The Dorsal Column Nuclei Scale Mechanical Sensitivity in Naive and Neuropathic Pain States.

Tactile perception relies on reliable transmission and modulation of low-threshold information as it travels from the periphery to the brain. During pathological conditions, tactile stimuli can aberrantly engage nociceptive pathways leading to the perception of touch as pain, known as mechanical allodynia. Two main drivers of peripheral tactile information, low-threshold mechanoreceptors (LTMRs) and postsynaptic dorsal column neurons (PSDCs), terminate in the brainstem dorsal column nuclei (DCN). Activity within the DRG, spinal cord, and DCN have all been implicated in mediating allodynia, yet the DCN remains understudied at the cellular, circuit, and functional levels compared to the other two. Here, we show that the gracile nucleus (Gr) of the DCN mediates tactile sensitivity for low-threshold stimuli and contributes to mechanical allodynia during neuropathic pain in mice. We found that the Gr contains local inhibitory interneurons in addition to thalamus-projecting neurons, which are differentially innervated by primary afferents and spinal inputs. Functional manipulations of these distinct Gr neuronal populations resulted in bidirectional changes to tactile sensitivity, but did not affect noxious mechanical or thermal sensitivity. During neuropathic pain, silencing Gr projection neurons or activating Gr inhibitory neurons was able to reduce tactile hypersensitivity, and enhancing inhibition was able to ameliorate paw withdrawal signatures of neuropathic pain, like shaking. Collectively, these results suggest that the Gr plays a specific role in mediating hypersensitivity to low-threshold, innocuous mechanical stimuli during neuropathic pain, and that Gr activity contributes to affective, pain-associated phenotypes of mechanical allodynia. Therefore, these brainstem circuits work in tandem with traditional spinal circuits underlying allodynia, resulting in enhanced signaling of tactile stimuli in the brain during neuropathic pain.

Review of the clinical diagnosis of sacroiliac dysfunction in horses - Challenges and limitations.

Sacroiliac dysfunction (SID) is a condition seen in horses associated with poor performance that affects hind limb gait and impulsion. The condition comprises pain and dysfunction but there lacks clarity around the aetiopathogenesis and whether SID encompasses abnormal joint pathology, abnormal joint movement, abnormal regional biomechanical function, joint laxity and pain, or various combinations of these that may vary over time. Clinical assessment remains challenging for equine clinicians due to the deep location of the sacroiliac joint (SIJ) and surrounding structures which limits access for palpation, diagnostic imaging and joint-specific injection. There is no recognised single reference standard diagnostic test for SID. Clinical diagnosis has been based on ruling out other causes of hind limb lameness, along with combinations of ultrasonography, scintigraphy and periarticular anaesthesia of the SIJ. Recent studies have highlighted the lack of specificity of injections targeting the SIJ, with significant dispersal of injectate into surrounding structures including around the lumbosacral joint (LSJ). Advanced imaging modalities such as computed tomography offers promise for assessment of the structure and pathology of the SIJ and surrounding bony structures. However, there is a need to improve the understanding of the significance of anatomic variation of the sacroiliac region structures, with recent studies reporting detailed anatomic variation in groups of horses with and without SID. There are also limitations around functional assessment of the joint which is still largely reliant on a thorough clinical examination. This review aims to present an update on clinical approaches to the diagnosis of horses with SID, and to consider the challenges and limitations.

Human Complement Inhibits Myophages against Pseudomonas aeruginosa.

Therapeutic bacteriophages (phages) are primarily chosen based on their in vitro bacteriolytic activity. Although anti-phage antibodies are known to inhibit phage infection, the influence of other immune system components is less well known. An important anti-bacterial and anti-viral innate immune system that may interact with phages is the complement system, a cascade of proteases that recognizes and targets invading microorganisms. In this research, we aimed to study the effects of serum components such as complement on the infectivity of different phages targeting Pseudomonas aeruginosa. We used a fluorescence-based assay to monitor the killing of P. aeruginosa by phages of different morphotypes in the presence of human serum. Our results reveal that several myophages are inhibited by serum in a concentration-dependent way, while the activity of four podophages and one siphophage tested in this study is not affected by serum. By using specific nanobodies blocking different components of the complement cascade, we showed that activation of the classical complement pathway is a driver of phage inhibition. To determine the mechanism of inhibition, we produced bioorthogonally labeled fluorescent phages to study their binding by means of microscopy and flow cytometry. We show that phage adsorption is hampered in the presence of active complement. Our results indicate that interactions with complement may affect the in vivo activity of therapeutically administered phages. A better understanding of this phenomenon is essential to optimize the design and application of therapeutic phage cocktails.

Protective effects of Saccharomyces boulardii CNCM I-745 in an experimental model of NSAID-induced enteropathy.

Nonsteroidal anti-inflammatory drugs (NSAIDs) induce a broad spectrum of gastro-intestinal adverse effects, including ulceration and bleeding. The pathophysiology of NSAID enteropathy is complex and incompletely understood, but some evidence showed that NSAIDs impair the intestinal barrier and cause a gut dysbiosis. Identifying new treatments aiming to reverse or attenuate NSAID-induced adverse effects would have a significant impact on a high number of patients. The aim of this work is to assess the effects of the probiotic yeast Saccharomyces boulardii CNCM I-745 (Sb) on a model of NSAID-induced enteropathy. Four groups of mice were tested: Control, Indomethacin, Sb, and Sb + Indomethacin. A clinical score was evaluated throughout the experiment. Faecal calprotectin, microbiota and haemoglobin analyses were performed. At the end of the treatments, the small intestine, colon, and caecum lengths, and intestinal permeability were measured. Sections of ileum and jejunum were observed to assess a histological score and ileal cytokines were measured by immunoassay. Indomethacin-treated animals showed an increase in their clinical scores, reflecting a worsening of their general state. Mice co-treated with Sb and indomethacin displayed an improvement of their clinical score in comparison with mice treated with indomethacin alone. Sb prevented the indomethacin-induced shortening of the small intestine and caecum, and significantly attenuated the severity of intestinal lesions. Sb also prevented the increase in faecal calprotectin, reduced faecal haemoglobin, and prevented the increase of intestinal permeability in mice treated with indomethacin. Sb also counteracted the increase of faecal bacteria associated with the pathogenesis of NSAID-enteropathy. In conclusion, our results show a protective effect of Sb in a model of indomethacin-induced enteropathy. Sb improved the intestinal barrier function and exerted a positive action on gut microbiota composition.

DREAMS: deep read-level error model for sequencing data applied to low-frequency variant calling and circulating tumor DNA detection.

Circulating tumor DNA detection using next-generation sequencing (NGS) data of plasma DNA is promising for cancer identification and characterization. However, the tumor signal in the blood is often low and difficult to distinguish from errors. We present DREAMS (Deep Read-level Modelling of Sequencing-errors) for estimating error rates of individual read positions. Using DREAMS, we develop statistical methods for variant calling (DREAMS-vc) and cancer detection (DREAMS-cc). For evaluation, we generate deep targeted NGS data of matching tumor and plasma DNA from 85 colorectal cancer patients. The DREAMS approach performs better than state-of-the-art methods for variant calling and cancer detection.

Structure and Dynamics of Three Escherichia coli NfsB Nitro-Reductase Mutants Selected for Enhanced Activity with the Cancer Prodrug CB1954.

Escherichia coli NfsB has been studied extensively for its potential for cancer gene therapy by reducing the prodrug CB1954 to a cytotoxic derivative. We have previously made several mutants with enhanced activity for the prodrug and characterised their activity in vitro and in vivo. Here, we determine the X-ray structure of our most active triple and double mutants to date, T41Q/N71S/F124T and T41L/N71S. The two mutant proteins have lower redox potentials than wild-type NfsB, and the mutations have lowered activity with NADH so that, in contrast to the wild-type enzyme, the reduction of the enzyme by NADH, rather than the reaction with CB1954, has a slower maximum rate. The structure of the triple mutant shows the interaction between Q41 and T124, explaining the synergy between these two mutations. Based on these structures, we selected mutants with even higher activity. The most active one contains T41Q/N71S/F124T/M127V, in which the additional M127V mutation enlarges a small channel to the active site. Molecular dynamics simulations show that the mutations or reduction of the FMN cofactors of the protein has little effect on its dynamics and that the largest backbone fluctuations occur at residues that flank the active site, contributing towards its broad substrate range.

An agent-based modelling framework for assessing SARS-CoV-2 indoor airborne transmission risk

We develop a framework for modelling the risk of infection from airborne Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) in well-mixed environments in the presence of interventions designed to reduce infection risk. Our framework allows development of models that are highly tailored to the specifics of complex indoor environments, including layout, people movements, and ventilation. We explore its utility through case studies, two of which are based on actual sites. Our results reflect previously quantified benefits of masks and vaccinations. We also produce quantitative estimates of the effects of air filters, and reduced indoor occupancy for which we cannot find quantitative estimates but for which positive benefits have been postulated. We find that increased airflow reduces risk due to dilution, even if that airflow is via recirculation in a large space. Our case studies have identified interventions which seem to generalise, and others which seem to be dependent on site-specific factors, such as occupant density.

Analyses of locomotion, wing morphology, and microbiome in Drosophila nigrosparsa after recovery from antibiotics.

Antibiotics, such as tetracycline, have been frequently used to cure arthropods of Wolbachia endosymbionts. After the symbionts have been removed, the hosts must recover for some generations from the side effects of the antibiotics. However, most studies do not assess the direct and indirect longer-term effects of antibiotics used to remove Wolbachia, which may question the exact contribution of this endosymbiont to the effects observed. Here, we used the fly Drosophila nigrosparsa treated or not with tetracycline for three generations followed by two generations of recovery to investigate the effects of this antibiotic on the fly locomotion, wing morphology, and the gut microbiome. We found that antibiotic treatment did not affect fly locomotion two generations after being treated with the antibiotic. In addition, gut-microbiome restoration was tested as a more efficient solution to reduce the potential side effects of tetracycline on the microbiome. There was no significant difference in alpha diversity between gut restoration and other treatments, but the abundance of some bacterial taxa differed significantly between the gut-restoration treatment and the control. We conclude that in D. nigrosparsa the recovery period of two generations after being treated with the antibiotic is sufficient for locomotion, and suggest a general assessment of direct and indirect effects of antibiotics after a particular recovery time.

Polymorphic Molecular Signatures in Variable Regions of the Plasmodium falciparum var2csa DBL3x Domain Are Associated with Virulence in Placental Malaria.

The Plasmodium falciparum protein VAR2CSA allows infected erythrocytes to accumulate within the placenta, inducing pathology and poor birth outcomes. Multiple exposures to placental malaria (PM) induce partial immunity against VAR2CSA, making it a promising vaccine candidate. However, the extent to which VAR2CSA genetic diversity contributes to immune evasion and virulence remains poorly understood. The deep sequencing of the var2csa DBL3X domain in placental blood from forty-nine primigravid and multigravid women living in malaria-endemic western Kenya revealed numerous unique sequences within individuals in association with chronic PM but not gravidity. Additional analysis unveiled four distinct sequence types that were variably present in mixed proportions amongst the study population. An analysis of the abundance of each of these sequence types revealed that one was inversely related to infant gestational age, another was inversely related to placental parasitemia, and a third was associated with chronic PM. The categorization of women according to the type to which their dominant sequence belonged resulted in the segregation of types as a function of gravidity: two types predominated in multigravidae whereas the other two predominated in primigravidae. The univariate logistic regression analysis of sequence type dominance further revealed that gravidity, maternal age, placental parasitemia, and hemozoin burden (within maternal leukocytes), reported a lack of antimalarial drug use, and infant gestational age and birth weight influenced the odds of membership in one or more of these sequence predominance groups. Cumulatively, these results show that unique var2csa sequences differentially appear in women with different PM exposure histories and segregate to types independently associated with maternal factors, infection parameters, and birth outcomes. The association of some var2csa sequence types with indicators of pathogenesis should motivate vaccine efforts to further identify and target VAR2CSA epitopes associated with maternal morbidity and poor birth outcomes.

Anti-inflammatory, Analgesic, and Cytotoxic Effects of The Phytexponent: A Polyherbal Formulation.

The Phytexponent is used to treat pain and inflammation in complementary and alternative medicine practices; however, empirical data supporting its pharmacological efficacy and safety is scanty, hence the present study. We used the carrageenan-induced paw oedema and the acetic acid-induced writhing techniques to determine the anti-inflammatory and analgesic efficacies, respectively, of the Phytexponent in Swiss albino mice models. The 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay technique was used to investigate the in vitro cytotoxic effects of the Phytexponent in the Vero E6 cell line. The Phytexponent exerted significant (P < .05) anti-inflammatory effects in the carrageenan-induced paw oedema mouse model in a dose- and time-dependent manner, with significantly higher efficacy at 250 mg/Kg BW, than indomethacin (4 mg/Kg BW), in the first, second, and third hour (P < .05). Besides, the Phytexponent significantly reduced the acetic acid-induced writhing frequency in mice (P < .05), in a dose-dependent manner, depicting its analgesic efficacy. Notably, the Phytexponent (at doses: 125 mg/Kg BW and 250 mg/Kg BW) exhibited significantly higher analgesic efficacy than the Indomethacin (P<.05). Moreover, the Phytexponent was not cytotoxic to Vero E6 cells (CC50 >1000 µg/ml) compared to cyclophosphamide (CC50 = 2.48 µg/ml). Thus, the Phytexponent has significant in vivo anti-inflammatory and analgesic efficacy in mice models and is not cytotoxic to Vero E6 cell line, depicting its therapeutic potential upon further empirical investigation.

Error Characterization and Statistical Modeling Improves Circulating Tumor DNA Detection by Droplet Digital PCR.

BACKGROUND: Droplet digital PCR (ddPCR) is a widely used and sensitive application for circulating tumor DNA (ctDNA) detection. As ctDNA is often found in low abundance, methods to separate low-signal readouts from noise are necessary. We aimed to characterize the ddPCR-generated noise and, informed by this, create a sensitive and specific ctDNA caller. METHODS: We built 2 novel complimentary ctDNA calling methods: dynamic limit of blank and concentration and assay-specific tumor load estimator (CASTLE). Both methods are informed by empirically established assay-specific noise profiles. Here, we characterized noise for 70 mutation-detecting ddPCR assays by applying each assay to 95 nonmutated samples. Using these profiles, the performance of the 2 new methods was assessed in a total of 9447 negative/positive reference samples and in 1311 real-life plasma samples from colorectal cancer patients. Lastly, performances were compared to 7 literature-established calling methods. RESULTS: For many assays, noise increased proportionally with the DNA input amount. Assays targeting transition base changes were more error-prone than transversion-targeting assays. Both our calling methods successfully accounted for the additional noise in transition assays and showed consistently high performance regardless of DNA input amount. Calling methods that were not noise-informed performed less well than noise-informed methods. CASTLE was the only calling method providing a statistical estimate of the noise-corrected mutation level and call certainty. CONCLUSIONS: Accurate error modeling is necessary for sensitive and specific ctDNA detection by ddPCR. Accounting for DNA input amounts ensures specific detection regardless of the sample-specific DNA concentration. Our results demonstrate CASTLE as a powerful tool for ctDNA calling using ddPCR.

Sexual and reproductive health factors associated with child, early and forced marriage and partnerships among refugee youth in a humanitarian setting in Uganda: Mixed methods findings.

Preventing early and forced marriage is a global priority, however, sexual and reproductive health (SRH) among youth remains understudied in humanitarian settings. This study examined child, early and forced marriage and partnership (CEFMP) among young refugees in Bidi Bidi refugee settlement, Uganda, and associations with SRH outcomes among young women. This mixed-methods study involved a qualitative phase with young (16-24 years) sexual violence survivors (n=58), elders (n=8) and healthcare providers (n=10), followed by a quantitative phase among refugee youth (16-24 years; n=120) during which sociodemographic and SRH data were collected. We examined SRH outcome differences by CEFMP using Fisher's exact test. Qualitative data showed that CEFMP was a significant problem facing refugee young women driven by stigma, gender norms and poverty. Among youth refugee survey participants, nearly one-third (31.7%) experienced CEFMP (57.9% women, 42.1% men). Among women in CEFMP compared to those who were not, a significantly higher proportion reported forced pregnancy (50.0% vs. 18.4%, p-value=0.018), forced abortion (45.4% vs. 7.0%, p-value=0.002), and missed school due to sexual violence (94.7% vs. 63.0%, p-value=0.016). This study illustrates the need for innovative community-engaged interventions to end CEFMP in humanitarian contexts in order to achieve sexual and reproductive health and rights for youth.

Local chickens in East African region: their production and potential.

Indigenous chickens (IC) contribute socioeconomically to household food security in the region of East Africa. However, their potential and improvement are not well documented. This review is aimed at exploring the production and potential of indigenous chickens in East Africa. The various tools for literature search such as google search and Google scholars, agricultural journals, animal sciences and health journals, poultry related journals, and country online databases were used to gather information. IC were primarily reared by women and were kept predominantly under scavenging systems where the conditions of management (feeding, housing, and health care) are poor. They presented a high variation in their reproduction and production characteristics. The products (meat and eggs) were of good quality and preferred by the local consumers. Despite the variation and potential of IC, improvements in the village system were constrained by diseases and loss due to Newcastle, Gumboro, and Ecto-endo parasites and predators. Farmers primarily used traditional methods to control the diseases, and some used conventional medications and vaccines. Due to the potential of IC, the exploration of various strategies for improvement supported by the details of their genetic variability and adaptation as well as different management conditions was a goal of this review.

Myeloperoxidase and Other Markers of Neutrophil Activation Associate With Malaria and Malaria/HIV Coinfection in the Human Placenta.

Introduction: Placental malaria (PM) is characterized by accumulation of inflammatory leukocytes in the placenta, leading to poor pregnancy outcomes. Understanding of the underlying mechanisms remains incomplete. Neutrophils respond to malaria parasites by phagocytosis, generation of oxidants, and externalization of Neutrophil Extracellular Traps (NETs). NETs drive inflammation in malaria but evidence of NETosis in PM has not been reported. Neutrophil activity in the placenta has not been directly investigated in the context of PM and PM/HIV-co-infection. Methods: Using peripheral and placental plasma samples and placental tissue collected from Kenyan women at risk for malaria and HIV infections, we assessed granulocyte levels across all gravidities and markers of neutrophil activation, including NET formation, in primi- and secundigravid women, by ELISA, western blot, immunohistochemistry and immunofluorescence. Results: Reduced peripheral blood granulocyte numbers are observed with PM and PM/HIV co-infection in association with increasing parasite density and placental leukocyte hemozoin accumulation. In contrast, placental granulocyte levels are unchanged across infection groups, resulting in enhanced placental: peripheral count ratios with PM. Within individuals, PM- women have reduced granulocyte counts in placental relative to peripheral blood; in contrast, PM stabilizes these relative counts, with HIV coinfection tending to elevate placental counts relative to the periphery. In placental blood, indicators of neutrophil activation, myeloperoxidase (MPO) and proteinase 3 (PRTN3), are significantly elevated with PM and, more profoundly, with PM/HIV co-infection, in association with placental parasite density and hemozoin-bearing leukocyte accumulation. Another neutrophil marker, matrix metalloproteinase (MMP9), together with MPO and PRTN3, is elevated with self-reported fever. None of these factors, including the neutrophil chemoattractant, CXCL8, differs in relation to infant birth weight or gestational age. CXCL8 and MPO levels in the peripheral blood do not differ with infection status nor associate with birth outcomes. Indicators of NETosis in the placental plasma do not vary with infection, and while structures consistent with NETs are observed in placental tissue, the results do not support an association with PM. Conclusions: Granulocyte levels are differentially regulated in the peripheral and placental blood in the presence and absence of PM. PM, both with and without pre-existing HIV infection, enhances neutrophil activation in the placenta. The impact of local neutrophil activation on placental function and maternal and fetal health remains unclear. Additional investigations exploring how neutrophil activation and NETosis participate in the pathogenesis of malaria in pregnant women are needed.

Four-Dimensional Flow MRI of Abdominal Veins: A Systematic Review.

The aim of this systematic review is to provide an overview of the use of Four-Dimensional Magnetic Resonance Imaging of vector blood flow (4D Flow MRI) in the abdominal veins. This study was composed according to the PRISMA guidelines 2009. The literature search was conducted in MEDLINE, Cochrane Library, EMBASE, and Web of Science. Quality assessment of the included studies was performed using the QUADAS-2 tool. The initial search yielded 781 studies and 21 studies were included. All studies successfully applied 4D Flow MRI in abdominal veins. Four-Dimensional Flow MRI was capable of discerning between healthy subjects and patients with cirrhosis and/or portal hypertension. The visual quality and inter-observer agreement of 4D Flow MRI were rated as excellent and good to excellent, respectively, and the studies utilized several different MRI data sampling strategies. By applying spiral sampling with compressed sensing to 4D Flow MRI, the blood flow of several abdominal veins could be imaged simultaneously in 18-25 s, without a significant loss of visual quality. Four-Dimensional Flow MRI might be a useful alternative to Doppler sonography for the diagnosis of cirrhosis and portal hypertension. Further clinical studies need to establish consensus regarding MRI sampling strategies in patients and healthy subjects.

Persistence of SARS-CoV-2 virus and viral RNA on hydrophobic and hydrophilic surfaces and investigating contamination concentration

The transmission of SARS-CoV-2 is likely to occur through a number of routes, including contact with contaminated surfaces. Many studies have used RT-PCR analysis to detect SARS-CoV-2 RNA on surfaces but seldom has viable virus been detected. This paper investigates the viability over time of SARS-CoV-2 dried onto a range of materials and compares viability of the virus to RNA copies recovered, and whether virus viability is concentration dependant. Viable virus persisted for the longest time on surgical mask material and stainless steel with a 99.9% reduction in viability by 124 and 113 hours respectively. Viability of SARS-CoV-2 reduced the fastest on a polyester shirt, with a 99.9% reduction within 2.5 hours. Viability on cotton was reduced second fastest, with 99.9% reduction in 72 hours. RNA on all the surfaces exhibited a one log reduction in genome copy recovery over 21 days. The findings show that SARS-CoV-2 is most stable on non-porous hydrophobic surfaces. RNA is highly stable when dried on surfaces with only one log reduction in recovery over three weeks. In comparison, SARS-CoV-2 viability reduced more rapidly, but this loss in viability was found to be independent of starting concentration. Expected levels of SARS-CoV-2 viable environmental surface contamination would lead to undetectable levels within two days. Therefore, when RNA is detected on surfaces it does not directly indicate presence of viable virus even at high CT values. ImportanceThis study shows the impact of material type on the viability of SARS-CoV-2 on surfaces. It demonstrates that the decay rate of viable SARS-CoV-2 is independent of starting concentration. However, RNA shows high stability on surfaces over extended periods. This has implications for interpretation of surface sampling results using RT-PCR to determine the possibility of viable virus from a surface. Unless sampled immediately after contamination it is difficult to align RNA copy numbers to quantity of viable virus on a surface.

Sugar, a powerful substitute for ethanol in ethanol postdependent rats: Relevance for clinical consideration?

Sugar has been shown to be a powerful substitute for drugs in preclinical studies on addiction. However, the link between sugar intake and alcohol use disorder (AUD) is poorly understood. We assessed the influence of sucrose on ethanol drinking in both nondependent (ND) and dependent (D) Long-Evans rats during acute withdrawal using the postdependent state model. Ethanol (10%-40%) and sucrose (1%-4%) solutions were offered in an operant paradigm either independently or concurrently under ratio schedules of reinforcement. We showed that D rats displayed an enhanced motivation for both 10% ethanol solution (10E) and 4% sucrose solution (4S) as compared with ND rats, and a clear preference for 4S was observed in both groups. During acute withdrawal, D rats showed a strong motivation for 30% ethanol (30E), even when adulterated with quinine, but still preferred 4S despite the fact that a high level of negative reinforcement could be expected. However, when a premix solution (30E4S) was offered concurrently with 4S, the preference for 4S was lost in D animals, which consumed as much premix as 4S, whereas ND animals displayed preference for 4S. Altogether, those results suggest that reinforcing properties of sucrose surpass those of ethanol in D rats under acute withdrawal, which indicates that sugar is a powerful substitute for ethanol. Our results suggest that craving for sugar may be increased in AUD patients during withdrawal and raise the issue of dependence transfer from alcohol to sugar.

Identification of additives in polymers from single-use bioprocessing bags by accelerated solvent extraction and ultra-high performance liquid chromatography coupled with high-resolution mass spectrometry.

Single-use technologies are increasingly used in biopharmaceutical manufacturing. Despite their advantages, these plastic assemblies draw concern because they are a potential source of contamination due to extractable and leachable compounds (E&Ls). Characterising E&Ls from such materials is a necessary step in establishing their suitability for use. Therefore, there is an urgent need for sensitive methods to identify and quantitatively assess compounds in plastic materials. Accelerated solvent extraction (ASE) is a powerful technique that can be reliably used for this purpose. In this study, ASE followed by liquid chromatography and Orbitrap-based High Resolution Accurate Mass (HRAM) mass analysis was found to be an efficient and versatile method for the determination of additives in different multilayer polymer systems from single-use bags. ASE optimisation was performed using a design of experiments approach. The type of solvent, temperature, swelling agent addition, static time and number of cycles were the selected variables. Optimum conditions were dependent on the type of plastic film. Ethyl acetate and cyclohexane were selected individually as optimum solvents. Optimum temperatures were 90-100 °C. Pressure was set at 1500 psi and extraction time was 30 min in 2 cycles. Swelling agent addition was necessary with polar extraction solvents. More than 100 additives and degradation products were confidently identified by HRAM MS. Correlations between the type and levels of identified additives and the type of polymer system were established. In addition, degradation behaviour and pathways for some additives can be addressed.

A distal regulatory region of a class I human histone deacetylase.

Histone deacetylases (HDACs) are key enzymes in epigenetics and important drug targets in cancer biology. Whilst it has been established that HDACs regulate many cellular processes, far less is known about the regulation of these enzymes themselves. Here, we show that HDAC8 is allosterically regulated by shifts in populations between exchanging states. An inactive state is identified, which is stabilised by a range of mutations and resembles a sparsely-populated state in equilibrium with active HDAC8. Computational models show that the inactive and active states differ by small changes in a regulatory region that extends up to 28 Å from the active site. The regulatory allosteric region identified here in HDAC8 corresponds to regions in other class I HDACs known to bind regulators, thus suggesting a general mechanism. The presented results pave the way for the development of allosteric HDAC inhibitors and regulators to improve the therapy for several disease states.