Neuronal Ndst1 depletion accelerates prion protein clearance and slows neurodegeneration in prion infection.

Select prion diseases are characterized by widespread cerebral plaque-like deposits of amyloid fibrils enriched in heparan sulfate (HS), a abundant extracellular matrix component. HS facilitates fibril formation in vitro, yet how HS impacts fibrillar plaque growth within the brain is unclear. Here we found that prion-bound HS chains are highly sulfated, and that the sulfation is essential for accelerating prion conversion in vitro. Using conditional knockout mice to deplete the HS sulfation enzyme, Ndst1 (N-deacetylase / N-sulfotransferase) from neurons or astrocytes, we investigated how reducing HS sulfation impacts survival and prion aggregate distribution during a prion infection. Neuronal Ndst1-depleted mice survived longer and showed fewer and smaller parenchymal plaques, shorter fibrils, and increased vascular amyloid, consistent with enhanced aggregate transit toward perivascular drainage channels. The prolonged survival was strain-dependent, affecting mice infected with extracellular, plaque-forming, but not membrane bound, prions. Live PET imaging revealed rapid clearance of recombinant prion protein monomers into the CSF of neuronal Ndst1- deficient mice, neuronal, further suggesting that HS sulfate groups hinder transit of extracellular prion protein monomers. Our results directly show how a host cofactor slows the spread of prion protein through the extracellular space and identify an enzyme to target to facilitate aggregate clearance.

Pilot Study of the Effects of Paced Breathing on Measures of Convergent and Divergent Thinking.

BACKGROUND: The ability of the autonomic nervous system's stress response to impair aspects of cognitive flexibility is known. However, the ability to modulate the sympathetic response and improve these cognitive impairments via nonpharmacological intervention, such as paced breathing (PB), requires further investigation. OBJECTIVE: To better elucidate the effects of PB on cognition. METHOD: We employed a PB protocol in a total of 52 healthy men and women and measured performance on convergent and divergent cognitive tasks, perceived stress, and physiological measures (eg, blood pressure, heart rate). Participants attended two experimental sessions consisting of either PB or normal breathing followed by cognitive assessments including convergent (compound remote associate, anagram) and divergent (alternate use, fluency) tasks. Experiment 2 consisted of more difficult versions of cognitive tasks compared with Experiment 1. RESULTS: In Experiment 1, PB significantly reduced the female participants' systolic and diastolic blood pressure immediately after the breathing protocol without affecting their cognition. In Experiment 2, PB significantly reduced perceived stress immediately after the breathing protocol, regardless of sex. There was no effect on cognition in Experiment 2, but a correlation was observed between perceived stress change and anagram number solved change. CONCLUSION: While PB modulates sympathetic activity in females, there was a lack of improvement in cognitive flexibility performance. At least for a single trial of PB, cognitive flexibility did not improve.

Structure and activity of native and thiolated α-chymotrypsin adsorbed onto gold nanoparticles.

A detailed understanding of protein-nanoparticle interactions is critical to realize the full potential of bioconjugate-enabled technologies. Parameters that lead to conformational changes in protein structure upon adsorption must be identified and controlled to mitigate loss of biological function. We hypothesized that the installation of thiol functional groups on a protein will facilitate robust adsorption to gold nanoparticles (AuNPs) and prevent protein unfolding to achieve thermodynamic stability. Here we investigated the adsorption behavior of α-chymotrypsin (ChT) and a thiolated analog of α-chymotrypsin (T-ChT) with AuNPs. ChT, which does not present any free thiols, was modified with 2-iminothiolane (Traut's reagent) to synthesize T-ChT consisting of two free thiols. Protein adsorption to AuNPs was monitored with dynamic light scattering and UV-vis spectrophotometry, and fluorescence spectra were acquired to assess changes in protein structure induced by interaction with the AuNP. The biological function of ChT, T-ChT, and respective bioconjugates were compared using a colorimetric enzymatic assay. The thiolated analog exhibited a greater affinity for the AuNP than the unmodified ChT, as determined from adsorption isotherms. The ChT protein formed a soft protein corona in which the enzyme denatures with prolonged exposure to AuNPs and, subsequently, lost enzymatic function. Conversely, the T-ChT formed a robust hard corona on the AuNP and retained structure and function. These data support the hypothesis, provide further insight into protein-AuNP interactions, and identify a simple chemical approach to synthesize robust and functional conjugates.

Virtual Reality Improves Patient Experience and Anxiety During In-office Carpal Tunnel Release.

Background: This study examined how wide- awake local anesthesia no tourniquet (WALANT) surgery in the office versus the standard operating room (OR) impacts patient experience, and the effect wide awake virtual reality (WAVR) has in conjunction with WALANT on patient experience. Methods: This is a patient-reported outcome study of patients undergoing carpal tunnel release by a single surgeon between August 2017 and March 2021. Patients were classified by location; traditional OR versus WALANT in-office. In-office patients were further classified by whether they chose to use WAVR or not. Patients rated overall experience, enjoyability, and anxiety using a Likert scale (1-7). Results: The online survey had a 44.8% response rate. OR patients were twice as likely to report a neutral or negative experience (23% versus 11%, P = 0.03), significantly lower enjoyment scores (44% versus 20%, P = 0.0007)' and higher anxiety (42% versus 26%, P = 0.04) compared with office-based WALANT patients. With the addition of WAVR, office patients reported higher enjoyment than those who did not use WAVR (85% versus 73%, P = 0.05). Patients reporting an anxiety disorder were more likely to choose WAVR when compared with patients without anxiety disorder (73.8% versus 56.4%). When they chose WAVR, they had greater anxiolysis (79% versus 47%, P = 0.01)' and increased enjoyment (90% versus 59%, P = 0.005). Conclusions: This study demonstrates improved patient experience in the office setting, further amplified by WAVR. Preexisting anxiety disorder is a positive predictive variable toward the patients' choice to use WAVR.

Rational use of diagnostic and screening tests.

Veterinarians have a vast and ever-expanding array of diagnostic tests available to them. However, this abundance can be an embarrassment of riches that confounds diagnosis and undermines patient care if we do not make critical and informed decisions about the selection and interpretation of the tests we employ. Effective use of diagnostic tests requires a deliberate and informed approach. We must consider the strengths and weaknesses of the tests themselves and the clinical context, and we must be wary of the many biases that skew our use and interpretation of diagnostic tests. Understanding sensitivity and specificity, likelihood, prevalence and predictive value, the basic principles of Bayesian reasoning, and the cognitive biases that drive inappropriate testing are all critical to ensuring our use of imaging and laboratory testing improves patient outcomes.

High-Affinity Points of Interaction on Antibody Allow Synthesis of Stable and Highly Functional Antibody-Gold Nanoparticle Conjugates.

Many emerging nanobiotechnologies rely on the proper function of proteins immobilized on gold nanoparticles. Often, the surface chemistry of the AuNP is engineered to control the orientation, surface coverage, and structure of the adsorbed protein to maximize conjugate function. Here, we chemically modified antibody to investigate the effect of protein surface chemistries on adsorption to AuNPs. A monoclonal anti-horseradish peroxidase IgG antibody (anti-HRP) was reacted with N-succinimidyl acrylate (NSA) or reduced dithiobissuccinimidyl propionate (DSP) to modify lysine residues. Zeta potential measurements confirmed that both chemical modifications reduced the localized regions of positive charge on the protein surface, while the DSP modification incorporated additional free thiols. Dynamic light scattering confirmed that native and chemically modified antibodies adsorbed onto AuNPs to form bioconjugates; however, adsorption kinetics revealed that the NSA-modified antibody required significantly more time to allow for the formation of a hard corona. Moreover, conjugates formed with the NSA-modified antibody lost antigen-binding function, whereas unmodified and DSP-modified antibodies adsorbed onto AuNPs to form functional conjugates. These results indicate that high-affinity functional groups are required to prevent protein unfolding and loss of function when adsorbed on the AuNP surface. The reduced protein charge and high-affinity thiol groups on the DSP-modified antibody enabled pH-dependent control of protein orientation and the formation of highly active conjugates at solution pHs (<7.5) that are inaccessible with unmodified antibody due to conjugate aggregation. This study establishes parameters for protein modification to facilitate the formation of highly functional and stable protein-AuNP conjugates.

Probing the Mechanism of Antibody-Triggered Aggregation of Gold Nanoparticles.

The unique physicochemical properties of gold nanoparticles (AuNPs) provide many opportunities to develop novel biomedical technologies. The surface chemistry of AuNPs can be engineered to perform a variety of functions, including targeted binding, cellular uptake, or stealthlike properties through the immobilization of biomolecules, such as proteins. It is well established that proteins can spontaneously adsorb onto AuNPs, to form a stable and functional bioconjugate; however, the protein-AuNP interaction may result in the formation of less desirable protein-AuNP aggregates. Therefore, it is imperative to investigate the protein-AuNP interaction and elucidate the mechanism by which protein triggers AuNP aggregation. Herein, we systematically investigated the interaction of immunoglobulin G (IgG) antibody with citrate-capped AuNPs as a function of solution pH. We found that the addition of antibody triggers the aggregation of AuNPs for pH < 7.5, whereas a monolayer of antibody adsorbs onto the AuNP to form a stable bioconjugate when the antibody is added to AuNPs at pH ≥ 7.5. Our data identifies electrostatic bridging between the antibody and the negatively charged AuNPs as the mechanism by which aggregation occurs and rules out protein unfolding and surface charge depletion as potential causes. Furthermore, we found that the electrostatic bridging of AuNPs is reversible within the first few hours of interaction, but the protein-AuNP interactions strengthen over 24 h, after which the protein-AuNP aggregate is irreversibly formed. From this data, we developed a straightforward approach to acrylate the basic residues on the antibody to prevent protein-induced aggregation of AuNP over a wide pH range. The results of this study provide additional insight into antibody-nanoparticle interactions and provide a pathway to control the interaction with the potential to enhance the conjugate function.

Absent in melanoma 2 regulates tumor cell proliferation in glioblastoma multiforme.

INTRODUCTION: Inflammation is a key aspect of glioblastoma multiforme (GBM) although it remains unclear how it contributes to GBM pathogenesis. Inflammasomes are intracellular multi-protein complexes that are involved in innate immunity and are activated by cellular stress, principally in macrophages. This study examined the expression of inflammasome-associated genes in GBM, particularly absent in melanoma 2 (AIM2). METHODS: Tissue samples from surgically-resected GBM tumors (n = 10) were compared to resected brain specimens from patients with epilepsy (age- and sex-matched Other Disease Controls (ODC, n=5)) by qRT-PCR, western blotting and immunofluorescence. Gene expression studies in human astrocytoma U251 cells were performed and the effects of deleting the absent in melanoma 2 (AIM2) gene using the CRISPR-Cas9 system were analyzed. RESULTS: GBM tissues showed significantly elevated expression of multiple immune (CD3E, CD163, CD68, MX1, ARG1) and inflammasome (AIM2, NLRP1, IL18, CASP1, and IL-33) genes compared to ODC tissues, without induction of IL1B, IFNG or TNFA. An insert-containing AIM2 variant transcript was highly expressed in GBM tissues and in U251 cells. AIM2 immunoreactivity was concentrated in the tumor core in the absence of PCNA immunodetection and showed a predominant 52 kDa immunoreactive band on western blot. Deletion of AIM2 resulted in significantly enhanced proliferation of U251 cells, which also displayed increased resistance to temozolomide treatment. CONCLUSIONS: GBM tumors express a distinct profile of inflammasome-associated genes in a tumor-specific manner. AIM2 expression in tumor cells suppressed cell proliferation while also conferring increased susceptibility to contemporary GBM therapy.

Liming impacts on soils, crops and biodiversity in the UK: A review.

Fertile soil is fundamental to our ability to achieve food security, but problems with soil degradation (such as acidification) are exacerbated by poor management. Consequently, there is a need to better understand management approaches that deliver multiple ecosystem services from agricultural land. There is global interest in sustainable soil management including the re-evaluation of existing management practices. Liming is a long established practice to ameliorate acidic soils and many liming-induced changes are well understood. For instance, short-term liming impacts are detected on soil biota and in soil biological processes (such as in N cycling where liming can increase N availability for plant uptake). The impacts of liming on soil carbon storage are variable and strongly relate to soil type, land use, climate and multiple management factors. Liming influences all elements in soils and as such there are numerous simultaneous changes to soil processes which in turn affect the plant nutrient uptake; two examples of positive impact for crops are increased P availability and decreased uptake of toxic heavy metals. Soil physical conditions are at least maintained or improved by liming, but the time taken to detect change varies significantly. Arable crops differ in their sensitivity to soil pH and for most crops there is a positive yield response. Liming also introduces implications for the development of different crop diseases and liming management is adjusted according to crop type within a given rotation. Repeated lime applications tend to improve grassland biomass production, although grassland response is variable and indirect as it relates to changes in nutrient availability. Other indicators of liming response in grassland are detected in mineral content and herbage quality which have implications for livestock-based production systems. Ecological studies have shown positive impacts of liming on biodiversity; such as increased earthworm abundance that provides habitat for wading birds in upland grasslands. Finally, understanding of liming impacts on soil and crop processes are explored together with functional aspects (in terms of ecosystems services) in a new qualitative framework that includes consideration of how liming impacts change with time. This holistic approach provides insights into the far-reaching impacts that liming has on ecosystems and the potential for liming to enhance the multiple benefits from agriculturally managed land. Recommendations are given for future research on the impact of liming and the implications for ecosystem services.

Enhancing a long-range salt bridge with intermediate aromatic and nonpolar amino acids.

The interaction of a positively charged amino acid residue with a negatively charged residue (i.e. a salt bridge) can contribute substantially to protein conformational stability, especially when two ionic groups are in close proximity. At longer distances, this stabilizing effect tends to drop off precipitously. However, several lines of evidence suggest that salt-bridge interaction could persist at longer distances if an aromatic amino acid residue were positioned between the anion and cation. Here we explore this possibility in the context of a peptide in which a Lys residue occupies the i + 8 position relative to an i-position Glu on the solvent-exposed surface of a helix-bundle homotrimer. Variable temperature circular dichroism (CD) experiments indicate that an i + 4-position Trp enables a favorable long-range interaction between Glu and the i + 8 Lys. A substantial portion of this effect relies on the presence of a hydrogen-bond donor on the arene; however, non-polar arenes, a cyclic hydrocarbon, and an acyclic Leu side-chain can also enhance the long-range salt bridge, possibly by excluding water and ions from the space between Glu and Lys.

Biogeographical factors affecting the distribution of stream salamanders on the Cumberland Plateau, USA.

Geophysical and climate conditions play an important role in the distribution of organisms at both fine and broad scales. Headwater streams integrate changes at broad geographical scales and serve as important regions of nutrient processing and support high biodiversity. Stream salamanders are important members of headwater aquatic communities as both predators and prey. Combined with their biphasic life histories and physiological requirements, stream amphibians may serve as indicators for headwater stream conditions. We surveyed 50 streams for salamander occupancy, across eight counties of the southern Cumberland Plateau in Tennessee for which we are unaware of any stream amphibian distribution data. Our objective was to determine what variables best-predicted stream amphibian occupancy among species with variable life histories and habitat needs. Models were generated to determine the effects of elevation, catchment forest cover, and total catchment size as indicators of stream condition. We found species showed no significant responses to forest cover. Though forest cover has previously been the primary predictor of stream amphibian occupancy in streams across the United States, stream amphibian occupancy of the southern Cumberland Plateau was most closely associated with elevation and catchment size. Thus, the unique topography of the southern Cumberland Plateau may instead be the more important factor driving occupancy patterns. Despite limited evidence in this study for negative human impacts on salamander occupancy, low occupancy and abundance relative to other ecoregions suggests either poor quality habitat or large historic impacts. Developing a more comprehensive understanding of regional stream salamander occupancy patterns is necessary to ensure effective management and habitat conservation in a region with limited description of stream ecology.

RIPK3 deficiency or catalytically inactive RIPK1 provides greater benefit than MLKL deficiency in mouse models of inflammation and tissue injury.

Necroptosis is a caspase-independent form of cell death that is triggered by activation of the receptor interacting serine/threonine kinase 3 (RIPK3) and phosphorylation of its pseudokinase substrate mixed lineage kinase-like (MLKL), which then translocates to membranes and promotes cell lysis. Activation of RIPK3 is regulated by the kinase RIPK1. Here we analyze the contribution of RIPK1, RIPK3, or MLKL to several mouse disease models. Loss of RIPK3 had no effect on lipopolysaccharide-induced sepsis, dextran sodium sulfate-induced colitis, cerulein-induced pancreatitis, hypoxia-induced cerebral edema, or the major cerebral artery occlusion stroke model. However, kidney ischemia-reperfusion injury, myocardial infarction, and systemic inflammation associated with A20 deficiency or high-dose tumor necrosis factor (TNF) were ameliorated by RIPK3 deficiency. Catalytically inactive RIPK1 was also beneficial in the kidney ischemia-reperfusion injury model, the high-dose TNF model, and in A20(-/-) mice. Interestingly, MLKL deficiency offered less protection in the kidney ischemia-reperfusion injury model and no benefit in A20(-/-) mice, consistent with necroptosis-independent functions for RIPK1 and RIPK3. Combined loss of RIPK3 (or MLKL) and caspase-8 largely prevented the cytokine storm, hypothermia, and morbidity induced by TNF, suggesting that the triggering event in this model is a combination of apoptosis and necroptosis. Tissue-specific RIPK3 deletion identified intestinal epithelial cells as the major target organ. Together these data emphasize that MLKL deficiency rather than RIPK1 inactivation or RIPK3 deficiency must be examined to implicate a role for necroptosis in disease.

Conjugation Strategy Strongly Impacts the Conformational Stability of a PEG-Protein Conjugate.

Site-specific PEGylation is an important strategy for enhancing the pharmacokinetic properties of protein drugs, and has been enabled by the recent development of many chemoselective reactions for protein side-chain modification. However, the impact of these different conjugation strategies on the properties of PEG-protein conjugates is poorly understood. Here we show that the ability of PEG to enhance protein conformational stability depends strongly on the identity of the PEG-protein linker, with the most stabilizing linkers involving conjugation of PEG to planar polar groups near the peptide backbone. We also find that branched PEGs provide superior stabilization relative to their linear counterparts, suggesting additional applications for branched PEGs in protein stabilization.

The evolution of bicontinuous polymeric nanospheres in aqueous solution.

Complex polymeric nanospheres in aqueous solution are desirable for their promising potential in encapsulation and templating applications. Understanding how they evolve in solution enables better control of the final structures. By unifying insights from cryoTEM and small angle X-ray scattering (SAXS), we present a mechanism for the development of bicontinuous polymeric nanospheres (BPNs) in aqueous solution from a semi-crystalline comb-like block copolymer that possesses temperature-responsive functionality. During the initial stages of water addition to THF solutions of the copolymer the aggregates are predominantly vesicles; but above a water content of 53% irregular aggregates of phase separated material appear, often microns in diameter and of indeterminate shape. We also observe a cononsolvency regime for the copolymer in THF-water mixtures from 22 to 36%. The structured large aggregates gradually decrease in size throughout dialysis, and the BPNs only appear upon cooling the fully aqueous dispersions from 35 °C to 5 °C. Thus, the final BPNs are ultimately the result of a reversible temperature-induced morphological transition.

Effect of ethnicity on care pathway and outcomes in patients hospitalized with influenza A(H1N1)pdm09 in the UK.

Data were extracted from the case records of UK patients admitted with laboratory-confirmed influenza A(H1N1)pdm09. White and non-White patients were characterized by age, sex, socioeconomic status, pandemic wave and indicators of pre-morbid health status. Logistic regression examined differences by ethnicity in patient characteristics, care pathway and clinical outcomes; multivariable models controlled for potential confounders. Whites (n = 630) and non-Whites (n = 510) differed by age, socioeconomic status, pandemic wave of admission, pregnancy, recorded obesity, previous and current smoking, and presence of chronic obstructive pulmonary disease. After adjustment for a priori confounders non-Whites were less likely to have received pre-admission antibiotics [adjusted odds ratio (aOR) 0·43, 95% confidence interval (CI) 0·28-0·68, P < 0·001) but more likely to receive antiviral drugs as in-patients (aOR 1·53, 95% CI 1·08-2·18, P = 0·018). However, there were no significant differences by ethnicity in delayed admission, severity at presentation for admission, or likelihood of severe outcome.

New method for assessing hand disinfection shows that pre-operative alcohol/chlorhexidine rub is as effective as a traditional surgical scrub.

BACKGROUND: Several studies have shown that rubbing hands with an alcohol/chlorhexidine solution provides equivalent microbial decontamination to a conventional surgical scrub using aqueous chlorhexidine. However, the authors believe that these studies have methodological flaws that limit their applicability to the operating theatre environment. As such, a method was developed to compare products in an everyday operating theatre environment using working operating theatre personnel. AIM: To determine whether or not an alcohol/chlorhexidine rub is as efficacious as a traditional surgical scrub using a novel method. METHODS: Bacterial counts at baseline were collected from 20 anaesthetists using the glove juice method. Subsequently, with sequential exchange of sterile gloves, one hand underwent a 3-min scrub using 4% aqueous chlorhexidine, and the other hand underwent a 60-s rub with a 70% isopropyl alcohol/0.5% chlorhexidine solution. The residual bacterial count was collected for each hand after 30 min using the glove juice method. These counts were converted to log10 values to compare the baseline counts of right and left hands, and efficacy between the treatment groups. FINDINGS: Mean [± standard deviation (SD)] bacterial counts at baseline were (log10) 4.42 ± 0.81 for left hands and 4.64 ± 0.60 for right hands (P > 0.05). The mean (± SD) reduction from baseline was (log10) 1.45 ± 0.50 for 4% chlorhexidine and 2.01 ± 0.98 for alcohol/chlorhexidine (P > 0.05). CONCLUSION: An alcohol/chlorhexidine hand rub was found to be as efficacious as a traditional scrub after 30 min; this study differs from previous work as it was undertaken in a population of practising anaesthetists in their working environment. The McKenzie method allows baseline and study evaluations to be performed contemporaneously on the same individual. Each subject was his/her own control. This method offers a more clinically relevant way to compare disinfectant solutions than standard methods.

Design and manufacture of a novel system to simulate the biomechanics of basic and pitching shoulder motion.

OBJECTIVES: Cadaveric models of the shoulder evaluate discrete motion segments using the glenohumeral joint in isolation over a defined trajectory. The aim of this study was to design, manufacture and validate a robotic system to accurately create three-dimensional movement of the upper body and capture it using high-speed motion cameras. METHODS: In particular, we intended to use the robotic system to simulate the normal throwing motion in an intact cadaver. The robotic system consists of a lower frame (to move the torso) and an upper frame (to move an arm) using seven actuators. The actuators accurately reproduced planned trajectories. The marker setup used for motion capture was able to determine the six degrees of freedom of all involved joints during the planned motion of the end effector. RESULTS: The testing system demonstrated high precision and accuracy based on the expected versus observed displacements of individual axes. The maximum coefficient of variation for displacement of unloaded axes was less than 0.5% for all axes. The expected and observed actual displacements had a high level of correlation with coefficients of determination of 1.0 for all axes. CONCLUSIONS: Given that this system can accurately simulate and track simple and complex motion, there is a new opportunity to study kinematics of the shoulder under normal and pathological conditions in a cadaveric shoulder model.

Low-hazard metallography of moisture-sensitive electrochemical cells.

A low-hazard approach is presented to prepare metallographic cross-sections of moisture-sensitive battery components. The approach is tailored for evaluation of thermal (molten salt) batteries composed of thin pressed-powder pellets, but has general applicability to other battery electrochemistries. Solution-cast polystyrene is used to encapsulate cells before embedding in epoxy. Nonaqueous grinding and polishing are performed in an industrial dry room to increase throughput. Lapping oil is used as a lubricant throughout grinding. Hexane is used as the solvent throughout processing; occupational exposure levels are well below the limits. Light optical and scanning electron microscopy on cross-sections are used to analyse a thermal battery cell. Spatially resolved X-ray diffraction on oblique angle cut cells complement the metallographic analysis.