Identification of a hydrodynamic threshold in karst rocks from the Biscayne Aquifer, south Florida, USA

A hydrodynamic threshold between Darcian and non-Darcian flow conditions was found to occur in cubes of Key Largo Limestone from Florida, USA (one cube measuring 0.2 m on each side, the other 0.3 m) at an effective porosity of 33% and a hydraulic conductivity of 10 m/day. Below these values, flow was laminar and could be described as Darcian. Above these values, hydraulic conductivity increased greatly and flow was non-laminar. Reynolds numbers (Re) for these experiments ranged fro

Understanding Conformational Regulation of the Integrin I-domain for Design of Chimeric Protein Switches

Within all complex biological processes intricate proteins are expressed to complete every niche and necessary task. Many express multiple allosterically regulated conformational states, with protein function regulated by effector molecules and other ligands. One such protein is the LFA-1 surface integrin protein and its inserted domain, the I-domain. We Isolated the I-domain for investigation of determining binding properties and understanding conformational regulations of affinity changes to its target ligand ICAM-1, for further use in chimeric protein switch design. A large change in binding affinity was found through the deletion of a sub-sequence of amino acids in I-domain known as the α7 helix. Our investigation shows that, when the α7 helix is deleted, I-domain converts into a permanent high affinity state in which binding affinity to ICAM-1 was increased, and this state can be reversed by co-expression with soluble α7 helix peptide. These results conclude that the α7 helix stabilizes the I domain in its low affinity conformation in a ligand-like manner, allowing relaxation to the high affinity conformation upon disruption of α7 helix interaction. While deletion of the α7 helix yields higher binding affinity in I-domain it cannot be applied in design of chimeric protein switches due to its permanent conformational state. Because of this, our switch design has a focus of allosterically regulating the I-domain and α7 helix through utilizing on/off switching of conformational states. I-domain is fused with EF3 and EF4 hands of calmodulin, which then regulates binding affinity to ICAM-1 through interaction with α7 helix, when the EF hands’ natural ligand peptides are present. Currently, mutant switches are being developed to alter EF hand binding specificity which, when bound to new target ligands, will cause an increase in I-domain-ICAM-1 binding affinity in switch molecules. The results of these allosteric regulations highlight the potential of chimeric protein switches for design of environmentally responsive targeting agents and suggest that, through directed evolution, regulated binding to a range of novel targets could be achieved for therapeutic intervention

Specialization in the Human Brain: The Case of Numbers

How numerical representation is encoded in the adult human brain is important for a basic understanding of human brain organization, its typical and atypical development, its evolutionary precursors, cognitive architectures, education, and rehabilitation. Previous studies have shown that numerical processing activates the same intraparietal regions irrespective of the presentation format (e.g., symbolic digits or non-symbolic dot arrays). This has led to claims that there is a single format-independent, numerical representation. In the current study we used a functional magnetic resonance adaptation paradigm, and effective connectivity analysis to re-examine whether numerical processing in the intraparietal sulci is dependent or independent on the format of the stimuli. We obtained two novel results. First, the whole brain analysis revealed that format change (e.g., from dots to digits), in the absence of a change in magnitude, activated the same intraparietal regions as magnitude change, but to a greater degree. Second, using dynamic causal modeling as a tool to disentangle neuronal specialization across regions that are commonly activated, we found that the connectivity between the left and right intraparietal sulci is format-dependent. Together, this line of results supports the idea that numerical representation is subserved by multiple mechanisms within the same parietal regions

Application of a genome-based predictive CHO model for increased mAb production and Glycosylation control

Monoclonal antibody therapeutics continue to grow in both number and market share with recent forecasts of global sales reaching ~$125MM by 2020. Most mAb products currently on the market are produced using cultured mammalian cells, typically Chinese Hamster Ovary (CHO) cells, which provide the necessary post-translational modifications to make the antibody efficacious. Many post-translational modifications such as the oligosaccharide profile are considered critical quality attributes (CQAs) that must be tightly controlled throughout the manufacturing process to ensure product safety and effectiveness. Therefore, the ability to predict how cell culture media components, including potential contaminants like trace metals, will affect product formation and glycosylation is important from both a process development and process control viewpoint. A detailed genome-based, predictive CHO model from the Insilico Cells™ library was adapted by the reconstruction software Insilico Discovery™ for a representative fed-batch process through a collaborative effort leveraging the computational and experimental expertise of two companies. The final, compartmentalized network model contained 1900 reactions (including transport reactions), 1300 compounds and contains stoichiometric descriptions of anabolic pathways for amino acids, lipids and carbohydrate species. The genome-scale model was constrained using several assumptions on the cell physiology and then used to compute time-resolved flux distributions by the software module Insilico Inspector™. The Insilico Designer™ module was then used to subsequently reduce the large model to a computationally manageable reduced model able to describe all flux distributions using 5 flux modes, of which 4 combined several metabolic functions and one is independently responsible for product synthesis. Using Insilico Designer™, the kinetic parameters of the reduced model were estimated by fitting the model-predicted metabolite concentrations to the experimentally determined values. The calibrated model was able to properly describe the time-dependent trajectories of biomass, product and most metabolites. Simulations using the reduced model were run and a media composition predicted to improve mAb production was identified and experimentally verified. Furthermore, experiments probing the effects of trace metals on product glycosylation were used to extend the model’s glycosylation predictability. The ability to identify both metabolic signatures, as well as media components, that correlate to specific glycan profiles will allow for fine-tuning of desired CQAs and enable more robust control strategies in upstream processes

Multiscale modeling of monoclonal antibody (mAb) production and glycosylation in a CHO cell culture process

The production of recombinant therapeutic monoclonal antibodies (mAbs) using cultured mammalian cells accounts for approximately $80 billion in global sales annually. These antibodies are often produced using Chinese hamster ovary (CHO) cell lines that execute the necessary post-translational modifications (e.g., glycosylation) for the drug to be therapeutically efficacious. Glycosylation is an intracellular, enzymatic process by which glycans (i.e., sugar molecules) are attached to a specific location on the antibody. The structure of the glycans attached to the mAb affects the therapeutic function of the molecule, making glycan distribution a critical quality attribute. Consequently, the ability to predict how variations in process parameters and/or media components affect both product formation and glycosylation is important from both a process development and process control viewpoint. A multiscale, mathematical model describing CHO cell growth and antibody production was developed in MATLAB to provide a quantitative understanding of how to manipulate a cell culture process to improve antibody titer and control glycosylation effectively. At the macro (bioreactor) scale, the model uses Monod growth kinetics to describe cell growth, nutrient/metabolite concentrations, and mAb production; at the micro scale, the glycosylation process in the Golgi apparatus is modeled using a glycosylation reaction network governed by Michaelis-Menten enzyme kinetics. Although both macro and micro scale processes are dynamic, disparate time scales makes it possible to solve the (fast) glycosylation model as a static function of the (slowly changing) macro scale state variables. In this multidisciplinary study, we will present a design of experiments approach for (1) identifying significant factors affecting glycosylation—including concentrations of asparagine, glutamine, and copper in the media, and (2) using these factors as macro scale “inputs” to the micro scale model. Model predictions are validated against an independent data set from a representative industrial mammalian cell culture process. Ultimately, the models we discuss will be valuable for biopharmaceutical process development and model-based control system design

Host Species Restriction of Middle East Respiratory Syndrome Coronavirus through Its Receptor, Dipeptidyl Peptidase 4

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in 2012. Recently, the MERS-CoV receptor dipeptidyl peptidase 4 (DPP4) was identified and the specific interaction of the receptor-binding domain (RBD) of MERS-CoV spike protein and DPP4 was determined by crystallography. Animal studies identified rhesus macaques but not hamsters, ferrets, or mice to be susceptible for MERS-CoV. Here, we investigated the role of DPP4 in this observed species tropism. Cell lines of human and nonhuman primate origin were permissive of MERS-CoV, whereas hamster, ferret, or mouse cell lines were not, despite the presence of DPP4. Expression of human DPP4 in nonsusceptible BHK and ferret cells enabled MERS-CoV replication, whereas expression of hamster or ferret DPP4 did not. Modeling the binding energies of MERS-CoV spike protein RBD to DPP4 of human (susceptible) or hamster (nonsusceptible) identified five amino acid residues involved in the DPP4-RBD interaction. Expression of hamster DPP4 containing the five human DPP4 amino acids rendered BHK cells susceptible to MERS-CoV, whereas expression of human DPP4 containing the five hamster DPP4 amino acids did not. Using the same approach, the potential of MERS-CoV to utilize the DPP4s of common Middle Eastern livestock was investigated. Modeling of the DPP4 and MERS-CoV RBD interaction predicted the ability of MERS-CoV to bind the DPP4s of camel, goat, cow, and sheep. Expression of the DPP4s of these species on BHK cells supported MERS-CoV replication. This suggests, together with the abundant DPP4 presence in the respiratory tract, that these species might be able to function as a MERS-CoV intermediate reservoir

Internal and external cooling methods and their effect on body temperature, thermal perception and dexterity

© 2018 The Authors. Published by PLOS. This is an open access article available under a Creative Commons licence. The published version can be accessed at the following link on the publisher’s website: https://doi.org/10.1371/journal.pone.0191416© 2018 Maley et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Objective The present study aimed to compare a range of cooling methods possibly utilised by occupational workers, focusing on their effect on body temperature, perception and manual dexterity. Methods Ten male participants completed eight trials involving 30 min of seated rest followed by 30 min of cooling or control of no cooling (CON) (34C, 58% relative humidity). The cooling methods utilised were: ice cooling vest (CV0), phase change cooling vest melting at 14C (CV14), evaporative cooling vest (CVEV), arm immersion in 10C water (AI), portable water-perfused suit (WPS), heliox inhalation (HE) and ice slushy ingestion (SL). Immediately before and after cooling, participants were assessed for fine (Purdue pegboard task) and gross (grip and pinch strength) manual dexterity. Rectal and skin temperature, as well as thermal sensation and comfort, were monitored throughout. Results Compared with CON, SL was the only method to reduce rectal temperature (P = 0.012). All externally applied cooling methods reduced skin temperature (P0.05). Conclusion The present study observed that ice ingestion or ice applied to the skin produced the greatest effect on rectal and skin temperature, respectively. AI should not be utilised if workers require subsequent fine manual dexterity. These results will help inform future studies investigating appropriate pre-cooling methods for the occupational worker.This project is financially supported by the US Government through the Technical Support Working Group within the Combating Terrorism Technical Support Office.Published versio

Inselect: Automating the Digitization of Natural History Collections

Copyright: © 2015 Hudson et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. The attached file is the published version of the article

The epidemiology of kidney disease in people of African ancestry with HIV in the UK

Background: Chronic kidney disease (CKD) is a leading cause of morbidity and mortality globally. The risk of CKD is increased in people of African ancestry and with Human Immunodeficiency Virus (HIV) infection. Methods: We conducted a cross-sectional study investigating the relationship between region of ancestry (East, Central, South or West Africa) and kidney disease in people of sub-Saharan African ancestry with HIV in the UK between May 2018 and February 2020. The primary outcome was renal impairment (estimated glomerular filtration rate [eGFR] of 50 mg/mmol), and biopsy-confirmed HIV-associated nephropathy (HIVAN), focal segmental glomerulosclerosis (FSGS) or arterionephrosclerosis. Multivariable robust Poisson regression estimated the effect of region of African ancestry on kidney disease outcomes. Findings: Of the 2468 participants (mean age 48.1 [SD 9.8] years, 62% female), 193 had renal impairment, 87 stage 5 CKD, 126 proteinuria, and 43 HIVAN/FSGS or arterionephrosclerosis. After adjusting for demographic characteristics, HIV and several CKD risk factors and with East African ancestry as referent, West African ancestry was associated with renal impairment (prevalence ratio [PR] 2.06 [95% CI 1.40–3.04]) and stage 5 CKD (PR 2.23 [1.23–4.04]), but not with proteinuria (PR 1.27 [0.78–2.05]). West African ancestry (as compared to East/South African ancestry) was also strongly associated with a diagnosis of HIVAN/FSGS or arterionephrosclerosis on kidney biopsy (PR 6.44 [2.42–17.14]). Interpretation: Our results indicate that people of West African ancestry with HIV are at increased risk of kidney disease. Although we cannot rule out the possibility of residual confounding, geographical region of origin appears to be a strong independent risk factor for CKD as the association did not appear to be explained by several demographic, HIV or renal risk factors