A study in glycation of a therapeutic recombinant humanized monoclonal antibody: where it is, how it got there, and how it affects charge-based behavior

The glycated form of a basic recombinant humanized monoclonal antibody (rhuMAb) was separated and quantitated by boronate affinity chromatography using optimized shielding reagents. Characterization on the isolated glycated material by peptide mapping analysis, using liquid chromatography-mass spectrometry (LC-MS) and tandem mass spectrometry (MS/MS) sequencing techniques, identified eight reactive lysine primary amine sites. The glycation reaction extent was similar among the various reactive sites, ranging from approximately 1 to 12%, and a single histidine residue separated the most and least reactive sites. Boronate chromatography run in a linear gradient mode separated monoglycated rhuMAb from higher order glycated species and indicated that the majority ( approximately 90%) of glycated rhuMAb is monoglycated. Low-level glycation on a heavy chain lysine located within a complementarity-determining region (CDR) did not significantly affect binding activity in potency measurements. The glycated forms also behaved as slightly more acidic than the nonglycated antibody in charge-based separation techniques, observable by capillary isoelectric focusing (cIEF) and ion exchange chromatography (IEC). The boronate column has significantly increased retention of aggregated rhuMAb material under separation conditions optimized for the monomer form. Recombinant protein glycation initially occurred during production in mammalian cell culture, where feed sugar and protein concentrations contribute to the total overall glycation on this antibody product.     

Plasticity in the timing of physiological development: Physiological heterokairy — What is it, how frequent is it, and does it matter?

The study of developmental sequences of physiological traits could be an important way of placing comparative developmental physiology (CDP) within the research agenda being forged by work on developmental plasticity. Here we focus on the concept of heterokairy defined by Spicer & Burggren in 2003 as changes in the timing of physiological development in an individual. The role of this concept in the future of the CDP is discussed. First we provide an historical perspective of the ideas that have led to the investigation of sequences in CDP. This is followed by a re-examination and clarification of the definition of physiological heterokairy before empirical case studies that (explicitly or implicitly) demonstrate physiological heterokairy are reviewed. We suggest that physiological heterokairy can be demonstrated through a wide range of invertebrate and vertebrate examples. However, care must be taken when inferring that heterokairy as a pattern is always the result of heterokairic processes as there is evidence that physiological heterokairy could result from the altered timing of both homologous or analogous physiological mechanisms. We conclude by discussing the potential link between heterokairy and heterochrony and suggest that the investigation of this link should be a major goal for workers in both CDP and developmental plasticity.     

Genome editing with the CRISPR‐Cas system: an art,ethics and global regulatory perspective

Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR‐Cas system, has led to significant progress in the field of plant and animal breeding. The CRISPR‐Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR‐Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre‐clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR‐Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR‐Cas‐modified organisms/products.     

Contested value and an ethics of resources: Water,mining and indigenous people in the Atacama Desert,Chile

The question of value is fundamental to contexts of resource scarcity given that contest over use and distribution of scarce resources centres on judgments about rights, interests and access. In mining processes, the use and extraction of water in great volumes commodifies and threatens supplies of what others understand to be a substance essential to all forms of life. In the Atacama, while industrial extraction and commodification by the mining industry are the basis for indigenous people's contestations over water resources, an analysis of everyday water practice and performance (as ‘ordinary ethics’) demonstrates that an indigenous ethics of resources includes commodity values under certain conditions. This paper examines a field of competing actors engaging in extraction and use of scarce waters in order to make an argument for the importance of considering the complexities and dynamics of ethical practice and water value.     

What is beyond a qRT‐PCR study on mesenchymal stem cell differentiation properties: how to choose the most reliable housekeeping genes

In the last years, mesenchymal stem cells (MSCs) have been identified as an attractive cell population in regenerative medicine. In view of future therapeutic applications, the study of specific differentiation‐related gene expression is a pivotal prerequisite to define the most appropriate MSC source for clinical translation. In this context, it is crucial to use stable housekeeping genes (HGs) for normalization of qRT‐PCR to obtain validated and comparable results. By our knowledge, an exhaustive validation study of HGs comparing MSCs from different sources under various differentiation conditions is still missing. In this pivotal study, we compared the expression levels of 12 genes (ACTB, Β2M, EF1alpha, GAPDH, GUSB, PPIA, RPL13A, RPLP0, TBP, UBC, YWHAZ and 18S rRNA) to assess their suitability as HGs in MSCs during adipogenic, osteogenic and chondrogenic differentiation. We demonstrated that many of the most popular HGs including 18S rRNA, B2M and ACTB were inadequate for normalization, whereas TBP/YWHAZ/GUSB were frequently identified among the best performers. Moreover, we showed the dramatic effects of suboptimal HGs choice on the quantification of cell differentiation markers, thus interfering with a reliable comparison of the lineage potential properties among various MSCs. Thus, in the emerging field of regenerative medicine, the identification of the most appropriate MSC source and cell line is so crucial for the treatment of patients that being inaccurate in the first step of the stem cell characterization can bring important consequences for the patients and for the promising potential of stem cell therapy.     

Invited review: Application of meta-omics to understand the dynamic nature of the rumen microbiome and how it responds to diet in ruminants

Ruminants are unique among livestock due to their ability to efficiently convert plant cell wall carbohydrates into meat and milk. This ability is a result of the evolution of an essential symbiotic association with a complex microbial community in the rumen that includes vast numbers of bacteria, methanogenic archaea, anaerobic fungi and protozoa. These microbes produce a diverse array of enzymes that convert ingested feedstuffs into volatile fatty acids and microbial protein which are used by the animal for growth. Recent advances in high-throughput sequencing and bioinformatic analyses have helped to reveal how the composition of the rumen microbiome varies significantly during the development of the ruminant host, and with changes in diet. These sequencing efforts are also beginning to explain how shifts in the microbiome affect feed efficiency. In this review, we provide an overview of how meta-omics technologies have been applied to understanding the rumen microbiome, and the impact that diet has on the rumen microbial community.     

Enhancing the learning of evolutionary anthropology skills by combining student‐active teaching with actual and virtual immersion of Master's students in fieldwork,laboratory practice,and dissemination

Higher education in evolutionary anthropology involves providing students with in‐depth knowledge of biological and cultural heritage sites and collections that are frequently inaccessible. Indeed, most sites, fossils, and archaeological remains can be visited or manipulated only rarely and solely by specialists with extensive experience. Owing to the development of 3D and medical imaging techniques, this fragile heritage is now more widely accessible, and in a dynamic way. However, exclusive adoption of virtual teaching and learning has a negative impact on student engagement and, naturally, on exchanges with instructors, and thus cannot be used without some reservations. In the ITAP (Immersion dans les Terrains de l’Anthropologie biologique et de la Préhistoire) project of the higher education STEP (Soutien à la Transformation et à l’Expérimentation Pédagogiques) transformation program at the University of Bordeaux, we combine student‐active teaching with Master''s students fully immersed in ongoing fieldwork, laboratory study, and dissemination of research results in order to develop more individually shaped learning curricula and to foster both professional and new interdisciplinary skills. Here, we present examples of experiments conducted in the ITAP project using both authentic and virtual collections of archaeological, experimental, and reference materials that help to break down the barriers between research activities and higher education, as well as providing a more general appraisal of the appropriate use of virtual tools in higher education by combining them with real‐life situations.     

土地流转背景下不同经营规模青田稻鱼共生系统的环境影响差异——基于碳足迹的实证研究

在快速工业化和城镇化的影响下,农业文化遗产的保护与管理正面临着适龄劳动力大量外流、土地抛荒、传统知识体系难以维持等诸多威胁与挑战。推动土地流转、进行适度规模经营,可在农业文化遗产的保护中产生积极作用。土地流转在给遗产地带来经济效益的同时,对当地生态环境产生的影响变化同样值得关注,但现有研究却少有涉及。本研究以全球重要农业文化遗产——浙江青田稻鱼共生系统为例,将不同经营规模的稻鱼共生系统分为小农户经营模式和规模化经营模式,运用生命周期法对两种模式的碳足迹进行核算。结果表明: 小农户经营模式和规模化经营模式的碳足迹分别为6510.80和5917.00 kg CO₂-eq·hm^-2,单位产值碳足迹分别为0.13和0.10 kg CO₂-eq·yuan^-1。与小农户经营模式相比,规模化经营模式温室气体排放更少,单位产值的环境影响更小。农户扩大经营规模后,当地温室气体排放减少了4097.20 kg CO₂-eq。农业生产过程中积累的CH₄在碳足迹中占比最大,农业生产资料中复合肥是仅次于CH₄的第二大温室气体排放来源。对于小农户经营模式,饲料中使用的玉米和小麦也对温室气体排放有重要的影响。因此,推动土地适度规模经营,有利于传统农业系统实现经济效益和环境效益的双赢,对于农业文化遗产保护具有重要作用。     

Benefits of teaching medical students how to communicate with patients having serious illness: comparison of two approaches to experiential, skill-based, and self-reflective learning

Innovative approaches are needed to teach medical students effective and compassionate communication with seriously ill patients. We describe two such educational experiences in the Yale Medical School curriculum for third-year medical students: 1) Communicating Difficult News Workshop and 2) Ward-Based End-of-Life Care Assignment. These two programs address educational needs to teach important clinical communication and assessment skills to medical students that previously were not consistently or explicitly addressed in the curriculum. The two learning programs share a number of educational approaches driven by the learning objectives, the students' development, and clinical realities. Common educational features include: experiential learning, the Biopsychosocial Model, patient-centered communication, integration into clinical clerkships, structured skill-based learning, self-reflection, and self-care. These shared features - as well as some differences - are explored in this paper in order to illustrate key issues in designing and implementing medical student education in these areas.     

Building and experimenting with an agent-based model to study the population-level impact of CommunityRx,a clinic-based community resource referral intervention

CommunityRx (CRx), an information technology intervention, provides patients with a personalized list of healthful community resources (HealtheRx). In repeated clinical studies, nearly half of those who received clinical “doses” of the HealtheRx shared their information with others (“social doses”). Clinical trial design cannot fully capture the impact of information diffusion, which can act as a force multiplier for the intervention. Furthermore, experimentation is needed to understand how intervention delivery can optimize social spread under varying circumstances. To study information diffusion from CRx under varying conditions, we built an agent-based model (ABM). This study describes the model building process and illustrates how an ABM provides insight about information diffusion through in silico experimentation. To build the ABM, we constructed a synthetic population (“agents”) using publicly-available data sources. Using clinical trial data, we developed empirically-informed processes simulating agent activities, resource knowledge evolution and information sharing. Using RepastHPC and chiSIM software, we replicated the intervention in silico, simulated information diffusion processes, and generated emergent information diffusion networks. The CRx ABM was calibrated using empirical data to replicate the CRx intervention in silico. We used the ABM to quantify information spread via social versus clinical dosing then conducted information diffusion experiments, comparing the social dosing effect of the intervention when delivered by physicians, nurses or clinical clerks. The synthetic population (N = 802,191) exhibited diverse behavioral characteristics, including activity and knowledge evolution patterns. In silico delivery of the intervention was replicated with high fidelity. Large-scale information diffusion networks emerged among agents exchanging resource information. Varying the propensity for information exchange resulted in networks with different topological characteristics. Community resource information spread via social dosing was nearly 4 fold that from clinical dosing alone and did not vary by delivery mode. This study, using CRx as an example, demonstrates the process of building and experimenting with an ABM to study information diffusion from, and the population-level impact of, a clinical information-based intervention. While the focus of the CRx ABM is to recreate the CRx intervention in silico, the general process of model building, and computational experimentation presented is generalizable to other large-scale ABMs of information diffusion.     

Single-locus species delimitation: a test of the mixed Yule-coalescent model, with an empirical application to Philippine round-leaf bats

Prospects for a comprehensive inventory of global biodiversity would be greatly improved by automating methods of species delimitation. The general mixed Yule-coalescent (GMYC) was recently proposed as a potential means of increasing the rate of biodiversity exploration. We tested this method with simulated data and applied it to a group of poorly known bats (Hipposideros) from the Philippines. We then used echolocation call characteristics to evaluate the plausibility of species boundaries suggested by GMYC. In our simulations, GMYC performed relatively well (errors in estimated species diversity less than 25%) when the product of the haploid effective population size (N(e)) and speciation rate (SR; per lineage per million years) was less than or equal to 10(5), while interspecific variation in N(e) was twofold or less. However, at higher but also biologically relevant values of N(e) × SR and when N(e) varied tenfold among species, performance was very poor. GMYC analyses of mitochondrial DNA sequences from Philippine Hipposideros suggest actual diversity may be approximately twice the current estimate, and available echolocation call data are mostly consistent with GMYC delimitations. In conclusion, we consider the GMYC model useful under some conditions, but additional information on N(e), SR and/or corroboration from independent character data are needed to allow meaningful interpretation of results.     

Plant growth and respiration re-visited: maintenance respiration defined - it is an emergent property of, not a separate process within, the system - and why the respiration : photosynthesis ratio is conservative

Background and Aims

Plant growth and respiration still has unresolved issues, examined here using a model. The aims of this work are to compare the model''s predictions with McCree''s observation-based respiration equation which led to the ‘growth respiration/maintenance respiration paradigm’ (GMRP) – this is required to give the model credibility; to clarify the nature of maintenance respiration (MR) using a model which does not represent MR explicitly; and to examine algebraic and numerical predictions for the respiration:photosynthesis ratio.

Methods

A two-state variable growth model is constructed, with structure and substrate, applicable on plant to ecosystem scales. Four processes are represented: photosynthesis, growth with growth respiration (GR), senescence giving a flux towards litter, and a recycling of some of this flux. There are four significant parameters: growth efficiency, rate constants for substrate utilization and structure senescence, and fraction of structure returned to the substrate pool.

Key Results

The model can simulate McCree''s data on respiration, providing an alternative interpretation to the GMRP. The model''s parameters are related to parameters used in this paradigm. MR is defined and calculated in terms of the model''s parameters in two ways: first during exponential growth at zero growth rate; and secondly at equilibrium. The approaches concur. The equilibrium respiration:photosynthesis ratio has the value of 0·4, depending only on growth efficiency and recycling fraction.

Conclusions

McCree''s equation is an approximation that the model can describe; it is mistaken to interpret his second coefficient as a maintenance requirement. An MR rate is defined and extracted algebraically from the model. MR as a specific process is not required and may be replaced with an approach from which an MR rate emerges. The model suggests that the respiration:photosynthesis ratio is conservative because it depends on two parameters only whose values are likely to be similar across ecosystems.     

The impact of an exotic social wasp (Vespula germanica) on the native arthropod community of north-west Patagonia, Argentina: an experimental study

Abstract 1. Biological invasions are usually thought to have a negative impact on native communities. However, data supporting this idea are often based on comparative studies between invaded and non‐invaded areas, and are spatially and temporally limited. 2. The present study experimentally assessed the impact of an exotic wasp, Vespula germanica, on the native arthropod community of north‐west Patagonia during 3 years in an area of 80 ha. Vespula germanica is an exotic social vespid that invaded north‐west Patagonia 20 years ago. It has been suggested that its populations affect native arthropods because of its broad diet and also because Patagonia lacks natural enemies and potential competitors for these wasps. 3. Using wasp‐specific toxic baits, V. germanica abundance was reduced in five sites of native woodlands during 3 consecutive years. The abundance, species richness, and composition of arthropods between non‐poisoned (control) and poisoned sites was then compared, both before and after the wasps were poisoned. 4. Wasp abundance represented 6% of the total arthropod catches in non‐poisoned sites and was reduced, on average, by 50% in the treated areas. The abundance, species richness, and composition of the arthropod community (305 species, 24 600 individuals) did not differ between control areas and areas where the abundance of V. germanica was reduced. Significant differences in response variables were found only before wasp poisoning had begun and were related to variations among sites. 5. These results suggest that V. germanica is not affecting the local arthropod assemblages, contradicting past work in other regions. The low relative abundance of wasps in Patagonia, when compared with other invaded regions, might explain the findings. 6. The present study provides further evidence for the importance of large‐scale experimental work with before/after comparisons to fully understand the impact of invaders on natural communities.     

The use of multi-parameter flow cytometry to study the impact of limiting substrate, agitation intensity, and dilution rate on cell aggregation during Bacillus licheniformis CCMI 1034 aerobic continuous culture fermentations

The main objective of this work was to establish those factors either physical (power input) or chemical (limiting substrate or dilution rate) that enhance cell aggregation (biofilm or floc formation) and cell physiological state during aerobic continuous cultures of Bacillus licheniformis. Glucose-limited steady-state continuous cultures growing at a dilution rate between 0.64 and 0.87/h and 1,000 rpm (mean specific energy dissipation rate (epsilonT) = 6.5 W/kg), led to the formation of a thin biofilm on the vessel wall characterized by the presence of a high proportion of healthy cells in the broth (after aggregate disruption by sonication) defined as having intact polarized cytoplasmic membranes. An increased epsilonT (from 6.5 W/kg to 38 W/kg) was found to hinder cell aggregation under carbon limitation. The carbon recovery calculated from glucose indicated that additional extracellular polymer was being produced at dilution rates >0.87/h. B. licheniformis growth under nitrogen limitation led to floc formation which increased in size with dilution rate. Counter-intuitively the flocs became more substantial with an increase in epsilonT from 6.5 W/kg to 38 W/kg under nitrogen limitation. Indeed the best culture conditions for enhanced metabolically active cell aggregate formation was under nitrogen limitation at epsilonT = 6.5 W/kg (leading to floc formation), and under carbon limitation at a dilution rate of between 0.64 and 0.87/h, at epsilonT = 6.5 W/kg (leading to vessel wall biofilm formation). This information could be used to optimize culture conditions for improved cell aggregation and hence biomass separation, during thermophilic aerobic bioremediation processes.