after clot induction, human cancer cells were introduced in the circulation by intracardiac injection. The number of cancer cells that homed to the intervention site was determined by quantitative real-time PCR and flow cytometry. Metastasis formation and longitudinal growth were evaluated by bioluminescence imaging. The number of cancer cells that homed to the intervention site after 24 hours was similar to the number of cells in the opposite tibia that did not undergo clot induction. This effect was confirmed using two more cancer cell lines. Furthermore, no difference in the number of macroscopic lesions or their growth could be detected. In the control group 72% developed a lesion in the left tibia. In the experimental groups with clot formation 79% and 65% developed lesions in the left tibia (p = ns when comparing each experimental group with the controls). Survival was similar too. In summary, the growth factors accumulating in a clot/hematoma are neither enough to promote cancer cell homing nor support growth in an experimental model of breast cancer bone metastasis. This suggests that blood clot formation, as occurs in traumatic fractures, surgical interventions, and bruises, does not increase the risk of metastasis formation. [ABSTRACT FROM AUTHOR]

A survey was conducted to determine the occurrence of fumonisin B1, B2 and B3 during 2007 in 186 samples of organic and conventional locally available corn products. Samples included baby food (n = 62), corn flour (11), cornflakes (23), pasta (14), cookies (17) and other corn products (59) were obtained from popular markets of Valencia (Spain) and Perugia (Italy). The analytical method used pressurized liquid extraction and liquid chromatography/electrospray ionization tandem mass spectrometry with a triple quadrupole (QqQ) analyser. Of the 104 Spanish samples, 22% contained levels in the range of 2-449 µg kg-1, 2-229 µg kg-1 and 6-105 µg kg-1 for FB1, FB2 and FB3, respectively, while 19 (23%) of the 82 Italian samples were positive with quantifiable levels between 2-235 µg kg-1, 3-187 µg kg-1, and 4-40 µg kg-1 for fumonisins B1, B2 and B3, respectively. Overall, none of the Italian samples and only one organic baby food sample from a Spanish market was above the maximum permitted levels established by European legislation. Fumonisins were found mostly in corn flour followed by cookies and cornflakes. Eleven samples from Spain and nine samples from Italy were organic products, being contaminated the 72% and 77% of the samples, respectively. Analysis of the results showed that levels of fumonisins in corn products were similar in Italy and Spain. The safety of fumonisin intake through corn products was demonstrated by the calculation of the estimated daily intake of both populations considering organic and conventional products separately, which ranged from 1.7 × 10-3 to 0.72 µg kg-1 bw day-1 and comparing them with the provisional maximum total daily intake (PMTDI) of 2 µg kg-1 bw day-1 established by the European Union. [ABSTRACT FROM AUTHOR]

Introducción: la pandemia de la COVID-19 ha acentuado las desigualdades sociales, económicas y relacionadas con la salud, afectando desproporcionadamente a las personas en situación de vulnerabilidad y perpetuando la inequidad en salud. En Argentina se implementó una campaña nacional gratuita de vacunación contra la COVID-19 con una perspectiva de equidad., Objetivo: identificar desigualdades territoriales en el acceso a la vacunación contra la COVID-19 en Quilmes., Métodos: se analizó la información referida a la vacunación contra la COVID-19 de personas residentes en el Municipio. Se efectuó la georreferenciación de cada vacunatorio y de cada persona a partir del domicilio declarado en el momento de la vacunación. Para caracterizar el grado de vulnerabilidad de las personas vacunadas, a cada una se le asignó el índice de carencias múltiples (ICM) correspondiente al radio censal de residencia., Resultados: al menos el 82 % de la población completó el esquema primario de vacunación (dosis 1 y dosis 2), porcentaje que alcanzó el 97 % en los mayores de 65 años. Analizando la media de dosis aplicadas se observa algo similar con un gradiente hacia los quintiles más altos pero con una mínima diferencia entre sí, situación que también se corrobora en todos los grupos etarios., Discusión: no se observaron brechas significativas entre los diferentes niveles socioeconómicos. Si bien se observó un mínimo gradiente en el promedio de dosis recibidas, el tiempo de acceso a las diferentes vacunas y el porcentaje de esquemas primarios completos recibidos, las mismas tienen escasa relevancia clínica y sanitaria., Competing Interests: Declaración de conflicto de interesesNingún conflicto declarado.

Parkinsonism is the primary type of movement disorder in adults, encompassing a set of clinical symptoms, including rigidity, tremors, dystonia, bradykinesia, and postural instability. These symptoms are primarily caused by a deficiency in dopamine (DA), an essential neurotransmitter in the brain. Currently, the DA precursor levodopa (synthetic L-DOPA) is the standard medication to treat DA deficiency, but it only addresses symptoms rather than provides a cure. In this review, we provide an overview of disorders associated with DA dysregulation and deficiency, particularly Parkinson's disease and rare inherited disorders leading predominantly to dystonia and/or parkinsonism, even in childhood. Although levodopa is relatively effective for the management of motor dysfunctions, it is less effective for severe forms of parkinsonism and is also associated with side effects and a loss of efficacy over time. We present ongoing efforts to reinforce the effect of levodopa and to develop innovative therapies that target the underlying pathogenic mechanisms affecting DA synthesis and transport, increasing neurotransmission through disease-modifying approaches, such as cell-based therapies, nucleic acid- and protein-based biologics, and small molecules., (© 2024 The Author(s).)

Enzymes catalyze the chemical reactions of life. For nearly half of known enzymes, catalysis requires the binding of small molecules known as cofactors. Polypeptide-cofactor complexes likely formed at a primordial stage and became starting points for the evolution of many efficient enzymes. Yet, evolution has no foresight so the driver for the primordial complex formation is unknown. Here, we use a resurrected ancestral TIM-barrel protein to identify one potential driver. Heme binding at a flexible region of the ancestral structure yields a peroxidation catalyst with enhanced efficiency when compared to free heme. This enhancement, however, does not arise from protein-mediated promotion of catalysis. Rather, it reflects the protection of bound heme from common degradation processes and a resulting longer lifetime and higher effective concentration for the catalyst. Protection of catalytic cofactors by polypeptides emerges as a general mechanism to enhance catalysis and may have plausibly benefited primordial polypeptide-cofactor associations., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Obligate symbionts typically exhibit high evolutionary rates. Consequently, their proteins may differ considerably from their modern and ancestral homologs in terms of both sequence and properties, thus providing excellent models to study protein evolution. Also, obligate symbionts are challenging to culture in the lab and proteins from uncultured organisms must be produced in heterologous hosts using recombinant DNA technology. Obligate symbionts thus replicate a fundamental scenario of metagenomics studies aimed at the functional characterization and biotechnological exploitation of proteins from the bacteria in soil. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its folding outside the original host is severely impaired, as shown by a very slow refolding in vitro and an inefficient expression in E. coli that leads mostly to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro (i.e., unassisted) folding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a minimal-perturbation, sequence-engineering approach to rescue inefficient heterologous expression which may potentially be useful in metagenomics efforts targeting recent adaptations., Competing Interests: Acknowledgements This work was supported by Human Frontier Science Program Grant RGP0041/2017 (J.M.S.-R. and E.A.G.), National Science Foundation Award #2032315 (E.A.G.), National Institutes of Health Award #R01AR069137 (E.A.G.), Department of Defense MURI Award #W911NF-16-1-0372 (E.A.G.), Spanish Ministry of Science and Innovation/FEDER Funds Grants RTI-2018-097142-B-100 (J.M.S.-R.) and BIO2016-74875-P (J.A.G.) and the Science, Engineering and Research Board (SERB, India) Grant MTR/2019/000392 (A.N.N.). We are grateful to the European Synchrotron Radiation Facility (ESRF), Grenoble, France, for the provision of time and the staff at ID23-1 beamline for assistance during data collection. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Objective: To measure SARS-CoV-2 RNA in sewage in a low-resource community in order to determine if it can be considered as an estimator of changes in the prevalence of COVID-19 in the population., Methods: In this descriptive observational study we collected samples of surface waters contaminated with sewage and optimized a method of purification of viral RNA using PEG concentration. We determined the amount of genetic material by quantitative real-time PCR using the CDC method for SARS-CoV-2 detection., Results: We quantified viral RNA in surface waters contaminated with sewage of a low resource community and determined that temporal trends of SARS-CoV-2 in wastewater samples mirrored trends in COVID-19 active cases., Conclusions: Measuring of SARS-CoV-2 RNA in sewage can be applied in low-resource communities without connection to sewers as an estimator of changes in the prevalence of COVID-19.

We present the case of a patient with recurrent episodes of ventricular fibrillation without evidence of structural cardiac diseases on imaging techniques and negative genetic testing for the most common primary arrhythmia syndromes. A new variant c.6023C>T p.Pro2008Leu of the SCN5A protein, responsible for the sodium inward current (I Na ) through the cardiomyocytes, was found. A likely pathogenic effect of this gene variant was hypothesized., Competing Interests: There are no conflicts of interest., (Copyright: © 2022 Journal of Cardiovascular Echography.)

Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the ∼5500 reported crystallographic structures of ∼1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

Evolution involves not only adaptation, but also the degradation of superfluous features. Many examples of degradation at the morphological level are known (vestigial organs, for instance). However, the impact of degradation on molecular evolution has been rarely addressed. Thioredoxins serve as general oxidoreductases in all cells. Here, we report extensive mutational analyses on the folding of modern and resurrected ancestral bacterial thioredoxins. Contrary to claims from recent literature, in vitro folding rates in the thioredoxin family are not evolutionarily conserved, but span at least a ∼100-fold range. Furthermore, modern thioredoxin folding is often substantially slower than ancestral thioredoxin folding. Unassisted folding, as probed in vitro, thus emerges as an ancestral vestigial feature that underwent degradation, plausibly upon the evolutionary emergence of efficient cellular folding assistance. More generally, our results provide evidence that degradation of ancestral features shapes, not only morphological evolution, but also the evolution of individual proteins., (© 2019 The Author(s).)

A high sensitive immunoassay-based lateral flow device for semi-quantitatively determine aflatoxin M1 (AFM1) in milk was developed. Investigation and optimization of the competitor design and of the gold-labelling strategy allowed the attainment of the ultra-sensitive assessment of AFM1 contamination at nanograms per litre level (LOD 20 ng L(-1), IC50 99 ng L(-1)), as requested by European regulations. A one order of magnitude detectability enhancement in comparison to previously reported gold colloid immunochromatographic assays for this toxin was obtained. Direct detection of the target toxin in milk could be obtained by acquiring images of the strips and correlating intensities of the coloured lines with analyte concentrations. The one-step assay can be completed in 17 min, including a very simple and rapid sample preparation, which allowed the application of the assay to milk samples which differ in fat and protein contents. Although imprecise (mean RSD about 30%), the method proved to be accurate and sensitive enough to allow the correct attribution of sample as compliant or non-compliant according to EU legislation in force. Agreeing results to those of a reference ELISA were obtained on 40 milk samples by matrix-matched calibration in pasteurized milk., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A 68-year-old woman developed tenosynovitis of the right second digit without a history of injury or animal bites. Apart from high titer anti-nuclear antibodies, serological studies were unremarkable. Tuberculin test and interferon gamma release assay were both negative. Several immunosuppressive therapies led only to partial relief of symptoms. Of note, clinical symptoms worsened significantly after introduction of adalimumab therapy. Tenosynovectomy was performed revealing a granulomatous inflammatory process. Seven weeks later, Mycobacterium malmoense could be cultured from the surgical specimen. A four drug antibiotic regimen was started and immunosuppressive therapy discontinued resulting in complete clinical remission. Our case highlights non-tuberculous mycobacterial (NTM) tenosynovitis as an important differential diagnosis of atypical arthritis. A negative tuberculin skin test as well as negative Ziehl-Neelsen stain does not argue against NTM infection. In fact, mycobacterial culture for extended periods remains the gold standard for diagnosis.

Natural toxin (for example mycotoxin and phycotoxin) contamination of food is of safety and economic concern, so much effort is devoted to the development of screening methods which enable the toxins to be continuously and widely monitored in food and feed. More generally speaking, rapid and non-instrumental assays for detection of a variety of food contaminants are generating ever-increasing scientific and technological interest because they enable high-throughput, economical, on-site monitoring of such contaminants. Among rapid methods for first-level screening of food contaminants, lateral-flow immunoassay (LFIA), also named immunochromatographic assay or immune-gold colloid immunoassay, has recently attracted scientific and industrial interest because of its attractive property of enabling very rapid, one-step, in-situ analysis. This review focuses on new aspects of the development and optimization of lateral-flow devices for mycotoxin and phycotoxin detection, including strategies for management of matrix interference and, particularly, for investigation of the improvements achieved by signal-enhancing strategies or by application of non-gold nanoparticle signal reporters.

Background: Rabbit meat has excellent nutritive properties. The purpose of this study was to characterize rabbit meat and offal; in particular, the lipid fraction was studied in order to evaluate total and positional fatty acid (FA) compositions of triacylglycerol (TAG) and phosphatidylcholine (PC) fractions. Eight samples of weaned and eight of fattened rabbits were considered., Results: Fattened rabbit meat contained slightly higher protein percentage content (P < 0.05) in comparison to weaned (20.1% versus 18.0%). Calcium content was higher in meat than in offal, unlike sodium, iron, zinc, manganese and copper. The cholesterol content in offal was much higher than in meat. FA profiles of total lipid showed a high percentage of unsaturated fatty acids and an n-6/n-3 ratio of 10.3 for fattened rabbit meat. Stereospecific analysis of TAG and PC was carried out on an eight-sample pool of each meat and offal from weaned and fattened rabbits. In all samples the sn-2-position was prevalently esterified with oleic and linoleic acids in TAG, with polyunsaturated fatty acids in PC., Conclusion: Lipids from rabbit meat presented higher content of monounsaturated FA and lower n-6/n-3 ratio in comparison to offal, which was characterized by higher cholesterol and mineral levels., (Copyright © 2011 Society of Chemical Industry.)

Stereospecific analysis is an important tool for the characterization of lipid fraction of food matrices, and also of milk samples. The results of a chemical-enzymatic-chromatographic analytical method were elaborated by chemometric procedures such as linear discriminant analysis (LDA) and artificial neural network (ANN). According to the total composition and intrapositional fatty acid distribution in the triacylglycerol (TAG) backbone, the obtained results were able to characterize pure milk samples and milk mixtures with 1, 3, 5% cow milk added to donkey milk. The resulting score was very satisfactory. Totally correct classified samples were obtained when the TAG stereospecific results of all the considered milk mixtures (donkey-cow) were elaborated by LDA and ANN chemometric procedures.

An immunoassay-based lateral flow device for the quantitative determination of four major aflatoxins in maize has been developed. The one-step assay has performance comparably with that of other screening methods, as confirmed by the intra- and the inter-day precision of the data (RSD 10-22%), and can be completed in 10 min. Quantification was obtained by acquiring images of the strip and correlating intensities of the coloured lines with analyte concentration by means of a stored calibration curve carried out by diluting aflatoxins in the extract from a blank maize sample. Limit of detection (1 µg kg⁻¹) and dynamic range (2-40 µg kg⁻¹) allows the direct assessment of aflatoxin contamination in maize at all levels of regulatory relevance. All reagents are immobilized on the lateral flow device. In addition, very simple sample preparation, using an aqueous buffered solution, has been demonstrated to allow the quantitative extraction of aflatoxins. Twenty-five maize samples were extracted with the aqueous medium and analyzed by the developed assay. A good correlation was observed (y = 0.97x + 0.07, r²= 0.980) when data was compared with that obtained through an official method. The developed method is reliable, rapid and allows for application outside the laboratory as a point-of-use test for screening purposes.

Large defects following resection of skin cancers are sometimes a challenge for the reconstructive surgeon. Although skin grafts are considered as the first choice for reconstruction of large skin defects at the trunk region, pedicled or free flaps provide sometimes a superior functional and aesthetic outcome. Perforator flaps represent a valuable option for these patients. The progress in understanding the perforator vessel system of the body facilitated the development of a plethora of novel pedicled flaps which could be transferred over long distances with minimal donor site morbidity. We present a patient suffering from a large exulcerated basalioma at the lumbar region. The skin defect after excision was reconstructed using a novel concept based on 2 independent pedicled perforator flaps, a lumbar artery perforator, and a lateral intercostal artery perforator.

A sensitive and reliable method using pressurized liquid extraction (PLE) and liquid chromatography (LC)/electrospray ionization (ESI) tandem mass spectrometry with a triple quadrupole (QqQ) analyzer has been developed for the analysis of fumonisin B(1) (FB(1)), fumonisin B(2) (FB(2)) and fumonisin B(3) (FB(3)) in corn-based baby foods. Influence of several extraction parameters that affect PLE efficiency such as temperature, pressure, solvent extraction, number of cycles and dispersant/clean-up agents were studied. The selected PLE operating method was: 3g of sample was packed into 11 ml stainless-steel cell and fumonisins were extracted with methanol at 40 degrees C, 34 atm in one cycle of 5 min at 60% flush. The analytes were ionized in ESI operating with positive ion mode and identified by selecting two monitoring transitions, permitting quantification and confirmation in a single injection. Recoveries ranged from 68% to 83% at fortification levels of 200 microg kg(-1) with relative standard deviation (RSD) from 4% to 12%. The limits of quantification were from 2 microg kg(-1) for FB(1) and FB(2), and 5 microg kg(-1) for FB(3), which are below the maximum residue level established by the European Union legislation in infant formulas. The proposed method was successfully applied to the analysis of twenty seven samples of baby food products collected from different markets, and one positive sample with a content of 15.9 microg kg(-1) for FB(1), 9.2 microg kg(-1)for FB(2) and 5.8 microg kg(-1) for FB(3) was obtained. Given the simplicity and potential of the proposed procedure, its application for safety control is recommended.

An innovative procedure to separate the 3 isomeric sn-monoacylglycerols (MAG) classes (sn-1-, sn-2-, sn-3-MAG) is described. MAGs, obtained by chemical deacylation of triacylglycerols (TAGs), have been derivatized with (S)-(+)-1-(1-naphtyl)ethyl-isocyanate, and the resulting urethane derivatives have been separated by normal-phase high-performance liquid chromatography. This procedure allows resolution as diasteroisomers of the 2 enantiomeric classes (sn-1-MAG and sn-3-MAG), without the need of a chiral column, and to separate also the isomeric sn-2-MAG class; moreover, by introducing a chromophoric moiety, this reagent makes possible the ultraviolet detection of the analyte molecules. This procedure has been used to obtain the stereospecific analysis of the TAG fraction of extra virgin olive oil samples. The use of a nondestructive detector permitted the collection of the individual urethane classes; the fatty acid composition of each was determined by high-resolution gas chromatography, obtaining directly from the data the fatty acid distribution within each sn- position of TAGs. To validate this new method, the results have been compared with those obtained by 2 other procedures for TAG stereospecific analysis, and the obtained results were satisfactory since the proposed method gave data very similar to the other procedures.