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Introduction: Irinotecan (CPT-11) is an inhibitor of DNA topoisomerase I and is clinically effective against several cancers. A major toxic effect of CPT-11 is delayed diarrhea; however, the exact mechanism by which the drug induces diarrhea has not been established.Purpose: Elucidate the mechanisms of induction of delayed diarrhea and determine the effects of the cytokine production inhibitor pentoxifylline (PTX) and thalidomide (TLD) in the experimental model of intestinal mucositis, induced by CPT-11.Materials and Methods: Intestinal mucositis was induced in male Swiss mice by intraperitoneal administration of CPT-11 (75 mg/kg) daily for 4 days. Animals received subcutaneous PTX (1.7, 5 and 15 mg/kg) or TLD (15, 30, 60 mg/kg) or 0.5 ml of saline daily for 5 and 7 days, starting 1 day before the first CPT-11 injection. The incidence of delayed diarrhea was monitored by scores and the animals were sacrificed on the 5th and 7th experimental day for histological analysis, immunohistochemistry for TNF-alpha and assay of myeloperoxidase (MPO) activity, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and KC ELISA.Results: CPT-11 caused significant diarrhea, histopathological alterations (inflammatory cell infiltration, loss of crypt architecture and villus shortening) and increased intestinal tissue MPO activity, TNF-alpha, IL-1beta and KC level and TNF-alpha immuno-staining. PTX inhibited delayed diarrhea of mice submitted to intestinal mucositis and reduced histopathological damage, intestinal MPO activity, tissue level of TNF-alpha, IL-1beta and KC and TNF-alpha immuno-staining. TLD significantly reduced the lesions induced by CPT-11 in intestinal mucosa, decreased MPO activity, TNF-alpha tissue level and TNF-alpha immuno-staining, but did not reduce the severity of diarrhea.Conclusion: These results suggest an important role of TNF-alpha, IL-1beta and KC in the pathogenesis of intestinal mucositis induced by CPT-11. [ABSTRACT FROM AUTHOR]

Host cell proteins (HCPs) are process-related impurities expressed by the host cells during biotherapeutics' manufacturing, such as monoclonal antibodies (mAbs). Some challenging HCPs evade clearance during the downstream processing and can be co-purified with the molecule of interest, which may impact product stability, efficacy, and safety. Therefore, HCP content is a critical quality attribute to monitor and quantify across the bioprocess. Here we explored a mass spectrometry (MS)-based proteomics tool, the sequential window acquisition of all theoretical fragment-ion spectra (SWATH) strategy, as an orthogonal method to traditional ELISA. The SWATH workflow was applied for high-throughput individual HCP identification and quantification, supporting characterization of a mAb purification platform. The design space of HCP clearance of two polishing resins was evaluated through a design of experiment study. Absolute quantification of high-risk HCPs was achieved (reaching 1.8 and 4.2 ppm limits of quantification, for HCP A and B respectively) using HCP-specific synthetic heavy labeled peptide calibration curves. Profiling of other HCPs was also possible using an average calibration curve (using labeled peptides from different HCPs). The SWATH approach is a powerful tool for HCP assessment during bioprocess development enabling simultaneous monitoring and quantification of different individual HCPs and improving process understanding of their clearance., Competing Interests: 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 © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Objective: To map evidence on technologies used by nurses to promote breastfeeding in Health Services., Method: This is a scoping review, based on the recommendations of the Joanna Briggs Institute and following the PRISMA Extension for Scoping Reviews, carried out in 2022. The searches took place in seven databases, using the following combined descriptors: "nurse", "technology", "breastfeeding", and "health services"., Results: Fifteen articles were found, the first from 2000 and the last from 2022, all published in English with a predominance of productions in the United States of America (n = 5) and Brazil (n = 3). The link was the technology present in most studies (n = 11). However, with regard to classification, educational and hard technologies were the most used in promotion strategies (n = 14 and n = 12), respectively., Conclusion: The articles showed a variety of technologies used to promote breastfeeding in health services, and thus, contributing for the maintenance and duration of breastfeeding.

Phylogenetic diversity is an increasingly applied metric used to maximize the representation of evolutionary history in spatial conservation planning. When following this approach, researchers commonly overlook sites with a relatively higher proportion of recently diverged endemic species, also known as centers of neo-endemism. Here we aim to demonstrate how targeting the conservation of different facets of diversity (taxonomic diversity, phylogenetic diversity and centers of endemism) can provide more cost-effective solutions to the conservation of the all evolutionary spectrum of biodiversity. We do so by using the mantellid frogs of Madagascar as a case study. Our results confirm that areas with high concentrations of neo-endemism can be effectively identified as conservation planning priorities only if we specifically target them. Neglecting areas that are poor in phylogenetic diversity may therefore compromise the maintenance of diversification processes, particularly when lesser proportions of the landscape are protected. This approach can be of particular interest to island ecosystems, since they often harbor unique and restricted evolutionary radiations., (© 2023. The Author(s).)

The majority of recombinant adeno-associated viruses (rAAV) approved for clinical use or in clinical trials areproduced by transient transfection using the HEK293 cell line. However, this platform has several manufacturing bottlenecks at commercial scales namely, low product quality (full to empty capsid ratio <20% in most rAAV serotypes), lower productivities obtained after scale-up and the high cost of raw materials, in particular of Good Manufacturing Practice grade plasmid DNA required for transfection. The HeLa-based stable cell line rAAV production system provides a robust and scalable alternative to transient transfection systems. Nevertheless, the time required to generate the producer cell lines combined with the complexity of rAAV production and purification processes still pose several barriers to the use of this platform as a suitable alternative to the HEK293 transient transfection. In this work we streamlined the cell line development and bioprocessing for the HeLaS3-based production of rAAV. By exploring this optimized approach, producer cell lines were generated in 3-4 months, and presented rAAV2 volumetric production (bulk) > 3 × 10 11  vg/mL and full to empty capsids ratio (>70%) at 2 L bioreactor scale. Moreover, the established downstream process, based on ion exchange and affinity-based chromatography, efficiently eliminated process related impurities, including the Adenovirus 5 helper virus required for production with a log reduction value of 9. Overall, we developed a time-efficient and robust rAAV bioprocess using a stable producer cell line achieving purified rAAV2 yields > 1 × 10 11  vg/mL. This optimized platform may address manufacturing challenges for rAAV based medicines., (© 2023 The Authors. Biotechnology and Bioengineering published by Wiley Periodicals LLC.)

Integrative and proactive conservation approaches are critical to the long-term persistence of biodiversity. Molecular data can provide important information on evolutionary processes necessary for conserving multiple levels of biodiversity (genes, populations, species, and ecosystems). However, molecular data are rarely used to guide spatial conservation decision-making. Here, we bridge the fields of molecular ecology (ME) and systematic conservation planning (SCP) (the 'why') to build a foundation for the inclusion of molecular data into spatial conservation planning tools (the 'how'), and provide a practical guide for implementing this integrative approach for both conservation planners and molecular ecologists. The proposed framework enhances interdisciplinary capacity, which is crucial to achieving the ambitious global conservation goals envisioned for the next decade., Competing Interests: Declaration of interests None declared by authors., (Copyright © 2022 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Understanding the ecological and evolutionary processes driving biodiversity patterns and allowing their persistence is of utmost importance. Many hypotheses have been proposed to explain spatial diversity patterns, including water-energy availability, habitat heterogeneity, and historical climatic refugia. The main goal of this study is to identify if general spatial drivers of species diversity patterns of phylogenetic diversity (PD) and phylogenetic endemism (PE) at the global scale are also predictive of PD and PE at regional scales, using Iberian amphibians as a case study. Our main hypothesis assumes that topography along with contemporary and historical climate are drivers of phylogenetic diversity and endemism, but that the strength of these predictors may be weaker at the regional scale than it tends to be at the global scale. We mapped spatial patterns of Iberian amphibians' phylogenetic diversity and endemism, using previously published phylogenetic and distribution data. Furthermore, we compiled spatial data on topographic and climatic variables related to the water-energy availability, topography, and historical climatic instability hypotheses. To test our hypotheses, we used Spatial Autoregressive Models and selected the best model to explain diversity patterns based on Akaike Information Criterion. Our results show that, out of the variables tested in our study, water-energy availability and historical climate instability are the most important drivers of amphibian diversity in Iberia. However, as predicted, the strength of these predictors in our case study is weaker than it tends to be at global scales. Thus, additional drivers should also be investigated and we suggest caution when interpreting these predictors as surrogates for different components of diversity., (© 2023 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Virus-based biopharmaceutical products are used in clinical applications such as vaccines, gene therapy, and immunotherapy. However, their manufacturing remains a challenge, hampered by the lack of appropriate analytical tools for purification monitoring or characterization of the final product. This paper describes the implementation of a highly sensitive method, capillary electrophoresis (CE)-sodium dodecyl sulfate (SDS) combined with a laser-induced fluorescence (LIF) detector to monitor the impact of various bioprocess steps on the quality of different viral vectors. The fluorescence labelling procedure uses the (3-(2-furoyl) quinoline-2-carboxaldehyde dye, and the CE-SDS LIF method enables the evaluation of in-process besides final product samples. This method outperforms other analytical methods, such as SDS-polyacrylamide gel electrophoresis with Sypro Ruby staining, in terms of sensitivity, resolution, and high-throughput capability. Notably, this CE-SDS LIF method was also successfully implemented to characterize enveloped viruses such as Maraba virus and lentivirus, whose development as biopharmaceuticals is now restricted by the lack of suitable analytical tools. This method was also qualified for quantification of rAAV2 according to the International Council for Harmonisation guidelines. Overall, our work shows that CE-SDS LIF is a precise and sensitive analytical platform for in-process sample analysis and quantification of different virus-based targets, with a great potential for application in biomanufacturing.

Rotavirus A infection is a global leading cause of severe acute gastroenteritis associated with life-threatening diarrheal episodes in infants and young children. The disease burden is being reduced, namely due to a wider access to rotavirus vaccines. However, there is a demand to expand rotavirus vaccination programs, and to achieve this, it is critical to improve high-throughput in-process product quality control and vaccine manufacturing monitoring. Here, we present the development of an analytical method for the quantification of rotavirus particles contained in a licensed vaccine. The binding of rotavirus proteins to distinct glycoconjugate receptors and monoclonal antibodies was evaluated using biolayer interferometry analysis, applied on an Octet platform. The antibody strategy presented the best results with a linear response range within 2.5 × 10 7 -1.0 × 10 8 particles·mL -1 and limits of detection and quantification of 2.5 × 10 6 and 7.5 × 10 6 particles·mL -1 , respectively. Method suitability for the quantification of in-process samples was shown using samples from different manufacturing stages and their titers were comparable with the approved CC ID(50) method. This cell-free method enables a fast and high-throughput analysis, compatible with time constraints during bioprocess development and it is suitable to be adapted to other viral particle-based drug products.

Recombinant adeno-associated (rAAV) vector-based gene therapy has been the focus of intense research driven by the safety profile and several recent clinical breakthroughs. As of April 2021, there are two rAAV-based gene therapies approved and more than two-hundred active clinical trials (approximately thirty in Phase III). However, the expected increase in demand for rAAV vectors still poses several challenges. Discussed herein are key aspects related to R&D needs and Chemistry, Manufacturing and Control (CMC) efforts required to attend this growing demand. Authors provide their perspective on strategic topics for rAAV-based therapies success: scalability and productivity; improved safety; increased process understanding combined with development of orthogonal bioanalytics that are able to identify, monitor and control Critical Quality Attributes (CQAs) during bioprocessing., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Virus-like particles (VLPs) are excellent platforms for the development of influenza vaccine candidates. Nonetheless, their characterization is challenging due to VLPs' unique biophysical and biochemical properties. To cope with such complexity, multiple analytical techniques have been developed to date (e.g., single-particle analysis, thermal stability, or quantification assays), most of which are rarely used or have been successfully demonstrated for being applicable for virus particle characterization. In this study, several biophysical and biochemical methods have been evaluated for thorough characterization of monovalent and pentavalent influenza VLPs from diverse groups (A and B) and subtypes (H1 and H3) produced in insect cells using the baculovirus expression vector system (IC-BEVS). Particle size distribution and purity profiles were monitored during the purification process using two complementary technologies - nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS). VLP surface charge at the selected process pH was also assessed by this last technique. The morphology of the VLP (size, shape, and presence of hemagglutinin spikes) was evaluated using transmission electron microscopy. Circular dichroism was used to assess VLPs' thermal stability. Total protein, DNA, and baculovirus content were also assessed. All VLPs analyzed exhibited similar size ranges (90-115 nm for NTA and 129-141 nm for TRPS), surface charges (average of -20.4 mV), and morphology (pleomorphic particles resembling influenza virus) exhibiting the presence of HA molecules (spikes) uniformly displayed on M1 protein scaffold. Our data shows that HA titers and purification efficiency in terms of impurity removal and thermal stability were observed to be particle dependent. This study shows robustness and generic applicability of the tools and methods evaluated, independent of VLP valency and group/subtype. Thus, they are most valuable to assist process development and enhance product characterization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Carvalho, Silva, Sousa, Peixoto, Roldão, Carrondo and Alves.)

Quality control of biopharmaceuticals such as monoclonal antibodies (mAbs) has been evolving and becoming more challenging as the requirements of the regulatory agencies increase due to the demanding complexity of products under evaluation. Mass Spectrometry (MS)-based methods such as the multi-attribute method (MAM) are being explored to achieve a deeper understanding of the attributes critical for the safety, efficacy, and quality of these products. MAM uses high mass accuracy/high-resolution MS data that enables the direct and simultaneous monitoring of relevant product quality attributes (PQAs, in particular, chemical modifications) in a single workflow, replacing several orthogonal methods, reducing time and costs associated with these assays. Here we describe a MAM implementation process using a QTOF high resolution platform. Method implementation was accomplished using NIST (National Institute for Standards and Technology) mAb reference material and an in-process mAb sample. PQAs as glycosylation profiles, methionine oxidation, tryptophan dioxidation, asparagine deamidation, pyro-Glu at N-terminal and glycation were monitored. Focusing on applications that require batch analysis and high-throughput, sample preparation and LC-MS parameters troubleshooting are discussed. This MAM workflow was successfully explored as reference analytical tool for comprehensive characterization of a downstream processing (DSP) polishing platform and for a comparability study following technology transfer between different laboratories., Competing Interests: Sanofi is also sponsor of this work. Anja Pfenninger, Martina Fischer, Michaela Strotbek and Nihal Tugçu are Sanofi employees and may hold shares and/or stock options in the company. The work described in this manuscript does not relate to any patients data, products in development or marketed products. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The relevant data to understand and replicate the method implementation reported in this work are within the manuscript and its Supporting Information files. Data containing antibody sequence (owned by Sanofi) cannot be shared publicly due to legal restrictions.

Marxan is the most common decision-support tool used to inform the design of protected-area systems. The original version of Marxan does not consider risk and uncertainty associated with threatening processes affecting protected areas, including uncertainty about the location and condition of species' populations and habitats now and in the future. We described and examined the functionality of a modified version of Marxan, Marxan with Probability. This software explicitly considers 4 types of uncertainty: probability that a feature exists in a particular place (estimated based on species distribution models or spatially explicit population models); probability that features in a site will be lost in the future due to a threatening process, such as climate change, natural catastrophes, and uncontrolled human interventions; probability that a feature will exist in the future due to natural successional processes, such as a fire or flood; and probability the feature exists but has been degraded by threatening processes, such as overfishing or pollution, and thus cannot contribute to conservation goals. We summarized the results of 5 studies that illustrate how each type of uncertainty can be used to inform protected area design. If there were uncertainty in species or habitat distribution, users could maximize the chance that these features were represented by including uncertainty using Marxan with Probability. Similarly, if threatening processes were considered, users minimized the chance that species or habitats were lost or degraded by using Marxan with Probability. Marxan with Probability opens up substantial new avenues for systematic conservation planning research and application by agencies., (© 2020 Society for Conservation Biology.)

Protected-area systems should conserve intraspecific genetic diversity. Because genetic data require resources to obtain, several approaches have been proposed for generating plans for protected-area systems (prioritizations) when genetic data are not available. Yet such surrogate-based approaches remain poorly tested. We evaluated the effectiveness of potential surrogate-based approaches based on microsatellite genetic data collected across the Iberian Peninsula for 7 amphibian and 3 reptilian species. Long-term environmental suitability did not effectively represent sites containing high genetic diversity (allelic richness). Prioritizations based on long-term environmental suitability had similar performance to random prioritizations. Geographic distances and resistance distances based on contemporary environmental suitability were not always effective surrogates for identification of combinations of sites that contain individuals with different genetic compositions. Our results demonstrate that population genetic data based on commonly used neutral markers can inform prioritizations, and we could not find an adequate substitute. Conservation planners need to weigh the potential benefits of genetic data against their acquisition costs., (© 2020 The Authors. Conservation Biology published by Wiley Periodicals LLC on behalf of Society for Conservation Biology.)

Reduced representation genome sequencing has popularized the application of single nucleotide polymorphisms (SNPs) to address evolutionary and conservation questions in nonmodel organisms. Patterns of genetic structure and diversity based on SNPs often diverge from those obtained with microsatellites to different degrees, but few studies have explicitly compared their performance under similar sampling regimes in a shared analytical framework. We compared range-wide patterns of genetic structure and diversity in two amphibians endemic to the Iberian Peninsula: Hyla molleri and Pelobates cultripes, based on microsatellite (18 and 14 loci) and SNP (15,412 and 33,140 loci) datasets of comparable sample size and spatial extent. Model-based clustering analyses with STRUCTURE revealed minor differences in genetic structure between marker types, but inconsistent values of the optimal number of populations (K) inferred. SNPs yielded more repeatable and less admixed ancestries with increasing K compared to microsatellites. Genetic diversity was weakly correlated between marker types, with SNPs providing a better representation of southern refugia and of gradients of genetic diversity congruent with the demographic history of both species. Our results suggest that the larger number of loci in a SNP dataset can provide more reliable inferences of patterns of genetic structure and diversity than a typical microsatellite dataset, at least at the spatial and temporal scales investigated., Competing Interests: The authors declare that they have no conflict of interest., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Ongoing global warming is disrupting several ecological and evolutionary processes, spanning different levels of biological organization. Species are expected to shift their ranges as a response to climate change, with relevant implications to peripheral populations at the trailing and leading edges. Several studies have analyzed the exposure of species to climate change but few have explored exposure at the intraspecific level. We introduce a framework to forecast exposure to climate change at the intraspecific level. We build on existing methods by combining correlative species distribution models, a model of species range dynamics, and a model of phylogeographic interpolation. We demonstrate the framework by applying it to 20 Iberian amphibian and reptile species. Our aims were to: (a) identify which species and intraspecific lineages will be most exposed to future climate change; (b) test if nucleotide diversity at the edges of species ranges are significantly higher or lower than on the overall range; and (c) analyze if areas of higher species gain, loss, and turnover coincide with those predicted for lineages richness and nucleotide diversity. We found that about 80% of the studied species are predicted to contract their range. Within each species, some lineages were predicted to contract their range, while others were predicted to maintain or expand it. Therefore, estimating the impacts of climate change at the species level only can underestimate losses at the intraspecific level. Some species had significant high amount of nucleotide at the trailing or leading edge, or both, but we did not find a consistent pattern across species. Spatial patterns of species richness, gain, loss, and turnover were fairly concurrent with lineages richness and nucleotide diversity. Our results support the need for increased attention to intraspecific diversity regarding monitoring and conservation strategies under climate change., (© 2019 John Wiley & Sons Ltd.)

Currently, marketed influenza vaccines are only efficient against homologous viruses, thus requiring a seasonal update based on circulating subtypes. This constant reformulation adds several challenges to manufacturing, particularly in purification due to the variation of the physicochemical properties of the vaccine product. A universal platform approach capable of handling such variation is therefore of utmost importance. In this work, a filtration-based approach is explored to purify influenza virus-like particles. Switching from adsorptive separation to size-based purification allows overcoming the differences in retention observed for different influenza strains. The proposed process employs a cascade of ultrafiltration and diafiltration steps, followed by a sterile filtration step. Different process parameters are assessed in terms of product recovery and impurities' removal. Membrane chemistry, pore size, operation modes, critical flux, transmembrane pressure, and permeate control strategies are evaluated. After membrane selection and parameter optimization, concentration factors and diafiltration volumes are also defined. By optimizing the filtration mode of operation, it is possible to achieve product recoveries of approximately 80%. Overall, the process time is decreased by 30%, its scalability is improved, and the costs are reduced due to the removal of chromatography and associated buffer consumptions, cleaning, and its validation steps., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Estimating geographical ranges of intra-specific evolutionary lineages is crucial to the fields of biogeography, evolution, and biodiversity conservation. Models of isolation mechanisms often consider multiple distances in order to explain genetic divergence. Yet, the available methods to estimate the geographical ranges of lineages are based on direct geographical distances, neglecting other distance metrics that can better explain the spatial genetic structure. We extended the phylogeographical interpolation method (phylin) in order to accommodate user-defined distance metrics and to incorporate the uncertainty associated with genetic distance calculation. These new features were tested with simulated and empirical data sets. Multiple distance matrices were generated including geographical, resistance, and environmental distances to derive maps of lineage occurrence. The new additions to this method improved the ability to predict lineage occurrence, even with low sample size. We used a regression framework to quantify the relationship between the genetic divergence and competing distance matrices representing potential isolation processes that are subsequently used in the interpolation process. Including uncertainty in tree topology and the different distance matrices improved the robustness of the variograms, allowing a better fit of the theoretical model of spatial dependence. The improvements to the method increase its potential application in other fields. Accurately mapping genetic divergence can help to locate potential contact zones between lineages as well as barriers to gene flow, which has a broad interest in biogeographical and evolutionary studies. Additionally, conservation efforts could benefit from the integration of genetic variation and landscape features in a spatially explicit framework., (© 2019 John Wiley & Sons Ltd.)

Mucins are high-molecular weight glycoproteins (0.25-20 MDa) containing one or more domains that are heavily O-glycosylated. Their implications as targets for cancer treatment have increased the interest in these glycoproteins, mainly in the fields of vaccines and antibodies. However, mucins present high heterogeneity, posing challenges that affect purification processes and quality control analysis. In that sense, it is necessary to develop and improve downstream processes and analytical methods to characterize these products. Here a tool based on biolayer interferometry analysis to improve mucin's detection and quantification in a fast, simple and label free-way is presented. Taking advantage of lectin recognition of mucins' carbohydrate structures, several lectins were evaluated and immobilized on streptavidin biosensors. Different assay conditions were optimized and the most suitable lectin, Aleuria aurantia lectin (AAL), was selected. Bovine Submaxillary Gland and human MUC5B mucins were used as proof of concept and were successfully detected and quantified at different stages of purification. High sensitivity levels were achieved with LOD and LOQ of 3.8 μg mL-1 and 11.7 μg mL-1 for BSM, and 0.2 μg mL-1 and 0.6 μg mL-1 for MUC5B. AAL binding specificity was also confirmed with fucose competition assays. Our method represents an advance on mucins detection and quantification since the existing methods present several disadvantages for process development. Hereafter, it can be applied to the optimization of new or already established downstream processes for mucins' purification.

Virus-like particles (VLPs) are becoming established as vaccines, in particular for influenza pandemics, increasing the interest in the development of VLPs manufacturing bioprocess. However, for complex VLPs, the analytical tools used for quantification are not yet able to keep up with the bioprocess progress. Currently, quantification for Influenza relies on traditional methods: hemagglutination assay or Single Radial Immunodiffusion. These analytical technologies are time-consuming, cumbersome, and not supportive of efficient downstream process development and monitoring. Hereby we report a label-free tool that uses Biolayer interferometry (BLI) technology applied on an Octet platform to quantify Influenza VLPs at all stages of bioprocess. Human (α2,6-linked sialic acid) and avian (α2,3-linked sialic acid) biotinylated receptors associated with streptavidin biosensors were used, to quantify hemagglutinin content in several mono- and multivalent Influenza VLPs. The applied method was able to quantify hemagglutinin from crude samples up to final bioprocessing VLP product. BLI technology confirmed its value as a high throughput analytical tool with high sensitivity and improved detection limits compared to traditional methods. This simple and fast method allowed for real-time results, which are crucial for in-line monitoring of downstream processing, improving process development, control and optimization., (© 2017 The Authors. Biotechnology Journal published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The use of phylogenies in ecology is increasingly common and has broadened our understanding of biological diversity. Ecological sub-disciplines, particularly conservation, community ecology and macroecology, all recognize the value of evolutionary relationships but the resulting development of phylogenetic approaches has led to a proliferation of phylogenetic diversity metrics. The use of many metrics across the sub-disciplines hampers potential meta-analyses, syntheses, and generalizations of existing results. Further, there is no guide for selecting the appropriate metric for a given question, and different metrics are frequently used to address similar questions. To improve the choice, application, and interpretation of phylo-diversity metrics, we organize existing metrics by expanding on a unifying framework for phylogenetic information. Generally, questions about phylogenetic relationships within or between assemblages tend to ask three types of question: how much; how different; or how regular? We show that these questions reflect three dimensions of a phylogenetic tree: richness, divergence, and regularity. We classify 70 existing phylo-diversity metrics based on their mathematical form within these three dimensions and identify 'anchor' representatives: for α-diversity metrics these are PD (Faith's phylogenetic diversity), MPD (mean pairwise distance), and VPD (variation of pairwise distances). By analysing mathematical formulae and using simulations, we use this framework to identify metrics that mix dimensions, and we provide a guide to choosing and using the most appropriate metrics. We show that metric choice requires connecting the research question with the correct dimension of the framework and that there are logical approaches to selecting and interpreting metrics. The guide outlined herein will help researchers navigate the current jungle of indices., (© 2016 The Authors. Biological Reviews published by John Wiley © Sons Ltd on behalf of Cambridge Philosophical Society.)

Accounting for evolutionary relationships between and within species is important for biodiversity conservation planning, but is rarely considered in practice. Here we introduce a novel framework to identify priority conservation areas accounting for phylogenetic and intraspecific diversity, integrating concepts from phylogeny, phylogeography, spatial statistics and spatial conservation prioritization. The framework allows planners to incorporate and combine different levels of evolutionary diversity and can be applied to any taxonomic group and to any region in the world. We illustrate our approach using amphibian and reptile species occurring in a biodiversity hotspot region, the Iberian Peninsula. We found that explicitly incorporating phylogenetic and intraspecific diversity in systematic conservation planning provides advantages in terms of maximizing overall biodiversity representation while enhancing its persistence and evolutionary potential. Our results emphasize the need to account for the evolutionary continuum in order to efficiently implement biodiversity conservation planning decisions.

To deliver the required cell numbers and doses to therapy, scaling-up production and purification processes (at least to the liter-scale) while maintaining cells' characteristics is compulsory. Therefore, the aim of this work was to prove scalability of an integrated streamlined bioprocess compatible with current good manufacturing practices (cGMP) comprised by cell expansion, harvesting and volume reduction unit operations using human mesenchymal stem cells (hMSC) isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). BM-MSC and AT-MSC expansion and harvesting steps were scaled-up from spinner flasks to 2L scale stirred tank single-use bioreactor using synthetic microcarriers and xeno-free medium, ensuring high cellular volumetric productivities (50×10 6 cellL -1 day -1 ), expansion factors (14-16 fold) and cell recovery yields (80%). For the concentration step, flat sheet cassettes (FSC) and hollow fiber cartridges (HF) were compared showing a fairly linear scale-up, with a need to slightly decrease the permeate flux (30-50 LMH, respectively) to maximize cell recovery yield. Nonetheless, FSC allowed to recover 18% more cells after a volume reduction factor of 50. Overall, at the end of the entire bioprocess more than 65% of viable (>95%) hMSC could be recovered without compromising cell's critical quality attributes (CQA) of viability, identity and differentiation potential. Alongside the standard quality assays, a proteomics workflow based on mass spectrometry tools was established to characterize the impact of processing on hMSC's CQA; These analytical tools constitute a powerful tool to be used in process design and development., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Cells release vesicles to the extracellular environment with characteristic nucleic acid, protein, lipid, and glycan composition. Here we have isolated and characterized extracellular vesicles (EVs) and total cell membranes (MBs) from ovarian carcinoma OVMz cells. EVs were enriched in specific markers, including Tsg101, CD63, CD9, annexin-I, and MBs contained markers of cellular membrane compartments, including calnexin, GRASP65, GS28, LAMP-1, and L1CAM. The glycoprotein galectin-3 binding protein (LGALS3BP) was strongly enriched in EVs and it contained sialylated complex N-glycans. Lectin blotting with a panel of lectins showed that EVs had specific glycosignatures relative to MBs. Furthermore, the presence of glycoproteins bearing complex N-glycans with α2,3-linked sialic acid, fucose, bisecting-GlcNAc and LacdiNAc structures, and O-glycans with the T-antigen were detected. The inhibition of N-glycosylation processing from high mannose to complex glycans using kifunensine caused changes in the composition of EVs and induced a decrease of several glycoproteins. In conclusion, the results showed that glycosignatures of EVs were specific and altered glycosylation within the cell affected the composition and/or dynamics of EVs release. Furthermore, the identified glycosignatures of EVs could provide novel biomarkers for ovarian cancer.

Genetic data are being generated at unprecedented rates. Policies of many journals, institutions and funding bodies aim to ensure that these data are publicly archived so that published results are reproducible. Additionally, publicly archived data can be 'repurposed' to address new questions in the future. In 2011, along with other leading journals in ecology and evolution, Molecular Ecology implemented mandatory public data archiving (the Joint Data Archiving Policy). To evaluate the effect of this policy, we assessed the genetic, spatial and temporal data archived for 419 data sets from 289 articles in Molecular Ecology from 2009 to 2013. We then determined whether archived data could be used to reproduce analyses as presented in the manuscript. We found that the journal's mandatory archiving policy has had a substantial positive impact, increasing genetic data archiving from 49 (pre-2011) to 98% (2011-present). However, 31% of publicly archived genetic data sets could not be recreated based on information supplied in either the manuscript or public archives, with incomplete data or inconsistent codes linking genetic data and metadata as the primary reasons. While the majority of articles did provide some geographic information, 40% did not provide this information as geographic coordinates. Furthermore, a large proportion of articles did not contain any information regarding date of sampling (40%). Although the inclusion of spatio-temporal data does require an increase in effort, we argue that the enduring value of publicly accessible genetic data to the molecular ecology field is greatly compromised when such metadata are not archived alongside genetic data., (© 2015 John Wiley & Sons Ltd.)

The quantification of realized niche overlap and the integration of species distribution models (SDMs) with calibrated phylogenies to study niche evolution are becoming not only powerful tools to understand speciation events, but can also be used as proxies regarding the delimitation of cryptic species. We applied these techniques in order to unravel how the fundamental niche evolved during cladogenesis within the Tarentola mauritanica species-complex. Our results suggest that diversification within this complex, during the Miocene and Pleistocene, is associated with both niche divergence and niche conservatism, with a pattern that varies depending on whether the variables involved are related to the mean or seasonality of temperature and humidity. Moreover, climatic variables related to humidity and temperature seasonality were involved in the niche shift and genetic diversification of the European/North African clade during the Pleistocene and in its maintenance in a fundamental niche distinct from that of the remaining members of the group. This study further highlights the need for a taxonomic revision of the T. mauritanica species-complex.

phylin is a package for the r programming environment which offers different methods to spatially interpolate genetic information from phylogeographic data. These interpolations can be used to predict the spatial occurrence of different lineages within a phylogeny using a modified method of kriging, which allows the usage of a genetic distance matrix to derive a model of spatial dependence. phylin improves the available methods to generate interpolated surfaces from a phylogenetic trees by assessing the autocorrelation structure of the genetic information, interpolating the genetic data based on a statistical model, estimating the uncertainty of the predictions and identifying lineage occurrence and contact zones probability without projection of pairwise genetic distances into mid-points between sample locations. The package also includes methods to plot interpolation surfaces and provide summary tables from the generated data and models. We provide an example of the usefulness of this tool by inferring the spatial occurrence of distinct historical evolutionary lineages of the Lataste's viper (Vipera latastei Boscá, 1878) in the Iberian Peninsula and identifying potential contact areas. The maps of phylogenetic patterns obtained with these methods provide a spatial context to test hypotheses related to processes underlying the geographic distribution of genetic diversity and to inform conservation planning., (© 2014 John Wiley & Sons Ltd.)

Relapsing polychondritis (RP) is a rare autoimmune disease that affects cartilage and multiple organ system. We describe a case of RP presenting with fever, conjunctivitis and inflammatory signs of the right knee. Infectious and neoplastic diseases were excluded by imaging and laboratory examinations. After 17 days of admission the patient reported coughing and odynophagia, and, a physical examination showed red ears. Otorhinolaryngological examination demonstrated arytenoid chondritis. RP was diagnosed based on four McAdam's criteria. The patient was medicated with oral prednisone, and a positive clinical response was noted. Severe disease may require high doses of corticosteroids or immunosuppressive agents and the biological treatment may be beneficial. Beginning manifestations, such as fever, are often unspecific, leading to RP misdiagnosis. In this case, close attention to the patient's clinical history and a detailed physical examination were fundamental in concluding the correct diagnosis, and consequently beginning the appropriate medication.

Deserts and arid regions are generally perceived as bare and rather homogeneous areas of low diversity. The Sahara is the largest warm desert in the world and together with the arid Sahel displays high topographical and climatic heterogeneity, and has experienced recent and strong climatic oscillations that have greatly shifted biodiversity distribution and community composition. The large size, remoteness and long-term political instability of the Sahara-Sahel, have limited knowledge on its biodiversity. However, over the last decade, there have been an increasing number of published scientific studies based on modern geomatic and molecular tools, and broad sampling of taxa of these regions. This review tracks trends in knowledge about biodiversity patterns, processes and threats across the Sahara-Sahel, and anticipates needs for biodiversity research and conservation. Recent studies are changing completely the perception of regional biodiversity patterns. Instead of relatively low species diversity with distribution covering most of the region, studies now suggest a high rate of endemism and larger number of species, with much narrower and fragmented ranges, frequently limited to micro-hotspots of biodiversity. Molecular-based studies are also unravelling cryptic diversity associated with mountains, which together with recent distribution atlases, allows identifying integrative biogeographic patterns in biodiversity distribution. Mapping of multivariate environmental variation (at 1 km × 1 km resolution) of the region illustrates main biogeographical features of the Sahara-Sahel and supports recently hypothesised dispersal corridors and refugia. Micro-scale water-features present mostly in mountains have been associated with local biodiversity hotspots. However, the distribution of available data on vertebrates highlights current knowledge gaps that still apply to a large proportion of the Sahara-Sahel. Current research is providing insights into key evolutionary and ecological processes, including causes and timing of radiation and divergence for multiple taxa, and associating the onset of the Sahara with diversification processes for low-mobility vertebrates. Examples of phylogeographic patterns are showing the importance of allopatric speciation in the Sahara-Sahel, and this review presents a synthetic overview of the most commonly hypothesised diversification mechanisms. Studies are also stressing that biodiversity is threatened by increasing human activities in the region, including overhunting and natural resources prospection, and in the future by predicted global warming. A representation of areas of conflict, landmines, and natural resources extraction illustrates how human activities and regional insecurity are hampering biodiversity research and conservation. Although there are still numerous knowledge gaps for the optimised conservation of biodiversity in the region, a set of research priorities is provided to identify the framework data needed to support regional conservation planning., (© 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society.)

Here we develop a framework to design multi-species monitoring networks using species distribution models and conservation planning tools to optimize the location of monitoring stations to detect potential range shifts driven by climate change. For this study, we focused on seven bat species in Northern Portugal (Western Europe). Maximum entropy modelling was used to predict the likely occurrence of those species under present and future climatic conditions. By comparing present and future predicted distributions, we identified areas where each species is likely to gain, lose or maintain suitable climatic space. We then used a decision support tool (the Marxan software) to design three optimized monitoring networks considering: a) changes in species likely occurrence, b) species conservation status, and c) level of volunteer commitment. For present climatic conditions, species distribution models revealed that areas suitable for most species occur in the north-eastern part of the region. However, areas predicted to become climatically suitable in the future shifted towards west. The three simulated monitoring networks, adaptable for an unpredictable volunteer commitment, included 28, 54 and 110 sampling locations respectively, distributed across the study area and covering the potential full range of conditions where species range shifts may occur. Our results show that our framework outperforms the traditional approach that only considers current species ranges, in allocating monitoring stations distributed across different categories of predicted shifts in species distributions. This study presents a straightforward framework to design monitoring schemes aimed specifically at testing hypotheses about where and when species ranges may shift with climatic changes, while also ensuring surveillance of general population trends.

S100 proteins are small dimeric calcium-binding proteins which control cell cycle, growth and differentiation via interactions with different target proteins. Intrinsic disorder is a hallmark among many signaling proteins and S100 proteins have been proposed to contain disorder-prone regions. Interestingly, some S100 proteins also form amyloids: S100A8/A9 forms fibrils in prostatic inclusions and S100A6 fibrillates in vitro and seeds SOD1 aggregation. Here we report a study designed to investigate whether β-aggregation is a feature extensive to more members of S100 family. In silico analysis of seven human S100 proteins revealed a direct correlation between aggregation and intrinsic disorder propensity scores, suggesting a relationship between these two independent properties. Averaged position-specific analysis and structural mapping showed that disorder-prone segments are contiguous to aggregation-prone regions and that whereas disorder is prominent on the hinge and target protein-interaction regions, segments with high aggregation propensity are found in ordered regions within the dimer interface. Acidic conditions likely destabilize the seven S100 studied by decreasing the shielding of aggregation-prone regions afforded by the quaternary structure. In agreement with the in silico analysis, hydrophobic moieties become accessible as indicated by strong ANS fluorescence. ATR-FTIR spectra support a structural inter-conversion from α-helices to intermolecular β-sheets, and prompt ThT-binding takes place with no noticeable lag phase. Dot blot analysis using amyloid conformational antibodies denotes a high diversity of conformers; subsequent analysis by TEM shows fibrils as dominant species. Altogether, our data suggests that β-aggregation and disorder-propensity are related properties in S100 proteins, and that the onset of aggregation is likely triggered by loss of protective tertiary and quaternary interactions.

This is a descriptive exploratory study with a quantitative approach, which aimed to characterize the physical violence suffered by prostitutes of Piauí and identify the prevalence of this disease. The study included 76 prostitutes registered in the Association of Sex Professionals of Picos, county of the state of Piauí, Brazil. The data collection was conducted in the areas of prostitution and in the association, from September to October 2010. The results showed that most prostitutes were young, with low income, low education, had more than two years in prostitution (50%), being mainly affected by psychological violence (60.5%) followed by physical (30.2%). The aggressors were known to the victims and women hardly made the complaint. The results were enlightening and led to affirm that it is common the occurrence of violence in women prostitutes, because they work in places determinants of violent acts.