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(R,S)-ketamine (ketamine) has rapid and sustained antidepressant (AD) efficacy at sub-anesthetic doses in depressed patients. A metabolite of ketamine, including (2R,6R)-hydroxynorketamine ((6)-HNKs) has been reported to exert antidepressant actions in rodent model of anxiety/depression. To further understand the specific role of ketamine's metabolism in the AD actions of the drug, we evaluated the effects of inhibiting hepatic cytochrome P450 enzymes on AD responses. We assessed whether pre-treatment with fluconazole (10 and 20 mg/kg, i. p.) 1 h prior to ketamine or HNKs (10 mg/kg, i. p.) administration would alter behavioral and neurochemical actions of the drugs in male BALB/cJ mice with a highly anxious phenotype. Extracellular microdialysate levels of glutamate and GABA (Glu ext , GABA ext ) were also measured in the medial prefrontal cortex (mPFC). Pre-treatment with fluconazole altered the pharmacokinetic profile of ketamine, by increasing both plasma and brain levels of ketamine and (R,S)-norketamine, while robustly reducing those of (6)-HNKs. At 24 h post-injection (t24 h), fluconazole prevented the sustained AD-like response of ketamine responses in the forced swim test and splash test, as well as the enhanced cortical GABA levels produced by ketamine. A single (2R,6R)-HNK administration resulted in prevention of the effects of fluconazole on the antidepressant-like activity of ketamine in mice. Overall, these findings are consistent with an essential contribution of (6)-HNK to the sustained antidepressant-like effects of ketamine and suggest potential interactions between pharmacological CYPIs and ketamine during antidepressant treatment in patients., Competing Interests: Declaration of competing interest DJD serves as a consultant for Lundbeck, Roche, and Servier. AMG serves as a consultant for Lundbeck and Servier. JM, IM-D, DJD, AMG, and CAD are named on provisional patent applications for the prophylactic use of ketamine and other compounds against stress-related psychiatric disorders. PZ, JH, RM, and TDG are co-inventors in patents and/or patent applications related to the pharmacology and synthesis, crystal structure and use of HNKs and related molecules for the treatment of addiction, depression, anxiety, anhedonia, suicidal ideation and post-traumatic stress disorders., (Copyright © 2024. Published by Elsevier Ltd.)

Extracellular vesicle (EV) cargo is known to change in response to stimuli such as muscle damage. This study aimed to assess particle size, concentration and microRNA (miR) content within small EV-enriched separations prepared from human blood taken before and after unaccustomed eccentric-biased exercise-induced muscle damage. Nine male volunteers underwent plyometric jumping and downhill running, with blood samples taken at baseline, 2, and 24 h post-exercise. EVs were separated using size exclusion chromatography (SEC) and their characteristics evaluated by nanoparticle tracking. No changes in EV size or concentration were seen following the muscle-damaging exercise. Small RNA sequencing identified 240 miRs to be consistently present within the EVs. RT-qPCR analysis was performed: specifically, for known muscle-enriched/important miRs, including miR-1, -206, -133a, -133b, -31, -208b, -451a, -486 and - 499 and the immune-important miR-21, -146a and - 155. Notably, none of the immune-important miRs within the EVs tested changed in response to the muscle damage. Of the muscle-associated miRs tested, only the levels of miR-31-5p were seen to change with decreased levels at 24 h compared to baseline and 2 h, indicating involvement in the damage response. These findings shed light on the dynamic role of EV miRs in response to exercise-induced muscle damage., (© 2024 The Author(s). Physiological Reports published by Wiley Periodicals LLC on behalf of The Physiological Society and the American Physiological Society.)

Introduction: Burkitt lymphoma (BL) is an aggressive non-Hodgkin lymphoma associated with Plasmodium falciparum and Epstein-Barr virus, both of which affect metabolic pathways. The metabolomic patterns of BL is unknown., Materials and Methods: We measured 627 metabolites in pre-chemotherapy treatment plasma samples from 25 male children (6-11 years) with BL and 25 cancer-free area- and age-frequency-matched male controls from the Epidemiology of Burkitt Lymphoma in East African Children and Minors study in Uganda using liquid chromatography-tandem mass spectrometry. Unconditional, age-adjusted logistic regression analysis was used to estimate odds ratios (ORs) and their 95% confidence intervals (CIs) for the BL association with 1-standard deviation increase in the log-metabolite concentration, adjusting for multiple comparisons using false discovery rate (FDR) thresholds and Bonferroni correction., Results: Compared to controls, levels for 42 metabolite concentrations differed in BL cases (FDR < 0.001), including triacylglyceride (18:0&#95;38:6), alpha-aminobutyric acid (AABA), ceramide (d18:1/20:0), phosphatidylcholine ae C40:6 and phosphatidylcholine C38:6 as the top signals associated with BL (ORs = 6.9 to 14.7, P < 2.4✕10 - 4 ). Two metabolites (triacylglyceride (18:0&#95;38:6) and AABA) selected using stepwise logistic regression discriminated BL cases from controls with an area under the curve of 0.97 (95% CI: 0.94, 1.00)., Conclusion: Our findings warrant further examination of plasma metabolites as potential biomarkers for BL risk/diagnosis., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Accumulating evidence shows a strong correlation between type 2 diabetes mellitus, mitochondrial dysfunction, and oxidative stress. We evaluated the effects of dietary peanut shell extract (PSE) supplementation on mitochondrial function and antioxidative stress/inflammation markers in diabetic mice. Fourteen db/db mice were randomly assigned to a diabetic group (DM in AIN-93G diet) and a PSE group (1% wt/wt PSE in AIN-93G diet) for 5 weeks. Six C57BL/6J mice were fed with an AIN-93G diet for 5 weeks (control group). Gene and protein expression in the liver, brain, and white adipose tissue (WAT) were determined using qRT-PCR and Immunoblot, respectively. Compared to the control group, the DM group had (i) increased gene and protein expression levels of DRP1 (fission), PINK1 (mitophagy), and TNFα (inflammation) and (ii) decreased gene and protein expression levels of MFN1, MFN2, OPA1 (fusion), TFAM, PGC-1α (biogenesis), NRF2 (antioxidative stress) and IBA1 (microglial activation) in the liver, brain, and WAT of db/db mice. Supplementation of PSE into the diet restored the DM-induced changes in the gene and protein expression of DRP1, PINK1, TNFα, MFN1, MFN2, OPA1, TFAM, PGC-1α, NRF2, and IBA1 in the liver, brain, and WAT of db/db mice. This study demonstrates that PSE supplementation improved mitochondrial function in the brain, liver, and WAT of db/db mice, in part due to suppression of oxidative stress and inflammation.

Emerging evidence shows that the gut microbiota plays an important role in neuropathic pain (NP) via the gut-brain axis. Male rats were divided into sham, spinal nerve ligation (SNL), SNL + 200 mg GEG/kg BW (GEG200), and SNL + 600 mg GEG/kg BW (GEG600) for 5 weeks. The dosages of 200 and 600 mg GEG/kg BW for rats correspond to 45 g and 135 g raw ginger for human daily consumption, respectively. Both GEG groups mitigated SNL-induced NP behavior. GEG-supplemented animals had a decreased abundance of Rikenella , Muribaculaceae , Clostridia UCG-014 , Mucispirillum schaedleri , RF39 , Acetatifactor , and Clostridia UCG-009 , while they had an increased abundance of Flavonifactor , Hungatella , Anaerofustis stercorihominis , and Clostridium innocuum group. Relative to sham rats, Fos and Gadd45g genes were upregulated, while Igf1 , Ccl2 , Hadc2 , Rtn4rl1 , Nfkb2 , Gpr84 , Pik3cg , and Abcc8 genes were downregulated in SNL rats. Compared to the SNL group, the GEG200 group and GEG600 group had increases/decreases in 16 (10/6) genes and 11 (1/10) genes, respectively. GEG downregulated Fos and Gadd45g genes and upregulated Hdac2 genes in the amygdala. In summary, GEG alleviates NP by modulating the gut microbiome and reversing a molecular neuroimmune signature.

The study of age-related biomarkers from different biofluids and tissues within the same individual might provide a more comprehensive understanding of age-related changes within and between compartments as these changes are likely highly interconnected. Understanding age-related differences by compartments may shed light on the mechanism of their reciprocal interactions, which may contribute to the phenotypic manifestations of aging. To study such possible interactions, we carried out a targeted metabolomic analysis of plasma, skeletal muscle, and urine collected from healthy participants, age 22-92 years, and identified 92, 34, and 35 age-associated metabolites, respectively. The metabolic pathways that were identified across compartments included inflammation and cellular senescence, microbial metabolism, mitochondrial health, sphingolipid metabolism, lysosomal membrane permeabilization, vascular aging, and kidney function., (Published 2023. This article is a U.S. Government work and is in the public domain in the USA. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Stroke is a common neurological emergency and although most cases are associated with traditional vascular risk factors leading to cerebral ischaemia by well-recognised pathophysiological mechanisms, around 4% of ischaemic strokes are due to rare conditions. These are important to recognise due to their different management, which is often specific and effective, and due to their different prognosis from otherwise cryptogenic ischaemic strokes. We outline a practical approach to identifying uncommon causes of ischaemic stroke by highlighting diagnostic 'red flags' and propose a structured approach to investigating them., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Declining nicotinamide adenine dinucleotide (NAD + ) concentration in the brain during aging contributes to metabolic and cellular dysfunction and is implicated in the pathogenesis of aging-associated neurological disorders. Experimental therapies aimed at boosting brain NAD + levels normalize several neurodegenerative phenotypes in animal models, motivating their clinical translation. Dietary intake of NAD + precursors, such as nicotinamide riboside (NR), is a safe and effective avenue for augmenting NAD + levels in peripheral tissues in humans, yet evidence supporting their ability to raise NAD + levels in the brain or engage neurodegenerative disease pathways is lacking. Here, we studied biomarkers in plasma extracellular vesicles enriched for neuronal origin (NEVs) from 22 healthy older adults who participated in a randomized, placebo-controlled crossover trial (NCT02921659) of oral NR supplementation (500 mg, 2x /day, 6 weeks). We demonstrate that oral NR supplementation increases NAD + levels in NEVs and decreases NEV levels of Aβ42, pJNK, and pERK1/2 (kinases involved in insulin resistance and neuroinflammatory pathways). In addition, changes in NAD(H) correlated with changes in canonical insulin-Akt signaling proteins and changes in pERK1/2 and pJNK. These findings support the ability of orally administered NR to augment neuronal NAD + levels and modify biomarkers related to neurodegenerative pathology in humans. Furthermore, NEVs offer a new blood-based window into monitoring the physiologic response of NR in the brain., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

We show that the sex of human experimenters affects mouse behaviors and responses following administration of the rapid-acting antidepressant ketamine and its bioactive metabolite (2R,6R)-hydroxynorketamine. Mice showed aversion to the scent of male experimenters, preference for the scent of female experimenters and increased stress susceptibility when handled by male experimenters. This human-male-scent-induced aversion and stress susceptibility was mediated by the activation of corticotropin-releasing factor (CRF) neurons in the entorhinal cortex that project to hippocampal area CA1. Exposure to the scent of male experimenters before ketamine administration activated CA1-projecting entorhinal cortex CRF neurons, and activation of this CRF pathway modulated in vivo and in vitro antidepressant-like effects of ketamine. A better understanding of the specific and quantitative contributions of the sex of human experimenters to study outcomes in rodents may improve replicability between studies and, as we have shown, reveal biological and pharmacological mechanisms., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Purpose: To evaluate the clinical and radiographic outcomes and complications following surgical treatment of adult posterior Monteggia fracture variants in which coexisting radial head and coronoid fractures were addressed through a single extensile posterior trans-olecranon approach., Methods: We performed a retrospective review from January 1, 2010, to January 1, 2021, of all adult patients who underwent fixation of posterior Monteggia variant fractures with coexisting radial head and/or coronoid fractures through a trans-olecranon approach by a single surgeon. Patient records were reviewed for postoperative complications and radiographic outcomes. All patients were contacted to participate in a repeat clinical examination and functional assessment., Results: Thirteen patients met the inclusion criteria, of which 7 agreed to participate. Final follow-up was conducted at an average of 49 months postoperatively. Six patients had a coexisting radial head fracture, 1 had a radial neck fracture, and all 7 patients had a coronoid fracture. All 7 coronoid fractures were repaired, 2 patients had internal fixation of the radial head, and 5 had radial head replacement. Four patients underwent reoperation. There were no cases of infection or heterotopic ossification, and all patients achieved radiographic bony union. The average final range of motion was 13°-133° flexion-extension (range: 0°-30° extension to 125°-145° flexion), 76° pronation (range: 70°-90°) and 72° supination (range: 60°-90°). The average Mayo Elbow Performance Score was 86 (range: 70-100), average Visual Analog Scale score was 1.4 (range: 0-3), and average Disabilities of the Arm, Shoulder, and Hand score was 9.8 (range: 0.83-35.83)., Conclusions: Use of a single extensile trans-olecranon exposure offers reliable visualization and access to all bony structures requiring repair while minimizing soft tissue disruption. Patients treated with this trans-olecranon surgical approach demonstrate satisfactory clinical and radiographic outcomes with low rates of heterotopic ossification, nonunion, and infection., Level of Evidence: Therapeutic IV., (© 2022 The Authors.)

Background: Adult Monteggia fracture variants are rare but potentially devastating injuries to the elbow generally requiring surgical treatment. Monteggia injuries with co- existing fractures of the radial head and coronoid present a surgical challenge due to the need to adequately expose all components of the injury for internal fixation or arthroplasty. In this study, we evaluated the clinical outcomes following surgical treatment of adult Monteggia fracture variants in which a coexisting radial head or coronoid fracture was ad- dressed through a single extensile posterior trans-olecranon approach., Methods: A retrospective review of all adult patients who underwent fixation of Monteggia variant fractures with coexisting radial head and coronoid fractures through a trans-olecranon approach by a single surgeon from January 1, 2012, to January 1, 2021, was performed. Patient records were reviewed for demographic information, radiographic outcomes, and postoperative complications leading to re- operation., Results: A total of 13 patients met the inclusion criteria for the study, three males and 10 females. The average age of the patients was 64 years old, and patients were followed for an average of 9.9 months postoperatively. Six patients underwent re-operation: one for flap coverage due to wound necrosis, two for revision fixation due to early loss of fixation, two for removal of painful hardware, and one for arthroscopic capsulectomy to treat a flexion contracture. Twelve patients achieved radiographic union and one patient had not achieved union by 1.3 months postoperatively and thereafter did not undergo further radiographic follow-up. No cases of heterotopic ossification, capitellar erosions, overstuffing of a radial head implant, or residual elbow subluxation were seen. There were two cases of lucency around a prosthetic radial head implant stem but without migration of the implant.

Subanesthetic-dose racemic (R,S)-ketamine (ketamine) produces rapid, robust, and sustained antidepressant effects in major depressive disorder (MDD) and bipolar disorder (BD) and has also been shown to effectively treat neuropathic pain, complex regional pain syndrome, and post-traumatic stress disorder (PTSD). However, to date, its mechanism of action remains unclear. Preclinical studies found that (2 R,6 R;2 S,6 S)-hydroxynorketamine (HNK), a major circulating metabolite of ketamine, elicits antidepressant effects similar to those of ketamine. To help determine how (2 R,6 R)-HNK contributes to ketamine's mechanism of action, an exploratory, targeted, metabolomic analysis was carried out on plasma and CSF of nine healthy volunteers receiving a 40-minute ketamine infusion (0.5 mg/kg). A parallel targeted metabolomic analysis in plasma, hippocampus, and hypothalamus was carried out in mice receiving either 10 mg/kg of ketamine, 10 mg/kg of (2 R,6 R)-HNK, or saline. Ketamine and (2 R,6 R)-HNK both affected multiple pathways associated with inflammatory conditions. In addition, several changes were unique to either the healthy human volunteers and/or the mouse arm of the study, indicating that different pathways may be differentially involved in ketamine's effects in mice and humans. Mechanisms of action found to consistently underlie the effects of ketamine and/or (2 R,6 R)-HNK across both the human metabolome in plasma and CSF and the mouse arm of the study included LAT1, IDO1, NAD + , the nitric oxide (NO) signaling pathway, and sphingolipid rheostat., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background: Ketamine is rapidly metabolized to norketamine and hydroxynorketamine (HNK) metabolites. In female mice, when compared to males, higher levels of ( 2R,6R;2S,6S )-HNK have been observed following ketamine treatment, and higher levels of ( 2R,6R )-HNK following the direct administration of ( 2R,6R )-HNK., Aim: The objective of this study was to evaluate the impact of sex in humans and mice, and gonadal hormones in mice on the metabolism of ketamine to form norketamine and HNKs and in the metabolism/elimination of ( 2R,6R )-HNK., Methods: In CD-1 mice, we utilized gonadectomy to evaluate the role of circulating gonadal hormones in mediating sex-dependent differences in ketamine and ( 2R,6R )-HNK metabolism. In humans (34 with treatment-resistant depression and 23 healthy controls) receiving an antidepressant dose of ketamine (0.5 mg/kg i.v. infusion over 40 min), we evaluated plasma levels of ketamine, norketamine, and HNKs., Results: In humans, plasma levels of ketamine and norketamine were higher in males than females, while ( 2R,6R;2S,6S )-HNK levels were not different. Following ketamine administration to mice (10 mg/kg i.p.), C max and total plasma concentrations of ketamine and norketamine were higher, and those of ( 2R,6R;2S,6S )-HNK were lower, in intact males compared to females. Direct ( 2R,6R )-HNK administration (10 mg/kg i.p.) resulted in higher levels of ( 2R,6R )-HNK in female mice. Ovariectomy did not alter ketamine metabolism in female mice, whereas orchidectomy recapitulated female pharmacokinetic differences in male mice, which was reversed with testosterone replacement., Conclusion: Sex is an important biological variable that influences the metabolism of ketamine and the HNKs, which may contribute to sex differences in therapeutic antidepressant efficacy or side effects.

Polyhydroxy compounds are secondary metabolites that are ubiquitous in plants of higher genera. They possess therapeutic properties against a wide spectrum of diseases, including cancers, neurodegenerative disorders, atherosclerosis, as well as cardiovascular disease. The phytochemical flavonol (a type of flavonoid) kaempferol (KMP) (3,5,7-trihydroxy-2-(4-hydroxyphenyl)- 4Hchromen-4-one) is abundant in cruciferous vegetables, including broccoli, kale, spinach, and watercress, as well as in herbs like dill, chives, and tarragon. KMP is predominantly hydrophobic in nature due to its diphenylpropane structure (a characteristic feature of flavonoids). Recent findings have indicated the promise of applying KMP in disease prevention due to its potential antioxidant, antimutagenic, antifungal, and antiviral activities. In the literature, there is evidence that KMP exerts its anticancer effects by modulating critical elements in cellular signal transduction pathways linked to apoptosis, inflammation, angiogenesis, and metastasis in cancer cells without affecting the viability of normal cells. It has been shown that KMP triggers cancer cell death by several mechanisms, including cell cycle arrest, caspase activation, metabolic alteration, and impacting human telomerase reverse-transcriptase gene expression. This review is aimed at providing critical insights into the influence of KMP on the intracellular cascades that regulate metabolism and signaling in breast, ovarian, and cervical cancer cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Modern biomarker and translational research as well as personalized health care studies rely heavily on powerful omics' technologies, including metabolomics and lipidomics. However, to translate metabolomics and lipidomics discoveries into a high-throughput clinical setting, standardization is of utmost importance. Here, we compared and benchmarked a quantitative lipidomics platform. The employed Lipidyzer platform is based on lipid class separation by means of differential mobility spectrometry with subsequent multiple reaction monitoring. Quantitation is achieved by the use of 54 deuterated internal standards and an automated informatics approach. We investigated the platform performance across nine laboratories using NIST SRM 1950-Metabolites in Frozen Human Plasma, and three NIST Candidate Reference Materials 8231-Frozen Human Plasma Suite for Metabolomics (high triglyceride, diabetic, and African-American plasma). In addition, we comparatively analyzed 59 plasma samples from individuals with familial hypercholesterolemia from a clinical cohort study. We provide evidence that the more practical methyl-tert-butyl ether extraction outperforms the classic Bligh and Dyer approach and compare our results with two previously published ring trials. In summary, we present standardized lipidomics protocols, allowing for the highly reproducible analysis of several hundred human plasma lipids, and present detailed molecular information for potentially disease relevant and ethnicity-related materials.

Aptamer-based proteomics revealed differentially abundant proteins in Alzheimer’s disease (AD) brains in the Baltimore Longitudinal Study of Aging and Religious Orders Study (mean age, 89 ± 9 years). A subset of these proteins was also differentially abundant in the brains of young APOE ε4 carriers relative to noncarriers (mean age, 39 ± 6 years). Several of these proteins represent targets of approved and experimental drugs for other indications and were validated using orthogonal methods in independent human brain tissue samples as well as in transgenic AD models. Using cell culture–based phenotypic assays, we showed that drugs targeting the cytokine transducer STAT3 and the Src family tyrosine kinases, YES1 and FYN, rescued molecular phenotypes relevant to AD pathogenesis. Our findings may accelerate the development of effective interventions targeting the earliest molecular triggers of AD.

Patients with refractory chronic migraine have substantial disability and have failed many acute and preventive medications. When aggressive intravenous therapy is indicated, both lidocaine and (R,S)-ketamine infusions have been used successfully to provide relief. Retrospective studies have shown that both agents may be associated with short-term analgesia. In this prospective, observational pilot study of 6 patients, we compared the effects of lidocaine and (R,S)-ketamine infusions and performed metabolite analyses of (R,S)-ketamine to determine its metabolic profile in this population. One of (R,S)-ketamine's metabolites, (2R,6R)-hydroxynorketamine, has been shown in animal studies to reduce pain, but human studies in patients undergoing continuous (R,S)-ketamine infusions for migraine are lacking. All 6 patients tolerated both infusions well with mild adverse effects. The baseline mean pain rating (0-10 numeric rating scale) decreased from 7.5 ± 2.2 to 4.7 ± 2.8 by end of lidocaine treatment ( P ≤ . 05 ) but increased to 7.0 ± 1.4 by the postdischarge visit at 4 weeks (P > .05 vs baseline). The baseline mean pain rating prior to ketamine treatment was 7.4 ± 1.4, which decreased to 3.7 ± 2.3 by the end of the hospitalization ( P ≤ . 05 ) but increased to 7.2 ± 1.7 by the postdischarge visit at 6 weeks (P > .05 vs baseline). For the primary outcome the change in pain from baseline to end of treatment was greater for ketamine than lidocaine (-3.7 vs -2.8; P ≤ . 05 ), but this has minimal clinical significance. Ketamine metabolite analysis revealed that (2R,6R)-hydroxynorketamine was the predominant metabolite during most of the infusion, consistent with previous studies., (© 2021, The American College of Clinical Pharmacology.)

The photophysical and photochemical properties of two Pt(IV)Re(I) conjugates were studied via both experimental and computational methods. Both conjugates exhibit modest photocytotoxicity against ovarian cancer cells. X-ray fluorescence microscopy showed that Pt and Re colocalize in cells whether they had been irradiated or not. This work demonstrates the potential of photoactivated multilimetallic agents for combating cancer.

Tropical forests store 40-50 per cent of terrestrial vegetation carbon 1 . However, spatial variations in aboveground live tree biomass carbon (AGC) stocks remain poorly understood, in particular in tropical montane forests 2 . Owing to climatic and soil changes with increasing elevation 3 , AGC stocks are lower in tropical montane forests compared with lowland forests 2 . Here we assemble and analyse a dataset of structurally intact old-growth forests (AfriMont) spanning 44 montane sites in 12 African countries. We find that montane sites in the AfriMont plot network have a mean AGC stock of 149.4 megagrams of carbon per hectare (95% confidence interval 137.1-164.2), which is comparable to lowland forests in the African Tropical Rainforest Observation Network 4 and about 70 per cent and 32 per cent higher than averages from plot networks in montane 2,5,6 and lowland 7 forests in the Neotropics, respectively. Notably, our results are two-thirds higher than the Intergovernmental Panel on Climate Change default values for these forests in Africa 8 . We find that the low stem density and high abundance of large trees of African lowland forests 4 is mirrored in the montane forests sampled. This carbon store is endangered: we estimate that 0.8 million hectares of old-growth African montane forest have been lost since 2000. We provide country-specific montane forest AGC stock estimates modelled from our plot network to help to guide forest conservation and reforestation interventions. Our findings highlight the need for conserving these biodiverse 9,10 and carbon-rich ecosystems., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Due to the abundance of lipoproteins in blood, it is challenging to characterize the biological functions and components of blood-derived extracellular vesicles. The aim of this study was to develop a multiple-step purification protocol to separate serum exosomes from serum proteins and lipoproteins and assess their regenerative potential. Exosomes were isolated by concentrating them in human serum using ultracentrifugation (UC), followed sequentially by density gradient (DG) UC and size exclusion chromatography (SEC). Purity and characterization were assessed by western blots, Lipoprint®, enzyme-linked immunosorbent assay, electron microscopy, mass spectrometry, and nanoparticle tracking analysis. Functionality was assessed by cell proliferation analysis and with an in vivo subcutaneous angiogenesis model. SEC alone isolated nano-sized vesicles possessing vesicle markers TSG101 and CD9, but there was a substantial presence of apolipoprotein B, predominantly derived from very-low- and intermediate-density lipoprotein particles. This was reduced to an undetectable level using the combined UC DG SEC approach. Mass spectrometry identified 224 proteins in UC DG SEC isolates relative to the 135 from SEC, with considerable increases in exosome-related proteins and reductions in lipoproteins. A consistent but limited increase in human dermal fibroblast proliferation and evidence of neovascularization enhancement were observed after exposure to UC DG SEC exosomes. An UC DG SEC purification protocol considerably improved the removal of lipoproteins during isolation of serum exosomes. The purified exosomes stimulated cell proliferation and potentially increased an in vivo angiogenic response. This multistep purification allows for more accurate identification of serum exosome functional activity and composition., (© 2020 John Wiley & Sons Ltd.)

Huntington's Disease (HD) is a progressive, fatal neurodegenerative condition. While generally considered for its devastating neurological phenotype, disturbances in other organ systems and metabolic pathways outside the brain have attracted attention for possible relevance to HD pathology, potential as therapeutic targets, or use as biomarkers of progression. In addition, it is not established how metabolic changes in the HD brain correlate to progression across the full spectrum of early to late-stage disease. In this pilot study, we sought to explore the metabolic profile across manifest HD from early to advanced clinical staging through metabolomic analysis by mass spectrometry in plasma and cerebrospinal fluid (CSF). With disease progression, we observed nominally significant increases in plasma arginine, citrulline, and glycine, with decreases in total and D-serine, cholesterol esters, diacylglycerides, triacylglycerides, phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins. In CSF, worsening disease was associated with nominally significant increases in NAD + , arginine, saturated long chain free fatty acids, diacylglycerides, triacylglycerides, and sphingomyelins. Notably, diacylglycerides and triacylglyceride species associated with clinical progression were different between plasma and CSF, suggesting different metabolic preferences for these compartments. Increasing NAD + levels strongly correlating with disease progression was an unexpected finding. Our data suggest that defects in the urea cycle, glycine, and serine metabolism may be underrecognized in the progression HD pathology, and merit further study for possible therapeutic relevance.

Chronic inflammation is associated with physical frailty and functional decline in older adults; however, the molecular mechanisms of this linkage are not understood. A mouse model of chronic inflammation showed reduced motor function and partial denervation at the neuromuscular junction. Metabolomic profiling of these mice and further validation in frail human subjects showed significant dysregulation in the tryptophan degradation pathway, including decreased tryptophan and serotonin, and increased levels of some neurotoxic kynurenines. In humans, kynurenine strongly correlated with age, frailty status, TNF-αR1 and IL-6, weaker grip strength, and slower walking speed. To study the effects of elevated neurotoxic kynurenines on motor neuronal cell viability and axonal degeneration, we used motor neuronal cells treated with 3-hydroxykynurenine and quinolinic acid and observed neurite degeneration in a dose-dependent manner and potentiation of toxicity between 3-hydroxykynurenine and quinolinic acid. These results suggest that kynurenines mediate neuromuscular dysfunction associated with chronic inflammation and aging.

A single, subanesthetic dose of (R,S)-ketamine (ketamine) exerts rapid and robust antidepressant effects. Several groups previously reported that (2S,6S;2R,6R)-hydroxynorketamine (HNK) had antidepressant effects in rodents, and that (2R,6R)-HNK increased cortical electroencephalographic gamma power. This exploratory study examined the relationship between ketamine metabolites, clinical response, psychotomimetic symptoms, and gamma power changes in 34 individuals (ages 18-65) with treatment-resistant depression (TRD) who received a single ketamine infusion (0.5 mg/kg) over 40 min. Plasma concentrations of ketamine, norketamine, and HNKs were measured at 40, 80, 120, and 230 min and at 1, 2, and 3 days post-infusion. Linear mixed models evaluated ketamine metabolites as mediators of antidepressant and psychotomimetic effects and their relationship to resting-state whole-brain magnetoencephalography (MEG) gamma power 6-9 h post-infusion. Three salient findings emerged. First, ketamine concentration positively predicted distal antidepressant response at Day 11 post-infusion, and an inverse relationship was observed between (2S,6S;2R,6R)-HNK concentration and antidepressant response at 3 and 7 days post-infusion. Norketamine concentration was not associated with antidepressant response. Second, ketamine, norketamine, and (2S,6S;2R,6R)-HNK concentrations at 40 min were positively associated with contemporaneous psychotomimetic symptoms; post-hoc analysis revealed that ketamine was the predominant contributor. Third, increased (2S,6S;2R,6R)-HNK maximum observed concentration (C max ) was associated with increased MEG gamma power. While contrary to preclinical observations and our a priori hypotheses, these exploratory results replicate those of a recently published study documenting a relationship between higher (2S,6S;2R,6R)-HNK concentrations and weaker antidepressant response in humans and provide further rationale for studying gamma power changes as potential biomarkers of antidepressant response.

Hypoplastic left heart syndrome (HLHS) is a clinically and anatomically severe form of congenital heart disease; however, its etiology remains largely unknown. We previously demonstrated that genetic variants in the MYH6 gene are significantly associated with HLHS. Additionally, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from an HLHS-affected family trio (affected parent, unaffected parent, affected proband) carrying an MYH 6-R443P head domain variant demonstrated dysmorphic sarcomere structure and increased compensatory MYH7 expression. Analysis of iPSC-CMs derived from the HLHS trio revealed that only beta myosin heavy chain expression was observed in CMs carrying the MYH6 -R443P variant after differentiation day 15 (D15). Functional assessments performed between D20-D23 revealed that MYH6 -R443P variant CMs contracted more slowly (40 ± 2 vs. 47 ± 2 contractions/min, P < 0.05), shortened less (5.6 ± 0.5 vs. 8.1 ± 0.7% of cell length, P < 0.05), and exhibited slower shortening rates (19.9 ± 1.7 vs. 28.1 ± 2.5 μm/s, P < 0.05) and relaxation rates (11.0 ± 0.9 vs. 19.7 ± 2.0 μm/s, P < 0.05). Treatment with isoproterenol had no effect on iPSC-CM mechanics. Using CRISPR/Cas9 gene editing technology, introduction of the R443P variant into the unaffected parent's iPSCs recapitulated the phenotype of the proband's iPSC-CMs, and conversely, correction of the R443P variant in the proband's iPSCs rescued the cardiomyogenic differentiation, sarcomere organization, slower contraction ( P < 0.05) and decreased velocity phenotypes ( P < 0.0001). This is the first report to identify that cardiac tissues from HLHS patients with MYH6 variants can exhibit sarcomere disorganization in atrial but not ventricular tissues. This new discovery was not unexpected, since MYH6 is expressed predominantly in the postnatal atria in humans. These findings demonstrate the feasibility of employing patient-derived iPSC-CMs, in combination with patient cardiac tissues, to gain mechanistic insight into how genetic variants can lead to HLHS. Results from this study suggest that decreased contractility of CMs due to sarcomere disorganization in the atria may effect hemodynamic changes preventing development of a normal left ventricle., (Copyright © 2020 Kim, Fleres, Lovett, Anfinson, Samudrala, Kelly, Teigen, Cavanaugh, Marquez, Geurts, Lough, Mitchell, Fitts and Tomita-Mitchell.)

The complex fac-[Re(CO)3(dmphen)(para-tolylisonitrile)]+ (TRIP), where dmphen = 2,9-dimethyl-1,10-phenanthroline, is an endoplasmic reticulum stress-inducing anticancer agent (A. P. King, S. C. Marker, R. V. Swanda, J. J. Woods, S.-B. Qian and J. J. Wilson, Chem. - Eur. J., 2019, 25, 9206-9210). A second-generation compound fac-[Re(CO)3(dmphen)(para-iodobenzeneisonitrile)]+ (I-TRIP) was synthesized, and its intracellular distribution was investigated using X-ray fluorescence microscopy to show that these complexes are highly stable in vitro.

The mitochondrial calcium uniporter (MCU) is the ion channel that mediates Ca 2+ uptake in mitochondria. Inhibitors of the MCU are valuable as potential therapeutic agents and tools to study mitochondrial Ca 2+ . The best-known inhibitor of the MCU is the ruthenium compound Ru360. Although this compound is effective in permeabilized cells, it does not work in intact biological systems. We have recently reported the synthesis and characterization of Ru265, a complex that selectively inhibits the MCU in intact cells. Here, the physical and biological properties of Ru265 and Ru360 are described in detail. Using atomic absorption spectroscopy and X-ray fluorescence imaging, we show that Ru265 is transported by organic cation transporter 3 (OCT3) and taken up more effectively than Ru360. As an explanation for the poor cell uptake of Ru360, we show that Ru360 is deactivated by biological reductants. These data highlight how structural modifications in metal complexes can have profound effects on their biological activities., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Aims and Objectives: To evaluate online information on dysmenorrhoea, including readability, credibility, quality and usability., Background: Menstrual pain impacts 45%-95% of women of reproductive age globally and is the leading cause of school and work absences among women. Women often seek online information on dysmenorrhoea; however, little is known about the information quality., Design: This was a descriptive study to evaluate online information on dysmenorrhoea., Methods: We imitated search strategies of the general public. Specifically, we employed the three most popular search engines worldwide-Google, Yahoo and Bing-and used lay search terms, "period pain" and "menstrual cramps." We screened 60 web pages. Following removal of duplicates and irrelevant web pages, 25 met the eligibility criteria. Two team members independently evaluated the included web pages using standardised tools. Readability was evaluated with the Flesch-Kincaid Reading Ease and Flesch-Kincaid Grade formulas; credibility, quality and usability were evaluated with established tools. We followed the STROBE checklist for reporting this study., Results: For readability, the mean Flesch-Kincaid level was 10th grade. For credibility, 8% of web pages referenced scientific literature and 28% stated the author's name and qualifications. For quality, no web page employed user-driven content production; 8% of web pages referenced evidence-based guidelines, 32% of web pages had accurate content, and 4% of web pages recommended shared decision-making. Most web pages were interactive and included nontextual information. Some nontextual information was inaccurate., Conclusion: Online information on dysmenorrhoea has generally low readability, mixed credibility and variable quality., Relevance to Clinical Practice: Strategies to improve health information on dysmenorrhoea include avoiding complex terms, incorporating visual aids, presenting evidence-based information and developing a decision aid to support shared decision-making. Healthcare providers should be aware of the problematic health information that individuals are exposed to and provide education about how to navigate online health information., (© 2019 John Wiley & Sons Ltd.)

Background and Purpose: (R)-Ketamine (arketamine) may have utility as a rapidly acting antidepressant. While (R)-ketamine has lower potency than (R,S)-ketamine to inhibit NMDA receptors in vitro, the extent to which (R)-ketamine shares the NMDA receptor-mediated adverse effects of (R,S)-ketamine in vivo has not been fully characterised. Furthermore, (R)-ketamine is metabolised to (2R,6R)-hydroxynorketamine (HNK), which may contribute to its antidepressant-relevant actions., Experimental Approach: Using mice, we compared (R)-ketamine with a deuterated form of the drug (6,6-dideutero-(R)-ketamine, (R)-d 2 -ketamine), which hinders its metabolism to (2R,6R)-HNK, in behavioural tests predicting antidepressant responses. We also examined the actions of intracerebroventricularly infused (2R,6R)-HNK. Further, we quantified putative NMDA receptor inhibition-mediated adverse effects of (R)-ketamine., Key Results: (R)-d 2 -Ketamine was identical to (R)-ketamine in binding to and functionally inhibiting NMDA receptors but hindered (R)-ketamine's metabolism to (2R,6R)-HNK. (R)-Ketamine exerted greater potency than (R)-d 2 -ketamine in several antidepressant-sensitive behavioural measures, consistent with a role of (2R,6R)-HNK in the actions of (R)-ketamine. There were dose-dependent sustained antidepressant-relevant actions of (2R,6R)-HNK following intracerebroventricular administration. (R)-Ketamine exerted NMDA receptor inhibition-mediated behaviours similar to (R,S)-ketamine, including locomotor stimulation, conditioned-place preference, prepulse inhibition deficits, and motor incoordination, with approximately half the potency of the racemic drug., Conclusions and Implications: Metabolism of (R)-ketamine to (2R,6R)-HNK increases the potency of (R)-ketamine to exert antidepressant-relevant actions in mice. Adverse effects of (R)-ketamine require higher doses than those necessary for antidepressant-sensitive behavioural changes in mice. However, our data revealing that (R)-ketamine's adverse effects are elicited at sub-anaesthetic doses indicate a potential risk for sensory dissociation and abuse liability., (© 2019 The British Pharmacological Society.)

The reduction-oxidation state of NAD + /NADH is critical for cellular health with NAD + and its metabolites playing critical roles in aging and pathologies. Given the inherent autooxidation of reduced dinucleotides (i.e. NADH/NADPH), and the well-established differential stability, the accurate measurement of NAD + and its metabolites is technically challenging. Moreover, sample processing, normalization and measurement strategies can profoundly alter results. Here we developed a rapid and sensitive liquid chromatography mass spectrometry-based method to quantify the NAD + metabolome with careful consideration of these intrinsic chemical instabilities. Utilizing this method we assess NAD + metabolite stabilities and determine the presence and concentrations of NAD + metabolites in clinically relevant human samples including cerebrospinal fluid, erythrocytes, and primate skeletal muscle., (Published by Elsevier Inc.)

Currently approved antidepressant drugs often take months to take full effect, and ∼30% of depressed patients remain treatment resistant. In contrast, ketamine, when administered as a single subanesthetic dose, exerts rapid and sustained antidepressant actions. Preclinical studies indicate that the ketamine metabolite ( 2R , 6R )-hydroxynorketamine [( 2R , 6R )-HNK] is a rapid-acting antidepressant drug candidate with limited dissociation properties and abuse potential. We assessed the role of group II metabotropic glutamate receptor subtypes 2 (mGlu 2 ) and 3 (mGlu 3 ) in the antidepressant-relevant actions of ( 2R , 6R )-HNK using behavioral, genetic, and pharmacological approaches as well as cortical quantitative EEG (qEEG) measurements in mice. Both ketamine and ( 2R , 6R )-HNK prevented mGlu 2/3 receptor agonist (LY379268)-induced body temperature increases in mice lacking the Grm3 , but not Grm2 , gene. This action was not replicated by NMDA receptor antagonists or a chemical variant of ketamine that limits metabolism to ( 2R , 6R )-HNK. The antidepressant-relevant behavioral effects and 30- to 80-Hz qEEG oscillation (gamma-range) increases resultant from ( 2R , 6R )-HNK administration were prevented by pretreatment with an mGlu 2/3 receptor agonist and absent in mice lacking the Grm2 , but not Grm3 -/- , gene. Combined subeffective doses of the mGlu 2/3 receptor antagonist LY341495 and ( 2R , 6R )-HNK exerted synergistic increases on gamma oscillations and antidepressant-relevant behavioral actions. These findings highlight that ( 2R , 6R )-HNK exerts antidepressant-relevant actions via a mechanism converging with mGlu 2 receptor signaling and suggest enhanced cortical gamma oscillations as a marker of target engagement relevant to antidepressant efficacy. Moreover, these results support the use of ( 2R , 6R )-HNK and inhibitors of mGlu 2 receptor function in clinical trials for treatment-resistant depression either alone or in combination., Competing Interests: Conflict of interest statement: P.Z., P.J.M., C.J.T., R.M., C.A.Z., and T.D.G. are listed as coauthors in patent applications related to the pharmacology and use of (2R,6R)-HNK in the treatment of depression, anxiety, anhedonia, suicidal ideation, and posttraumatic stress disorders. R.M. and C.A.Z. are listed as coinventors on a patent for the use of ketamine in major depression and suicidal ideation. T.D.G. has received research funding from Janssen, Allergan, and Roche Pharmaceuticals and was a consultant for FSV7 LLC during the preceding 3 years. All of the other authors report no conflict of interest.

Preclinical studies indicate that (2 R ,6 R )-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine's antidepressant and adverse effects, the potency of (2 R ,6 R )-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2 R ,6 R )-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2 R ,6 R )-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2 R ,6 R )-HNK administration in mice. The effects of ketamine, (2 R ,6 R )-HNK, and, in some cases, the (2 S ,6 S )-HNK stereoisomer were evaluated on the following: ( i ) NMDA-induced lethality in mice, ( ii ) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, ( iii ) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and ( iv ) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2 R ,6 R )-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED 50 of (2 R ,6 R )-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2 R ,6 R )-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2 S ,6 S )-HNK was more potent than (2 R ,6 R )-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2 R ,6 R ;2 S ,6 S )-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2 R ,6 R )-HNK., Competing Interests: Conflict of interest statement: T.D.G. has received research funding from Janssen, Roche, and Allergan Pharmaceuticals, and served as a consultant for FSV7 LLC during the preceding 3 years. The authors declare competing financial interests: T.D.G., P.Z., R.M., P.J.M., C.J.T., and C.A.Z. are listed as co-inventors on a patent application for the use of (2R,6R)-hydroxynorketamine and (2S,6S)-hydroxynorketamine in the treatment of depression, anxiety, anhedonia, suicidal ideation, and post-traumatic stress disorders. C.A.Z. and R.M. are listed as co-inventors on a patent for the use of (2R,6R)-hydroxynorketamine, (S)-dehydronorketamine, and other stereoisomeric dehydro- and hydroxylated metabolites of ketamine metabolites in the treatment of depression and neuropathic pain. R.M., P.J.M., C.J.T., and C.A.Z. have assigned patent rights to the US Government but will share a percentage of any royalties that may be received by the Government. P.Z. and T.D.G. have assigned their patent rights to the University of Maryland, Baltimore, but will share a percentage of any royalties that may be received by the University of Maryland, Baltimore. S.F.T. received research support from Janssen, is a consultant for Janssen, is a member of the Scientific Advisory Board for Sage Therapeutics, is a co-founder of NeurOp, Inc., and co-inventor on Emory-owned IP. S.J.M. owns stock in NeurOp, Inc., which is developing NMDAR inhibitors for use in treating neurological disease and disorders. All other authors declare no competing interests.

Background: ( R,S )-ketamine has gained attention for its rapid-acting antidepressant actions in patients with treatment-resistant depression. However, widespread use of ketamine is limited by its side effects, abuse potential, and poor oral bioavailability. The ketamine metabolite, ( 2R,6R )-hydroxynorketamine, exerts rapid antidepressant effects, without ketamine's adverse effects and abuse potential, in rodents., Methods: We evaluated the oral bioavailability of ( 2R,6R )-hydroxynorketamine in three species (mice, rats, and dogs) and also evaluated five candidate prodrug modifications for their capacity to enhance the oral bioavailability of ( 2R,6R )-hydroxynorketamine in mice. Oral administration of ( 2R,6R )-hydroxynorketamine was assessed for adverse behavioral effects and for antidepressant efficacy in the mouse forced-swim and learned helplessness tests., Results: ( 2R,6R )-hydroxynorketamine had absolute bioavailability between 46-52% in mice, 42% in rats, and 58% in dogs. Compared to intraperitoneal injection in mice, the relative oral bioavailability of ( 2R,6R )-hydroxynorketamine was 62%, which was not improved by any of the candidate prodrugs tested. Following oral administration, ( 2R,6R )-hydroxynorketamine readily penetrated the brain, with brain to plasma ratios between 0.67-1.2 in mice and rats. Oral administration of ( 2R,6R )-hydroxynorketamine to mice did not alter locomotor activity or precipitate behaviors associated with discomfort, sickness, or stereotypy up to a dose of 450 mg/kg. Oral ( 2R,6R )-hydroxynorketamine reduced forced-swim test immobility time (15-150 mg/kg) and reversed learned helplessness (50-150 mg/kg) in mice., Conclusions: These results demonstrate that ( 2R,6R )-hydroxynorketamine has favorable oral bioavailability in three species and exhibits antidepressant efficacy following oral administration in mice.

The last decade has seen a sharp increase in the number of scientific publications describing physiological and pathological functions of extracellular vesicles (EVs), a collective term covering various subtypes of cell-released, membranous structures, called exosomes, microvesicles, microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names. However, specific issues arise when working with these entities, whose size and amount often make them difficult to obtain as relatively pure preparations, and to characterize properly. The International Society for Extracellular Vesicles (ISEV) proposed Minimal Information for Studies of Extracellular Vesicles ("MISEV") guidelines for the field in 2014. We now update these "MISEV2014" guidelines based on evolution of the collective knowledge in the last four years. An important point to consider is that ascribing a specific function to EVs in general, or to subtypes of EVs, requires reporting of specific information beyond mere description of function in a crude, potentially contaminated, and heterogeneous preparation. For example, claims that exosomes are endowed with exquisite and specific activities remain difficult to support experimentally, given our still limited knowledge of their specific molecular machineries of biogenesis and release, as compared with other biophysically similar EVs. The MISEV2018 guidelines include tables and outlines of suggested protocols and steps to follow to document specific EV-associated functional activities. Finally, a checklist is provided with summaries of key points.

Poly( N -vinylpyrrolidone) (PNVP) is a well-known, highly polar, nonionic water-soluble polymer. However, N -vinylpyrrolidone (NVP) usually exhibits strongly non-ideal behavior when copolymerized with methacrylic or styrenic monomers. Moreover, NVP is not particularly well-controlled under living radical polymerization conditions. For these reasons, alternative pyrrolidone-based monomers have been investigated. For example, the reversible addition-fragmentation chain transfer (RAFT) polymerization of 2-( N -methacryloyloxy)ethylpyrrolidone (NMEP) has been recently investigated using various polymerization formulations. However, PNMEP homopolymers are significantly less hydrophilic than PNVP and exhibit inverse temperature solubility in aqueous solution. In the present work, we studied the RAFT aqueous solution polymerization of 2-( N -acryloyloxy)ethylpyrrolidone (NAEP) using either AIBN at 70 °C or a low-temperature redox initiator at 30 °C. PNAEP homopolymers are obtained in high yield (>99%) with good control ( M w / M n < 1.20) for target degrees of polymerization (DP) of up to 400 using the latter initiator, which produced relatively fast rates of polymerization. However, targeting DPs above 400 led to lower NAEP conversions and broader molecular weight distributions. 2-Hydroxyethyl acrylate (HEA) and oligo(ethylene glycol) methyl ether acrylate (OEGA) were chain-extended using a PNAEP x macro-CTA via RAFT aqueous solution polymerization, yielding double-hydrophilic acrylic diblock copolymers with high conversions (>99%) and good control ( M w / M n < 1.31). In addition, a PNAEP 95 macro-CTA was chain-extended via RAFT aqueous solution polymerization of N -isopropylacrylamide (NIPAM) at 22 °C. Dynamic light scattering (DLS) analysis indicated that heating above the lower critical solution temperature of PNIPAM led to so-called "anomalous micellization" at 35 °C and the formation of near-monodisperse spherical micelles at 40 °C. Finally, 2-(diethylamino)ethyl methacrylate (DEA) was polymerized using an N -morpholine-functionalized trithiocarbonate-based RAFT chain transfer agent and subsequently chain-extended using NAEP to form a novel pH-responsive diblock copolymer. Above the p K a of PDEA (∼7.3), DLS and 1 H NMR studies indicated the formation of well-defined PDEA-core spherical micelles., Competing Interests: The authors declare the following competing financial interest(s): A patent application has been filed to protect the results reported in this manuscript.

(R,S)-Ketamine produces rapid, robust, and sustained antidepressant effects in major depressive disorder. Specifically, its pharmacological efficacy in treatment refractory depression is considered a major breakthrough in the field. However, the mechanism of action of ketamine's rapid effect remains to be determined. In order to identify pathways that are responsible for ketamine's effect, a targeted metabolomic approach was carried out using a double-blind, placebo-controlled crossover design, with infusion order randomized with medication-free patients with treatment-resistant major depressive disorder (29 subjects) and healthy controls (25 subjects). The metabolomic profile of these subjects was characterized at multiple time points, and a comprehensive analysis was investigated between the following: MDD and healthy controls, treatment and placebo in both groups and the corresponding response to ketamine treatment. Ketamine treatment resulted in a general increase in circulating sphingomyelins, levels which were not correlated with response. Ketamine response resulted in more pronounced effects in the kynurenine pathway and the arginine pathway at 4 h post-infusion, where a larger decrease in circulating kynurenine levels and a larger increase in the bioavailability of arginine were observed in responders to ketamine treatment, suggesting possible mechanisms for response to ketamine treatment.

Characterisation of 27 types of biomass was performed together with an assessment of regional resource availability. Charcoal was produced under two conditions from all samples and their yields were compared. Sugarcane bagasse, sal and pine produced the best charcoal with a low volatile matter and high calorific value. The amount of high-quality charcoal which can be made within Nepal from the biomass types tested is equivalent to 8,073,000 tonnes of firewood a year or 51% of the yearly demand. The areas which would benefit the most from charcoal making facilities are the Mid-hills of the Western, Central and Eastern Development Regions, as well as the Terai in the Central and Eastern Development Regions. The main potential benefit is to convert agricultural residues which are underutilised because, in their original form, produce large quantities of smoke, to cleaner burning charcoal. The conversion of agricultural residues to charcoal is also a viable alternative to anaerobic digestion in the Mid-hills., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Knowledge about the biogeographic affinities of the world's tropical forests helps to better understand regional differences in forest structure, diversity, composition, and dynamics. Such understanding will enable anticipation of region-specific responses to global environmental change. Modern phylogenies, in combination with broad coverage of species inventory data, now allow for global biogeographic analyses that take species evolutionary distance into account. Here we present a classification of the world's tropical forests based on their phylogenetic similarity. We identify five principal floristic regions and their floristic relationships: ( i ) Indo-Pacific, ( ii ) Subtropical, ( iii ) African, ( iv ) American, and ( v ) Dry forests. Our results do not support the traditional neo- versus paleotropical forest division but instead separate the combined American and African forests from their Indo-Pacific counterparts. We also find indications for the existence of a global dry forest region, with representatives in America, Africa, Madagascar, and India. Additionally, a northern-hemisphere Subtropical forest region was identified with representatives in Asia and America, providing support for a link between Asian and American northern-hemisphere forests., Competing Interests: Conflict of interest statement: V.A.-R., K.B.-G., B.B., F.Q.B., N.F., M.K., W.F.L., S. G. Letcher, C.B.S., D.S., T. Stevart, and S. Wiser have coauthored papers with Jens-Christian Svenning in the past 48 months. A.M.L. and Jens-Christian Svenning are both affiliated with Aarhus University., (Copyright © 2018 the Author(s). Published by PNAS.)

Reduced insulin action develops naturally during the peripartum to ensure maternal nutrient delivery to the fetus and neonate. However, increased insulin resistance can facilitate excessive lipolysis which in turn promotes metabolic disease in overweight dairy cattle. Increased fatty acid availability favors the accumulation of the sphingolipid ceramide and is implicated in the pathogenesis of insulin resistance, however, the relationship between sphingolipid metabolism and insulin resistance during the peripartum remains largely unknown. Our objectives were to characterize temporal responses in plasma and tissue sphingolipids in lean and overweight peripartal cows and to establish the relationships between sphingolipid supply and lipolysis, hepatic lipid deposition, and systemic insulin action. Twenty-one multiparous lean and overweight Holstein cows were enrolled in a longitudinal study spanning the transition from gestation to lactation (d -21 to 21, relative to parturition). Plasma, liver, and skeletal muscle samples were obtained, and sphingolipids were profiled using LC/MS/MS. Insulin sensitivity was assessed utilizing intravenous insulin and glucose challenges. Our results demonstrated the following: first, insulin resistance develops postpartum concurrently with increased lipolysis and hepatic lipid accumulation; second, ceramides and glycosylated ceramides accumulate during the transition from gestation to lactation and are further elevated in overweight cows; third, ceramide accrual is associated with lipolysis and liver lipid accumulation, and C16:0- and C24:0-ceramide are inversely associated with systemic insulin sensitivity postpartum; fourth, plasma sphingomyelin, a potential source of ceramides reaches a nadir at parturition and is closely associated with feed intake; fifth, select sphingomyelins are lower in the plasma of overweight cows during the peripartal period. Our results demonstrate that dynamic changes occur in peripartal sphingolipids that are influenced by adiposity, and are associated with the onset of peripartal insulin resistance. These observations are in agreement with a putative potential role for sphingolipids in facilitating the physiological adaptations of peripartum.

Reduced insulin action is a key adaptation that facilitates glucose partitioning to the mammary gland for milk synthesis and enhances adipose tissue lipolysis during early lactation. The progressive recovery of insulin sensitivity as cows advance toward late lactation is accompanied by reductions in circulating nonesterified fatty acids (NEFA) and milk yield. Because palmitic acid can promote insulin resistance in monogastrics through sphingolipid ceramide-dependent mechanisms, palmitic acid (C16:0) feeding may enhance milk production by restoring homeorhetic responses. We hypothesized that feeding C16:0 to mid-lactation cows would enhance ceramide supply and ceramide would be positively associated with milk yield. Twenty multiparous mid-lactation Holstein cows were enrolled in a study consisting of a 5-d covariate, 49-d treatment, and 14-d posttreatment period. All cows were randomly assigned to a sorghum silage-based diet containing no supplemental fat (control; n=10; 138±45 d in milk) or C16:0 at 4% of ration dry matter (PALM; 98% C16:0; n=10; 136±44 d in milk). Blood and milk were collected at routine intervals. Liver and skeletal muscle tissue were biopsied at d 47 of treatment. Intravenous glucose tolerance tests (300mg/kg of body weight) were performed at d -1, 24, and 49 relative to start of treatment. The plasma and tissue concentrations of ceramide and glycosylated ceramide were determined using liquid chromatography coupled with tandem mass spectrometry. Data were analyzed as repeated measures using a mixed model with fixed effects of treatment and time, and milk yield served as a covariate. The PALM treatment increased milk yield, energy-corrected milk, and milk fat yield. The most abundant plasma and tissue sphingolipids detected were C24:0-ceramide, C24:0-monohexosylceramide (GlcCer), and C16:0-lactosylceramide. Plasma concentrations of total ceramide and GlcCer decreased as lactation advanced, and ceramide and GlcCer were elevated in cows fed PALM. Palmitic acid feeding increased hepatic ceramide levels, a response not observed in skeletal muscle tissue. Plasma ceramides (e.g., C24:0-ceramide) were positively correlated with plasma NEFA and milk yield, and positively correlated with NEFA levels following a glucose challenge. Our data demonstrate a remodeled plasma and hepatic sphingolipidome in mid-lactation dairy cows fed PALM. The potential involvement in ceramide in homeorhetic nutrient partitioning to support lactation requires further consideration., (Copyright © 2016 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

Objective: We present seven cases of patients with severe respiratory failure refractory to conventional ventilation who were safely transported in the prone position., Methods: We describe all cases of patients transported by a regional critical care network in the prone position from January 2010-June 2015. All patients were mechanically ventilated for respiratory failure and transported by specialized nonphysician critical care teams. Utilizing direct medical oversight and real-time technical support from the clinical department, each patient underwent a thorough bedside evaluation, transport ventilator trial, and transfer to a transport stretcher either with the endotracheal tube secured by an anesthesia pillow or overhanging the stretcher., Results: Seven patients with acute respiratory distress syndrome were transported in the prone position. Four were female (57%), with a median weight of 78 kg (range 58-131) and median age of 53 years (range 37-78). Initial vital signs demonstrated a median oxygen saturation of 94% (range 90-97%) supported with a FiO2 of 100% for all patients with a median positive end-expiratory pressure (PEEP) of 16 (range 14-20). Seven patients were transported, six by helicopter and one transported by ground ambulance. The median transport time was 36 minutes (IQR 19, 51). There were no deaths or major incidents (tube dislodgement or line displacement) during patient transport., Conclusion: The transport of mechanically ventilated patients with respiratory failure in the prone position is feasible and safe, with minimal complications identified in this case series.

Hydro-meteorological data is an important asset that can enhance management of water resources. But existing data often contains gaps, leading to uncertainties and so compromising their use. Although many methods exist for infilling data gaps in hydro-meteorological time series, many of these methods require inputs from neighbouring stations, which are often not available, while other methods are computationally demanding. Computing techniques such as artificial intelligence can be used to address this challenge. Self-organizing maps (SOMs), which are a type of artificial neural network, were used for infilling gaps in a hydro-meteorological time series in a Sudano-Sahel catchment. The coefficients of determination obtained were all above 0.75 and 0.65 while the average topographic error was 0.008 and 0.02 for rainfall and river discharge time series, respectively. These results further indicate that SOMs are a robust and efficient method for infilling missing gaps in hydro-meteorological time series.

A poly(glycerol monomethacrylate) (PGMA) chain transfer agent is chain-extended by reversible addition-fragmentation chain transfer (RAFT) statistical copolymerization of 2-hydroxypropyl methacrylate (HPMA) with glycidyl methacrylate (GlyMA) in concentrated aqueous solution via polymerization-induced self-assembly (PISA). A series of five free-standing worm gels is prepared by fixing the overall degree of polymerization of the core-forming block at 144 while varying its GlyMA content from 0 to 20 mol %. 1 H NMR kinetics indicated that GlyMA is consumed much faster than HPMA, producing a GlyMA-rich sequence close to the PGMA stabilizer block. Temperature-dependent oscillatory rheological studies indicate that increasing the GlyMA content leads to progressively less thermoresponsive worm gels, with no degelation on cooling being observed for worms containing 20 mol % GlyMA. The epoxy groups in the GlyMA residues can be ring-opened using 3-aminopropyltriethoxysilane (APTES) in order to prepare core cross-linked worms via hydrolysis-condensation with the siloxane groups and/or hydroxyl groups on the HPMA residues. Perhaps surprisingly, 1 H NMR analysis indicates that the epoxy-amine reaction and the intermolecular cross-linking occur on similar time scales. Cross-linking leads to stiffer worm gels that do not undergo degelation upon cooling. Dynamic light scattering studies and TEM analyses conducted on linear worms exposed to either methanol (a good solvent for both blocks) or anionic surfactant result in immediate worm dissociation. In contrast, cross-linked worms remain intact under such conditions, provided that the worm cores comprise at least 10 mol % GlyMA.

This article investigates the impact of an early intervention program, which experimentally modifies the parenting and home environment of disadvantaged families, on child physical health in the first 3 years of life. We recruited and randomized 233 (115 intervention, 118 control) pregnant women from a socioeconomically disadvantaged community in Dublin, Ireland into an intervention or control group. The treatment includes regular home visits commencing antenatally and an additional parenting course commencing at 2 years. Maternal reports of child health are assessed at 6, 12, 18, 24, and 36 months. Treatment effects are estimated using permutation testing to account for small sample size, inverse probability weighting to account for differential attrition, and both the stepdown procedure and an indices approach to account for multiple hypothesis testing. Following adjustment for multiple testing and attrition, we observe a positive and statistically significant main treatment effect for wheezing/asthma. The intervention group are 15.5 percentage points (pp) less likely to require medical attention for wheezing/asthma compared to the control group. Subgroup analysis reveals more statistically significant adjusted treatment effects for boys than girls regarding fewer health problems (d=0.63), accidents (23.9pp), and chest infections (22.8-37.9pp). Our results suggest that a community-based home visiting program may have favorable impacts on early health conditions., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Viruses are highly abundant in soils with their numbers exceeding those of cooccurring bacterial cells by 10- to over 1000-fold. Water and organic matter content influence the magnitude of the viral-to-bacterial ratio in soils; thus, ecosystem type and land use shape interactions between viral and host microbial communities in soils. Less understood are the shorter term interactions between viral and host communities that ultimately maintain the large viral standing stock within soils. This study examined short-term dynamics of viral and bacterial communities in soils to determine whether the growth of soil bacterial communities results in the production of soil viruses, and if viral community responses occur within specific populations. In microcosms amended with different carbon sources, increases in viral abundance (VA) accompanied increases in bacterial abundance (BA) and bacterial respiration rate (BRR). The timing and intensity of increases in BA, VA and BRR were different across C sources suggesting differences in the predominant mode of viral replication within growth-stimulated bacterial populations. Moreover, compositional changes occurred in soil bacterial and viral communities indicating that new viral production arose from a subset of host populations. To our knowledge, these are the first observations of soil viral populations responding to short-term changes in soil bacterial communities., (© FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Repetitive armed conflicts may be directly and indirectly responsible for severe biophysical modification to the environment. This, in turn, makes land more susceptible to degradation. Mapping and monitoring land degradation are essential for designing and implementing post-conflict recovery plans and informed policy decisions. The aim of this work was to evaluate the effect of repetitive armed conflicts on land degradation along the coastal zone of North Lebanon using multi-temporal satellite data. The specific objectives were to (1) identify a list of indicators for use in conjunction with satellite remote sensing, (2) monitor land cover change throughout repetitive events of armed conflicts and (3) model the effect of repetitive armed conflicts on land degradation. The methodology of work comprised the use of multi-temporal Landsat images and literature review data in GEographic Object-Based Image Analysis (GEOBIA) approach. The work resulted in the development of (1) a list of indicators to be employed, (2) land cover change detection maps with the use of multi-temporal Landsat images and, consequently, a fire risk associated with changes in vegetation cover throughout repetitive armed conflict events, and (3) an integrated approach for modelling the effect of repetitive armed conflicts on land degradation with the use of a composite land degradation index (CLDI). The final synthetic map showed four classes of exposure to land degradation associated with repetitive armed conflicts. Data collected from field visits showed that the final classification results highly reflected (average of 90 %) the effect of repetitive armed conflicts on the different classes of exposure to land degradation.

The OP9/OP9-DL1 co-culture system has become a well-established method for deriving differentiated blood cell types from embryonic and hematopoietic progenitors of both mouse and human origin. It is now used to address a growing variety of complex genetic, cellular and molecular questions related to hematopoiesis, and is at the cutting edge of efforts to translate these basic findings to therapeutic applications. The procedures are straightforward and routinely yield robust results. However, achieving successful hematopoietic differentiation in vitro requires special attention to the details of reagent and cell culture maintenance. Furthermore, the protocol features technique sensitive steps that, while not difficult, take care and practice to master. Here we focus on the procedures for differentiation of T lymphocytes from mouse embryonic stem cells (mESC). We provide a detailed protocol with discussions of the critical steps and parameters that enable reproducibly robust cellular differentiation in vitro. It is in the interest of the field to consider wider adoption of this technology, as it has the potential to reduce animal use, lower the cost and shorten the timelines of both basic and translational experimentation.

Viruses are the most abundant and diverse biological entities within soils, yet their ecological impact is largely unknown. Defining how soil viral communities change with perturbation or across environments will contribute to understanding the larger ecological significance of soil viruses. A new approach to examining the composition of soil viral communities based on random PCR amplification of polymorphic DNA (RAPD-PCR) was developed. A key methodological improvement was the use of viral metagenomic sequence data for the design of RAPD-PCR primers. This metagenomically informed approach to primer design enabled the optimization of RAPD-PCR sensitivity for examining changes in soil viral communities. Initial application of RAPD-PCR viral fingerprinting to soil viral communities demonstrated that the composition of autochthonous soil viral assemblages noticeably changed over a distance of meters along a transect of Antarctic soils and across soils subjected to different land uses. For Antarctic soils, viral assemblages segregated upslope from the edge of dry valley lakes. In the case of temperate soils at the Kellogg Biological Station, viral communities clustered according to land use treatment. In both environments, soil viral communities changed along with environmental factors known to shape the composition of bacterial host communities. Overall, this work demonstrates that RAPD-PCR fingerprinting is an inexpensive, high-throughput means for addressing first-order questions of viral community dynamics within environmental samples and thus fills a methodological gap between narrow single-gene approaches and comprehensive shotgun metagenomic sequencing for the analysis of viral community diversity.

Locus control regions (LCRs) are cis-acting gene regulatory elements with the unique, integration site-independent ability to transfer the characteristics of their locus-of-origin's gene expression pattern to a linked transgene in mice. LCR activities have been discovered in numerous T cell lineage-expressed gene loci. These elements can be adapted to the design of stem cell gene therapy vectors that direct robust therapeutic gene expression to the T cell progeny of engineered stem cells. Currently, transgenic mice provide the only experimental approach that wholly supports all the critical aspects of LCR activity. In this study, we report the manifestation of all key features of mouse TCR-α gene LCR function in T cells derived in vitro from mouse embryonic stem cells. High-level, copy number-related TCR-α LCR-linked reporter gene expression levels are cell type restricted in this system, and upregulated during the expected stage transition of T cell development. We also report that de novo introduction of TCR-α LCR-linked transgenes into existing T cell lines yields incomplete LCR activity. These data indicate that establishing full TCR-α LCR activity requires critical molecular events occurring prior to final T lineage determination. This study also validates a novel, tractable, and more rapid approach for the study of LCR activity in T cells, and its translation to therapeutic genetic engineering.