Recombinant human epidermal growth factor inclusion body solubilization and refolding at large scale using expanded-bed adsorption chromatography from Escherichia coli

Amongst the various endogenous growth factors, epidermal growth factor (EGF) plays an important role in normal wound healing of tissue such as skin, cornea and gastrointestinal tract. Various studies have proved that supplementing recombinant human EGF (rhEGF) results in significant augmentation of wound healing. In the present work, a high level expression system with poly-arginine sequences was used for the production of recombinant human EGF (rhEGF) as inclusion bodies. The inclusion bodies were solubilized and the protein was refolded by using expanded-bed adsorption chromatography. The renatured protein was digested with appropriate concentration of trypsin and subsequently the digested rhEGF is purified by passing through ion-exchange chromatography (Toyopearl-SP) to obtain a biologically active protein. This process is the shortest process with reduced number of steps of purification, eliminates the usage of preparative reversed phase HPLC (RP-HPLC) for final purification, which is an expensive technique. The purified protein was analyzed by RP-HPLC, showing a purity >99% and size exclusion chromatography profile shows that there are minimal aggregates, with 99% renatured active protein. The purified rhEGF showed a specific activity of 5 × 10⁵ IU/mg protein, in comparison with NIBSC standard (1st International Standard of rDNA-derived EGF, Code 91/530). The process has been successfully adopted at 100 L fermentation scale and the rhEGF based formulation has been commercialized with brand name REGEN D, with excellent clinical results.     

Rapid review in cervical cytology: a retrospective review of cases detected on rapid review within a DGH cytology department and subsequent outcome

We retrospectively reviewed smears detected by rapid review within a district general hospital (DGH) laboratory over a period of 33 months and the subsequent histological or cytological outcome. Sixty-three cases had adequate follow-up data: 32 subsequently had two negative smears and 31 had a histological abnormality on subsequent biopsy. Twenty were high-grade lesions (CIN2, CIN3 or a glandular lesion) and eight of these were preceded by a low-grade smear abnormality. We reviewed and compared the pattern and distribution of smear abnormalities in these 63 cases. Abnormalities were often present within few cells (76%, n = 48 with 50 abnormal cells or less) or in micro-biopsies (27%, n = 17). There was no statistical difference in the pattern and distribution of smear abnormality between the rapid review-detected smears with a biopsy-proven abnormality and those with negative follow-up smears. Overall, the positive predictive value for high-grade CIN detected by rapid review (75%) was within NHSCSP achievable standards targets. This review of rapid review-detected abnormalities and the biopsy and cytological follow-up reiterates the importance of the method in cervical screening.     

A cross-system synthesis of consumer and nutrient resource control on producer biomass

Nutrient availability and herbivory control the biomass of primary producer communities to varying degrees across ecosystems. Ecological theory, individual experiments in many different systems, and system-specific quantitative reviews have suggested that (i) bottom-up control is pervasive but top-down control is more influential in aquatic habitats relative to terrestrial systems and (ii) bottom-up and top-down forces are interdependent, with statistical interactions that synergize or dampen relative influences on producer biomass. We used simple dynamic models to review ecological mechanisms that generate independent vs. interactive responses of community-level biomass. We calibrated these mechanistic predictions with the metrics of factorial meta-analysis and tested their prevalence across freshwater, marine and terrestrial ecosystems with a comprehensive meta-analysis of 191 factorial manipulations of herbivores and nutrients. Our analysis showed that producer community biomass increased with fertilization across all systems, although increases were greatest in freshwater habitats. Herbivore removal generally increased producer biomass in both freshwater and marine systems, but effects were inconsistent on land. With the exception of marine temperate rocky reef systems that showed positive synergism of nutrient enrichment and herbivore removal, experimental studies showed limited support for statistical interactions between nutrient and herbivory treatments on producer biomass. Top-down control of herbivores, compensatory behaviour of multiple herbivore guilds, spatial and temporal heterogeneity of interactions, and herbivore-mediated nutrient recycling may lower the probability of consistent interactive effects on producer biomass. Continuing studies should expand the temporal and spatial scales of experiments, particularly in understudied terrestrial systems; broaden factorial designs to manipulate independently multiple producer resources (e.g. nitrogen, phosphorus, light), multiple herbivore taxa or guilds (e.g. vertebrates and invertebrates) and multiple trophic levels; and - in addition to measuring producer biomass - assess the responses of species diversity, community composition and nutrient status.     

Comparison of sample preparation techniques for large‐scale proteomics

Numerous workflows exist for large‐scale bottom‐up proteomics, many of which achieve exceptional proteome depth. Herein, we evaluated the performance of several commonly used sample preparation techniques for proteomic characterization of HeLa lysates [unfractionated in‐solution digests, SDS‐PAGE coupled with in‐gel digestion, gel‐eluted liquid fraction entrapment electrophoresis (GELFrEE) technology, SCX StageTips and high‐/low‐pH reversed phase fractionation (HpH)]. HpH fractionation was found to be superior in terms of proteome depth (>8400 proteins detected) and fractionation efficiency compared to other techniques. SCX StageTip fractionation required minimal sample handling and was also a substantial improvement over SDS‐PAGE separation and GELFrEE technology. Sequence coverage of the HeLa proteome increased to 38% when combining all workflows, however, total proteins detected improved only slightly to 8710. In summary, HpH fractionation and SCX StageTips are robust techniques and highly suited for complex proteome analysis.     

Segmented linear modeling of CHO fed‐batch culture and its application to large scale production

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed‐batch cultures. Using the model structure and parameter values from a small‐scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed‐batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785–797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Fabrication of elastomeric silk fibers

Methods to generate fibers from hydrogels, with control over mechanical properties, fiber diameter, and crystallinity, while retaining cytocompatibility and degradability, would expand options for biomaterials. Here, we exploited features of silk fibroin protein for the formation of tunable silk hydrogel fibers. The biological, chemical, and morphological features inherent to silk were combined with elastomeric properties gained through enzymatic crosslinking of the protein. Postprocessing via methanol and autoclaving provided tunable control of fiber features. Mechanical, optical, and chemical analyses demonstrated control of fiber properties by exploiting the physical cross‐links, and generating double network hydrogels consisting of chemical and physical cross‐links. Structure and chemical analyses revealed crystallinity from 30 to 50%, modulus from 0.5 to 4 MPa, and ultimate strength 1–5 MPa depending on the processing method. Fabrication and postprocessing combined provided fibers with extensibility from 100 to 400% ultimate strain. Fibers strained to 100% exhibited fourth order birefringence, revealing macroscopic orientation driven by chain mobility. The physical cross‐links were influenced in part by the drying rate of fabricated materials, where bound water, packing density, and microstructural homogeneity influenced cross‐linking efficiency. The ability to generate robust and versatile hydrogel microfibers is desirable for bottom‐up assembly of biological tissues and for broader biomaterial applications.     

Persistence of psychosis spectrum symptoms in the Philadelphia Neurodevelopmental Cohort: a prospective two‐year follow‐up

Prospective evaluation of youths with early psychotic‐like experiences can enrich our knowledge of clinical, biobehavioral and environmental risk and protective factors associated with the development of psychotic disorders. We aimed to investigate the predictors of persistence or worsening of psychosis spectrum features among US youth through the first large systematic study to evaluate subclinical symptoms in the community. Based on Time 1 screen of 9,498 youth (age 8‐21) from the Philadelphia Neurodevelopmental Cohort, a subsample of participants was enrolled based on the presence (N=249) or absence (N=254) of baseline psychosis spectrum symptoms, prior participation in neuroimaging, and current neuroimaging eligibility. They were invited to participate in a Time 2 assessment two years on average following Time 1. Participants were administered the Structured Interview for Prodromal Syndromes, conducted blind to initial screen status, along with the Schizotypal Personality Questionnaire and other clinical measures, computerized neurocognitive testing, and neuroimaging. Clinical and demographic predictors of symptom persistence were examined using logistic regression. At Time 2, psychosis spectrum features persisted or worsened in 51.4% of youths. Symptom persistence was predicted by higher severity of subclinical psychosis, lower global functioning, and prior psychiatric medication at baseline. Youths classified as having psychosis spectrum symptoms at baseline but not at follow‐up nonetheless exhibited comparatively higher symptom levels and lower functioning at both baseline and follow‐up than typically developing youths. In addition, psychosis spectrum features emerged in a small number of young people who previously had not reported significant symptoms but who had exhibited early clinical warning signs. Together, our findings indicate that varying courses of psychosis spectrum symptoms are evident early in US youth, supporting the importance of investigating psychosis risk as a dynamic developmental process. Neurocognition, brain structure and function, and genomics may be integrated with clinical data to provide early indices of symptom persistence and worsening in youths at risk for psychosis.     

Using triage for environmental remediation in Antarctica

Remediation of sites impacted by human activity in Antarctica is a difficult and resource intensive process. With increasing activity and climate change, the extent of damage from human activities is expected to increase and it will not be feasible to protect the environment entirely. We recommend a triage process be used to provide informed and transparent management decisions for comprehensive and adequate environmental remediation in Antarctica. We provide examples that demonstrate realistic outcomes where we have avoided tying up resources on disturbed sites that will recover naturally, are stable, or too damaged to recover, and that also incorporate feasible operational practices. Not all disturbed sites will be remediated and many of those that are, are unlikely to be returned to pristine condition. The decisions around remediation are not based solely on the desired environmental outcome. In the absence of effective legal obligation, we recognize that financial, social, policy, health and safety, technological confidence, and operational feasibility are part of the decision‐making process.     

Don't forget to look down – collaborative approaches to predator conservation

Finding effective ways of conserving large carnivores is widely recognised as a priority in conservation. However, there is disagreement about the most effective way to do this, with some favouring top‐down ‘command and control’ approaches and others favouring collaboration. Arguments for coercive top‐down approaches have been presented elsewhere; here we present arguments for collaboration. In many parts of the developed world, flexibility of approach is built into the legislation, so that conservation objectives are balanced with other legitimate goals. In the developing world, limited resources, poverty and weak governance mean that collaborative approaches are likely to play a particularly important part in carnivore conservation. In general, coercive policies may lead to the deterioration of political legitimacy and potentially to non‐compliance issues such as illegal killing, whereas collaborative approaches may lead to psychological ownership, enhanced trust, learning, and better social outcomes. Sustainable hunting/trapping plays a crucial part in the conservation and management of many large carnivores. There are many different models for how to conserve carnivores effectively across the world, research is now required to reduce uncertainty and examine the effectiveness of these approaches in different contexts.