Enhanced Cell Viability and Migration of Primary Bovine Annular Fibrosus Fibroblast-like Cells Induced by Microsecond Pulsed Electric Field Exposure.

This study is the first to report the enhancement of cell migration and proliferation induced by in vitro microsecond pulsed electric field (µsPEF) exposure of primary bovine annulus fibrosus (AF) fibroblast-like cells. AF primary cells isolated from fresh bovine intervertebral disks (IVDs) are exposed to 10 and 100 µsPEFs with different numbers of pulses and applied electric field strengths. The results indicate that 10 µs-duration pulses induce reversible electroporation, while 100 µs pulses induce irreversible electroporation of the cells. Additionally, µsPEF exposure increased AF cell proliferation up to 150% while increasing the average migration speed by 0.08 µm/min over 24 h. The findings suggest that the effects of PEF exposure on cells are multifactorial-depending on the duration, intensity, and number of pulses used in the stimulation. This highlights the importance of optimizing the µsPEF parameters for specific cell types and applications. For instance, if the goal is to induce cell death for cancer treatment, then high numbers of pulses can be used to maximize the lethal effects. On the other hand, if the goal is to enhance cell proliferation, a combination of the number of pulses and the applied electric field strength can be tuned to achieve the desired outcome. The information gleaned from this study can be applied in the future to in vitro cell culture expansion and tissue regeneration.

Intrinsic disorder and conformational coexistence in auxin coreceptors.

AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) transcriptional repressor proteins and the TRANSPORT INHIBITOR RESISTANT 1/AUXIN SIGNALING F-BOX (TIR1/AFB) proteins to which they bind act as auxin coreceptors. While the structure of TIR1 has been solved, structural characterization of the regions of the Aux/IAA protein responsible for auxin perception has been complicated by their predicted disorder. Here, we use NMR, CD and molecular dynamics simulation to investigate the N-terminal domains of the Aux/IAA protein IAA17/AXR3. We show that despite the conformational flexibility of the region, a critical W-P bond in the core of the Aux/IAA degron motif occurs at a strikingly high (1:1) ratio of cis to trans isomers, consistent with the requirement of the cis conformer for the formation of the fully-docked receptor complex. We show that the N-terminal half of AXR3 is a mixture of multiple transiently structured conformations with a propensity for two predominant and distinct conformational subpopulations within the overall ensemble. These two states were modeled together with the C-terminal PB1 domain to provide the first complete simulation of an Aux/IAA. Using MD to recreate the assembly of each complex in the presence of auxin, both structural arrangements were shown to engage with the TIR1 receptor, and contact maps from the simulations match closely observations of NMR signal-decreases. Together, our results and approach provide a platform for exploring the functional significance of variation in the Aux/IAA coreceptor family and for understanding the role of intrinsic disorder in auxin signal transduction and other signaling systems.

Phospholipid headgroup composition modulates the molecular interactions and antimicrobial effects of sulfobetaine zwitterionic detergents against the "ESKAPE" pathogen Pseudomonas aeruginosa.

We determine the efficacy for three known structurally related, membrane active detergents against multidrug resistant and wild type strains of Pseudomonas aeruginosa. Accessible solution state NMR experiments are used to quantify phospholipid headgroup composition of the microbial membranes and to gain molecular level insight into antimicrobial mode of action.

Threshold Microsecond Pulsed Electric Field Exposures for Change in Spinach Quality.

Pulsed electric fields (PEFs) are often used to pretreat foods to enhance subsequent processes, such as drying, where maintaining food product quality is important for consumer satisfaction. This study aims to establish a threshold PEF exposure to determine the doses at which electroporation is viable for use on spinach leaves, wherein integrity is maintained postexposure. Three numbers of consecutive pulses (1, 5, 50) and two pulse durations (10 and 100 µs) have been examined herein at a constant pulse repetition of 10 Hz and 1.4 kV/cm field strength. The data indicate that pore formation in itself is not a cause for loss of spinach leaf food quality, i.e., significant changes in color and water content. Rather, cell death, or the rupture of the cell membrane from a high-intensity treatment, is necessary to significantly alter the exterior integrity of the plant tissue. PEF exposures thus can be used on leafy greens up until the point of inactivation before consumers would see any alterations, making reversible electroporation a viable treatment for consumer-intended products. These results open up future opportunities to use emerging technologies based on PEF exposures and provide useful information in setting parameters to avoid food quality diminishment.

Yields and product comparison between Escherichia coli BL21 and W3110 in industrially relevant conditions: anti-c-Met scFv as a case study.

INTRODUCTION: In the biopharmaceutical industry, Escherichia coli is one of the preferred expression hosts for large-scale production of therapeutic proteins. Although increasing the product yield is important, product quality is a major factor in this industry because greatest productivity does not always correspond with the highest quality of the produced protein. While some post-translational modifications, such as disulphide bonds, are required to achieve the biologically active conformation, others may have a negative impact on the product's activity, effectiveness, and/or safety. Therefore, they are classified as product associated impurities, and they represent a crucial quality parameter for regulatory authorities. RESULTS: In this study, fermentation conditions of two widely employed industrial E. coli strains, BL21 and W3110 are compared for recombinant protein production of a single-chain variable fragment (scFv) in an industrial setting. We found that the BL21 strain produces more soluble scFv than the W3110 strain, even though W3110 produces more recombinant protein in total. A quality assessment on the scFv recovered from the supernatant was then performed. Unexpectedly, even when our scFv is correctly disulphide bonded and cleaved from its signal peptide in both strains, the protein shows charge heterogeneity with up to seven distinguishable variants on cation exchange chromatography. Biophysical characterization confirmed the presence of altered conformations of the two main charged variants. CONCLUSIONS: The findings indicated that BL21 is more productive for this specific scFv than W3110. When assessing product quality, a distinctive profile of the protein was found which was independent of the E. coli strain. This suggests that alterations are present in the recovered product although the exact nature of them could not be determined. This similarity between the two strains' generated products also serves as a sign of their interchangeability. This study encourages the development of innovative, fast, and inexpensive techniques for the detection of heterogeneity while also provoking a debate about whether intact mass spectrometry-based analysis of the protein of interest is sufficient to detect heterogeneity in a product.

Electrical impedance decreases in annulus fibrosus cartilage exposed to microsecond pulsed electric fieldsex vivo.

Electropermeabilization of biomembranes often is measured by microscopic imaging of a membrane-impermeable fluorophore that penetrates the cells following pulsed electric field (PEF) exposure. PEF exposure subsequently changes physiological properties of tissue. One way to probe these changes in tissue is measuring electrical properties by way of electrochemical impedance spectroscopy (EIS). In this study, we analyse impedance and conductivity of bovine annulus fibrosus (AF) cartilage before and after exposures to PEF of 100µs duration. Two PEF parameters-electric field amplitude and number of pulses-are varied, and total specific dose of PEF is calculated. AF tissue conductivity increases with both amplitude and number of pulses, indicating electropermeabilization of the AF cells. A Live/Dead cell imaging assay validates the EIS measurements, indicating intratissue cell permeabilization byµsPEF exposure. These results support the extension of EIS to monitor extent of electropermeabilization of cells within cartilage tissue.

Histone deacetylase 4 and 5 translocation elicited by microsecond pulsed electric field exposure is mediated by kinase activity.

Electroporation-based technologies using microsecond pulsed electric field (µsPEF) exposures are established as laboratory and clinical tools that permeabilize cell membranes. We demonstrate a µsPEF bioeffect on nucleocytoplasmic import and export of enzymes that regulate genetic expression, histone deacetylases (HDAC) -4 and -5. Their µsPEF-induced nucleocytoplasmic transport depends on presence and absence of extracellular calcium ions (Ca2+) for both MCF7 and CHO-K1 cells. Exposure to 1, 10, 30 and 50 consecutive square wave pulses at 1 Hz and of 100 µs duration with 1.45 kV/cm magnitude leads to translocation of endogenous HDAC4 and HDAC5. We posit that by eliciting a rise in intracellular Ca2+ concentration, a signaling pathway involving kinases, such as Ca2+/CaM-dependent protein kinase II (CaMKII), is activated. This cascade causes nuclear export and import of HDAC4 and HDAC5. The potential of µsPEF exposures to control nucleocytoplasmic transport unlocks future opportunities in epigenetic modification.

Te Aho o Te Kahu: weaving equity into national-level cancer control.

The purpose of this manuscript was to consider how mainstream health organisations can develop structures, processes, and functions to address inequity, using the New Zealand Cancer Control Agency (Te Aho o Te Kahu) as an example. In New Zealand (Aotearoa), as in other countries, inequities in cancer incidence and outcomes exist between population groups, including for indigenous populations. Despite much discussion regarding the need to address racial inequities, often the proposed solutions are at operational or programmatic levels, and disadvantaged communities are unable to have much of a say in the system design and service delivery of these solutions. The establishment of a dedicated cancer control agency has created a unique opportunity to centralise principles and approaches to achieving equity within the core functions of the agency, and enabled a new method of approaching cancer control with the aim of achieving equity for the most disadvantaged populations. Using a framework based on the founding agreement between New Zealand's Indigenous Maori people and the British Government (Te Tiriti o Waitangi), we consider how health system organisations can develop structures, processes, and functions to achieve equity, and summarise how this new agency has been shaped to achieve these objectives for Maori people in particular, including the innovative and equity-first approach to organisational structure and focus. Within this framework, we highlight the key equity-focused work programmes, initiatives, and other actions taken since the inception of the agency. Finally, we discuss the ongoing equity-related challenges the agency faces, as well as the current and future opportunities for achieving equity in health outcomes.

Staphylococcus hominis Infective Endocarditis Presenting with Embolic Splenic and Renal Infarcts and Spinal Discitis.

Staphylococcus hominis (S. hominis) is a Gram-positive, coagulase-negative bacteria that occurs as a normal commensal organism on the skin and may rarely cause native valve endocarditis (NVE). We present a 62-year-old male with type 2 diabetes mellitus, coronary artery disease, and hypertension presenting with fever and abdominal pain. CT (computerized tomography) of the abdomen revealed splenic and renal infarcts; further imaging with MRI (magnetic resonance imaging) revealed enhancements consistent with discitis in T5-6 and L1-2. Three sets of blood cultures were positive for S. hominis sensitive to methicillin on antimicrobial susceptibility tests, and echocardiogram showed posterior mitral valve vegetation. The patient was initially treated with 10 weeks of nafcillin IV (intravenous) 2 g q4 hours. He had recurrent bouts of S. hominis bacteremia that was treated with IV vancomycin. His clinical course was complicated by new-onset atrial fibrillation with rapid ventricular response and congestive heart failure. Once bacteremia was cleared, his infective endocarditis was successfully definitively treated with mitral valve replacement and tricuspid repair.

Localization and functional characterization of the alternative oxidase in Naegleria.

The alternative oxidase (AOX) is a protein involved in supporting enzymatic reactions of the Krebs cycle in instances when the canonical (cytochrome-mediated) respiratory chain has been inhibited, while allowing for the maintenance of cell growth and necessary metabolic processes for survival. Among eukaryotes, alternative oxidases have dispersed distribution and are found in plants, fungi, and protists, including Naegleria ssp. Naegleria species are free-living unicellular amoeboflagellates and include the pathogenic species of N. fowleri, the so-called "brain-eating amoeba." Using a multidisciplinary approach, we aimed to understand the evolution, localization, and function of AOX and the role that plays in Naegleria's biology. Our analyses suggest that AOX was present in last common ancestor of the genus and structure prediction showed that all functional residues are also present in Naegleria species. Using cellular and biochemical techniques, we also functionally characterize N. gruberi's AOX in its mitochondria, and we demonstrate that its inactivation affects its proliferation. Consequently, we discuss the benefits of the presence of this protein in Naegleria species, along with its potential pathogenicity role in N. fowleri. We predict that our findings will spearhead new explorations to understand the cell biology, metabolism, and evolution of Naegleria and other free-living relatives.

Metabolic Fluctuations in the Human Stool Obtained from Blastocystis Carriers and Non-Carriers.

Blastocystis is an obligate anaerobic microbial eukaryote that frequently inhabits the gastrointestinal tract. Despite this prevalence, very little is known about the extent of its genetic diversity, pathogenicity, and interaction with the rest of the microbiome and its host. Although the organism is morphologically static, it has no less than 28 genetically distinct subtypes (STs). Reports on the pathogenicity of Blastocystis are conflicting. The association between Blastocystis and intestinal bacterial communities is being increasingly explored. Nonetheless, similar investigations extending to the metabolome are non-existent.Using established NMR metabolomics protocols in 149 faecal samples from individuals from South Korea (n = 38), Thailand (n = 44) and Turkey (n = 69), we have provided a snapshot of the core metabolic compounds present in human stools with (B+) and without (B-) Blastocystis. Samples included hosts with gastrointestinal symptoms and asymptomatics. A total of nine, 62 and 98 significant metabolites were associated with Blastocystis carriage in the South Korean, Thai and Turkish sample sets respectively, with a number of metabolites increased in colonised groups. The metabolic profiles of B+ and B- samples from all countries were distinct and grouped separately in the partial least squares-discriminant analysis (PLS-DA). Typical inflammation-related metabolites negatively associated with Blastocystis positive samples. This data will assist in directing future studies underlying the involvement of Blastocystis in physiological processes of both the gut microbiome and the host. Future studies using metabolome and microbiome data along with host physiology and immune responses information will contribute significantly towards elucidating the role of Blastocystis in health and disease.

Establishing a Metabolite Extraction Method to Study the Metabolome of Blastocystis Using NMR.

Blastocystis is an opportunistic parasite commonly found in the intestines of humans and other animals. Despite its high prevalence, knowledge regarding Blastocystis biology within and outside the host is limited. Analysis of the metabolites produced by this anaerobe could provide insights that can help map its metabolism and determine its role in both health and disease. Due to its controversial pathogenicity, these metabolites could define its deterministic role in microbiome's "health" and/or subsequently resolve Blastocystis' potential impact in gastrointestinal health. A common method for elucidating the presence of these metabolites is through 1H nuclear magnetic resonance (NMR). However, there are currently no described benchmarked methods available to extract metabolites from Blastocystis for 1H NMR analysis. Herein, several extraction solvents, lysis methods and incubation temperatures were compared for their usefulness as an extraction protocol for this protozoan. Following extraction, the samples were freeze-dried, re-solubilized and analysed with 1H NMR. The results demonstrate that carrying out the procedure at room temperature using methanol as an extraction solvent and bead bashing as a lysis technique provides a consistent, reproducible and efficient method to extract metabolites from Blastocystis for NMR.

A quantitative assay to study the lipid selectivity of membrane-associated systems using solution NMR.

The activity of membrane proteins and compounds that interact with the membrane is modulated by the surrounding lipid composition. However, there are no simple methods that determine the composition of these annular phospholipids in eukaryotic systems. Herein, we describe a simple methodology that enables the identification and quantification of the lipid composition around membrane-associated compounds using SMA-nanodiscs and routine 1H-31P NMR.

Code S: Redesigning Hospital-Wide Peer Review Processes to Identify System Errors.

Hospital medical errors that result in patient harm and death are largely identified as system failures. Most hospitals lack the tools to effectively identify most system errors. Traditional methods used in many hospitals, such as incident reporting (IR), departmental morbidity and mortality conferences, and root cause analysis committees, are often flawed by under reporting. We introduced the Code S designation into our hospital's ongoing physician peer review process as an additional and innovative way to identify system errors that contributed to adverse clinical outcomes. The authors conducted a retrospective review of all peer review cases from January 2008 to December 2011 and determined the quantity and type of system errors that occurred. System errors were categorized based on a modified 5M model which was adapted to reflect system errors encountered in healthcare. The Code S designation discovered 204 system errors that otherwise may not have previously been identified. The addition of the Code S designation to the peer review process can be readily adopted by other healthcare organizations as another tool to help identify, quantify and categorize system errors, and promote hospital-wide process improvements to decrease errors and improve patient safety.

The elucidation of phospholipid bilayer-small molecule interactions using a combination of phospholipid nanodiscs and solution state NMR techniques.

Quantifying phospholipid bilayer-small molecule interactions is vital to the development of new drug candidates and/or medicinal therapies. However, obtaining these data remains problematic. Herein, we detail a phospholipid nanodisc assay which enables the elucidation of these interactions using conventional solution state NMR spectroscopy techniques.

Structure-Based Design, Synthesis and Bioactivity of a New Anti-TNFα Cyclopeptide.

As opposed to small molecules, macrocyclic peptides possess a large surface area and are recognised as promising candidates to selectively treat diseases by disrupting specific protein-protein interactions (PPIs). Due to the difficulty in predicting cyclopeptide conformations in solution, the de novo design of bioactive cyclopeptides remains significantly challenging. In this study, we used the combination of conformational analyses and molecular docking studies to design a new cyclopeptide inhibitor of the interaction between the human tumour necrosis factor alpha (TNFα) and its receptor TNFR-1. This interaction is a key in mediating the inflammatory response to tissue injury and infection in humans, and it is also an important causative factor of rheumatoid arthritis, psoriasis and inflammatory bowel disease. The solution state NMR structure of the cyclopeptide was determined, which helped to deduce its mode of interaction with TNFα. TNFα sensor cells were used to evaluate the biological activity of the peptide.

Cell encapsulation in gelatin bioink impairs 3D bioprinting resolution.

Recent advances in three-dimensional (3D) bioprinting technologies have enabled precise patterning of cellular components along with biomimetic constructs for tissue engineering and regenerative medicine. The viscoelasticity of bioinks regulate printability and the smallest feature size in 3D bioprinted constructs. The impact of cellular components is typically neglected when choosing 3D bioprinting parameters. In this short communication, we quantified the effect of cell densities on the printability of hydrogel bioinks. Unexpectedly, our results show that encapsulated cells reduced the steady shear viscosity of gelatin-based bioinks by approximately 50% and the minimum force for onset of flow by approximately 30%. These results may justify the lower spatial resolution in 3D bioprinted cell-laden hydrogels.

Nicotinamide Inhibits Aphid Fecundity and Impacts Survival.

Nicotinamide (NAM) alters behavior in C. elegans and Drosophila, serving as an agonist of TRPV channels affecting sensory neurons and mimicking the mode of action of insecticides used to control phloem-feeding insects. The impact of NAM on green peach aphid (Myzus persicae) behaviors was assessed in artificial diet assays and foliar applications to Arabidopsis plants. Aphids feeding on artificial diets supplemented with NAM impaired stylet movement causing feeding interruptions and ultimately starvation and death. Aphid feeding behaviors were negatively impacted on NAM sprayed plants at concentrations as low as 2.5 mM leading to increased mortality. In choice assays with NAM sprayed leaves aphids showed clear preference for untreated control leaves. NAM is an intermediate in the NAD salvage pathway that should accumulate in nicotinamidase (nic) mutants. LC-MS analysis showed NAM accumulates 60-fold in nic-1-1 Arabidopsis mutants as compared with Col-0. Aphid reproductive potential was significantly decreased on nic-1-1 mutant plants, resulting in a smaller colony size and arrested population development. The results support the hypothesis that dietary NAM causes behavioral changes in aphids, including altered feeding, reduced reproduction, and increased mortality. NAM is thought to bind to TRPV channels causing overstimulation of sensory neurons in the aphid feeding apparatus.

Structural mapping of oligomeric intermediates in an amyloid assembly pathway.

Transient oligomers are commonly formed in the early stages of amyloid assembly. Determining the structure(s) of these species and defining their role(s) in assembly is key to devising new routes to control disease. Here, using a combination of chemical kinetics, NMR spectroscopy and other biophysical methods, we identify and structurally characterize the oligomers required for amyloid assembly of the protein ΔN6, a truncation variant of human ß2-microglobulin (ß2m) found in amyloid deposits in the joints of patients with dialysis-related amyloidosis. The results reveal an assembly pathway which is initiated by the formation of head-to-head non-toxic dimers and hexamers en route to amyloid fibrils. Comparison with inhibitory dimers shows that precise subunit organization determines amyloid assembly, while dynamics in the C-terminal strand hint to the initiation of cross-ß structure formation. The results provide a detailed structural view of early amyloid assembly involving structured species that are not cytotoxic.