Direct HCN synthesis via plasma-assisted conversion of methane and nitrogen.

Hydrogen cyanide (HCN) is synthesized from ammonia (NH3) and methane (CH4) at ~1200°C over a Pt catalyst. Ammonia synthesis entails several complex, highly emitting processes. Plasma-assisted HCN synthesis directly from CH4 and nitrogen (N2) could be pivotal for on-demand HCN production. Here, we evaluate the potential of dielectric barrier discharge (DBD) N2/CH4 plasma for decentralized catalyst-free selective HCN production. We demonstrate a single-step conversion of methane and nitrogen to HCN with a 72% yield at <300°C. HCN is favored at low CH4 concentrations with ethane (C2H6) as the secondary product. We propose a first-principles microkinetic model with few electron impact reactions. The model accurately predicts primary product yields and elucidates that methyl radical (·CH3) is a common intermediate in HCN and C2H6 synthesis. Compared to current industrial processes, N2/CH4 DBD plasma can achieve minimal CO2 emissions.

Activated phosphoinositide 3-kinase δ syndrome: Update from the ESID Registry and comparison with other autoimmune-lymphoproliferative inborn errors of immunity.

BACKGROUND: Activated phosphoinositide-3-kinase δ syndrome (APDS) is an inborn error of immunity (IEI) with infection susceptibility and immune dysregulation, clinically overlapping with other conditions. Management depends on disease evolution, but predictors of severe disease are lacking. OBJECTIVES: This study sought to report the extended spectrum of disease manifestations in APDS1 versus APDS2; compare these to CTLA4 deficiency, NFKB1 deficiency, and STAT3 gain-of-function (GOF) disease; and identify predictors of severity in APDS. METHODS: Data was collected from the ESID (European Society for Immunodeficiencies)-APDS registry and was compared with published cohorts of the other IEIs. RESULTS: The analysis of 170 patients with APDS outlines high penetrance and early onset of APDS compared to the other IEIs. The large clinical heterogeneity even in individuals with the same PIK3CD variant E1021K illustrates how poorly the genotype predicts the disease phenotype and course. The high clinical overlap between APDS and the other investigated IEIs suggests relevant pathophysiological convergence of the affected pathways. Preferentially affected organ systems indicate specific pathophysiology: bronchiectasis is typical of APDS1; interstitial lung disease and enteropathy are more common in STAT3 GOF and CTLA4 deficiency. Endocrinopathies are most frequent in STAT3 GOF, but growth impairment is also common, particularly in APDS2. Early clinical presentation is a risk factor for severe disease in APDS. CONCLUSIONS: APDS illustrates how a single genetic variant can result in a diverse autoimmune-lymphoproliferative phenotype. Overlap with other IEIs is substantial. Some specific features distinguish APDS1 from APDS2. Early onset is a risk factor for severe disease course calling for specific treatment studies in younger patients.

Functional and structural insight into the flexibility of cytochrome P450 reductases from Sorghum bicolor and its implications for lignin composition.

Plant NADPH-dependent cytochrome P450 reductase (CPR) is a multidomain enzyme that donates electrons for hydroxylation reactions catalyzed by class II cytochrome P450 monooxygenases involved in the synthesis of many primary and secondary metabolites. These P450 enzymes include trans-cinnamate-4-hydroxylase, p-coumarate-3'-hydroxylase, and ferulate-5-hydroxylase involved in monolignol biosynthesis. Because of its role in monolignol biosynthesis, alterations in CPR activity could change the composition and overall output of lignin. Therefore, to understand the structure and function of three CPR subunits from sorghum, recombinant subunits SbCPR2a, SbCPR2b, and SbCPR2c were subjected to X-ray crystallography and kinetic assays. Steady-state kinetic analyses demonstrated that all three CPR subunits supported the oxidation reactions catalyzed by SbC4H1 (CYP73A33) and SbC3'H (CYP98A1). Furthermore, comparing the SbCPR2b structure with the well-investigated CPRs from mammals enabled us to identify critical residues of functional importance and suggested that the plant flavin mononucleotide-binding domain might be more flexible than mammalian homologs. In addition, the elucidated structure of SbCPR2b included the first observation of NADP+ in a native CPR. Overall, we conclude that the connecting domain of SbCPR2, especially its hinge region, could serve as a target to alter biomass composition in bioenergy and forage sorghums through protein engineering.

Catalytic resonance of ammonia synthesis by simulated dynamic ruthenium crystal strain.

Ammonia affords dense storage for renewable energy as a fungible liquid fuel, provided it can be efficiently synthesized from hydrogen and nitrogen. In this work, the catalysis of ammonia synthesis was computationally explored beyond the Sabatier limit by dynamically straining a ruthenium crystal (±4%) at the resonant frequencies (102 to 105+ Hz) of N2 surface dissociation and hydrogenation. Density functional theory calculations at different strain conditions indicated that the energies of NHx surface intermediates and transition states scale linearly, allowing the description of ammonia synthesis at a continuum of strain conditions. A microkinetic model including multiple sites and surface diffusion between step and Ru(0001) terrace sites of varying ratios for nanoparticles of differing size revealed that dynamic strain yields catalytic ammonia synthesis conversion and turnover frequency comparable to industrial reactors (400°C, 200 atm) but at lower temperature (320°C) and an order of magnitude lower pressure (20 atm).

MicroRNA antagonist therapy during normothermic machine perfusion of donor kidneys.

Normothermic machine perfusion (NMP) is a novel clinical approach to overcome the limitations of traditional hypothermic organ preservation. NMP can be used to assess and recondition organs prior to transplant and is the subject of clinical trials in solid organ transplantation. In addition, NMP provides an opportunity to deliver therapeutic agents directly to the organ, thus avoiding many limitations associated with systemic treatment of the recipient. We report the delivery of oligonucleotide-based therapy to human kidneys during NMP, in this case to target microRNA function (antagomir). An antagomir targeting mir-24-3p localized to the endothelium and proximal tubular epithelium. Endosomal uptake during NMP conditions facilitated antagomir co-localization with proteins involved in the RNA-induced silencing complex (RISC) and demonstrated engagement of the miRNA target. This pattern of uptake was not seen during cold perfusion. Targeting mir-24-3p action increased expression of genes controlled by this microRNA, including heme oxygenase-1 and sphingosine-1-phosphate receptor 1. The expression of genes not under the control of mir-24-3p was unchanged, indicating specificity of the antagomir effect. In summary, this is the first report of ex vivo gymnotic delivery of oligonucleotide to the human kidney and demonstrates that NMP provides the platform to bind and block detrimental microRNAs in donor kidneys prior to transplantation.

Management of bilateral Wilms' tumour: A case report.

INTRODUCTION: Wilms' tumour remains the most common renal tumour in children (6% of all pediatric malignancies) and present as one of the most challenging tasks for paediatric urologists as its management requires an advanced procedure. The ultimate goal in these cases is to preserve as much renal parenchyma as possible whilst still achieving complete tumour resection. PRESENTATION OF CASE: Here we present a six year follow up report of a bilateral Wilms' tumour case in a 19-months old boy. This patient underwent neoadjuvant chemotherapy regimen, followed by right partial nephrectomy and left radical nephrectomy. Adjuvant radiotherapy was performed following the surgery. Follow-up imaging 5 months afterward revealed a firmly heterogeneous cystic lesion consist of fat and calcification at the upper pole of the right kidney, none of which created any problem for the patient. MRI was later performed on the 19th month after the surgery, showing marked decrease in the size of the cyst. DISCUSSION: According to SIOP and NWTSG classification, the patient presented as stage V of the disease. The patient was on neoadjuvant chemotherapy (Regimen I) as recommended by NWTSG. This strategy was shown to be effective, as the tumour on the left kidney was reduced to less than 70% of the initial size. A routine follow-up using chest x-ray, abdominal ultrasonography (USG), and contrast studies such as MRI and MSCT scan, was performed in our reports. CONCLUSION: From our experience, the combination of neo-adjuvant chemotherapy, renal salvage surgery and adjuvant radiotherapy is a feasible, safe and effective option for bilateral Wilms' tumour cases.

Time-resolved measurement of space-charge evolution in dielectric films or slabs by means of repeatable laser-induced pressure pulses.

A new variant of the Laser-Induced Pressure-Pulse (LIPP) method for repeatable, time-resolved space-charge profile measurements is proposed and demonstrated. Automated deposition of a fresh laser-target film before each illumination leads to good repeatability of the LIPP and thus allows for the detection of time-resolved changes in the space-charge distribution over many hours. We describe and discuss the experimental setup and its features, compare the repeatability of the LIPP measurements on the same sample without and with re-preparation of the test cell, and present the time-resolved evolution of the space-charge profile in a two-layer arrangement of a silicone-grease and a silicone-elastomer film as an example. Finally, the temperature dependence of the space-charge evolution during polarization under high voltage and during depolarization in short circuit is shown. Possible uses and future developments of the new LIPP approach are also discussed.

Seeking mTORC1 Inhibitors Through Molecular Dynamics Simulation of Arginine Analogs Inhibiting CASTOR1.

BACKGROUND: Hyperactivity of the mechanistic target of rapamycin complex 1 (mTORC1) is implicated in a variety of diseases such as cancer and diabetes. Treatment may benefit from effective mTORC1 inhibition, which can be achieved by preventing arginine from disrupting the cytosolic arginine sensor for mTORC1 subunit 1 (CASTOR1)-GTPase-activating proteins toward RAGS subcomplex 2 (GATOR2) complex through binding with CASTOR1. An attractive idea is to determine analogues of arginine that are as competent as arginine in binding with CASTOR1, but without disrupting the CASTOR1-GATOR2 interaction. MATERIALS AND METHODS: Molecular dynamics simulations were performed for binding of arginine analogues with CASTOR1 and binding free energy, hydrogen bond formation, and root mean squared deviation and root mean square fluctuation kinetics were then calculated. RESULTS: The binding free energy calculations revealed that Nα-acetyl-arginine, citrulline, and norarginine have sufficient binding affinity with CASTOR1 to compete with arginine. The hydrogen bond analysis revealed that norarginine, Nα-acetyl-arginine and D-arginine have proficient H-bonds that can facilitate their entering the narrow binding pocket. CONCLUSION: Norarginine and Nα-acetyl-arginine are the top drug candidates for mTORC1 inhibition, with Nα-acetyl-arginine being the best choice.

The Importance of the Autopsy in Medicine: Perspectives of Pathology Colleagues.

This article presents a perspective on the importance of the autopsy in medical practice and science based on experiences of the authors as physician-scientists involved in autopsy practice. Our perspectives are presented on the seminal contributions of the autopsy in the areas of cardiovascular disease, including congenital heart disease, atherosclerosis, coronary artery disease, and myocardial infarction, and infectious disease, including tuberculosis and viral infections. On the positive side of the future of the autopsy, we discuss the tremendous opportunities for important research to be done by application of advanced molecular biological techniques to formalin-fixed, paraffin-embedded tissue blocks obtained at autopsy. We also note with concern the countervailing forces impacting the influence of pathology in education and clinical practice at our academic medical centers, which also present impediments to increasing autopsy rates. Our challenge as academic pathologists, whose careers have been molded by involvement in the autopsy, is to counter these trends. The challenges are great but the benefits for medicine and society are enormous.

Heparan sulfate in chronic kidney diseases: Exploring the role of 3-O-sulfation.

One of the main feature of chronic kidney disease is the development of renal fibrosis. Heparan Sulfate (HS) is involved in disease development by modifying the function of growth factors and cytokines and creating chemokine gradients. In this context, we aimed to understand the function of HS sulfation in renal fibrosis. Using a mouse model of renal fibrosis, we found that total HS 2-O-sulfation was increased in damaged kidneys, whilst, tubular staining of HS 3-O-sulfation was decreased. The expression of HS modifying enzymes significantly correlated with the development of fibrosis with HS3ST1 demonstrating the strongest correlation. The pro-fibrotic factors TGFß1 and TGFß2/IL1ß significantly downregulated HS3ST1 expression in both renal epithelial cells and renal fibroblasts. To determine the implication of HS3ST1 in growth factor binding and signalling, we generated an in vitro model of renal epithelial cells overexpressing HS3ST1 (HKC8-HS3ST1). Heparin Binding EGF like growth factor (HB-EGF) induced rapid, transient STAT3 phosphorylation in control HKC8 cells. In contrast, a prolonged response was demonstrated in HKC8-HS3ST1 cells. Finally, we showed that both HS 3-O-sulfation and HB-EGF tubular staining were decreased with the development of fibrosis. Taken together, these data suggest that HS 3-O-sulfation is modified in fibrosis and highlight HS3ST1 as an attractive biomarker of fibrosis progression with a potential role in HB-EGF signalling.

Ischaemia reperfusion injury: mechanisms of progression to chronic graft dysfunction.

The increasing use of extended criteria organs to meet the demand for kidney transplantation raises an important question of how the severity of early ischaemic injury influences long-term outcomes. Significant acute ischaemic kidney injury is associated with delayed graft function, increased immune-associated events and, ultimately, earlier deterioration of graft function. A comprehensive understanding of immediate molecular events that ensue post-ischaemia and their potential long-term consequences are key to the discovery of novel therapeutic targets. Acute ischaemic injury primarily affects tubular structure and function. Depending on the severity and persistence of the insult, this may resolve completely, leading to restoration of normal function, or be sustained, resulting in persistent renal impairment and progressive functional loss. Long-term effects of acute renal ischaemia are mediated by several mechanisms including hypoxia, HIF-1 activation, endothelial dysfunction leading to vascular rarefaction, sustained pro-inflammatory stimuli involving innate and adaptive immune responses, failure of tubular cells to recover and epigenetic changes. This review describes the biological relevance and interaction of these mechanisms based on currently available evidence.

Active HHV-6 Infection of Cerebellar Purkinje Cells in Mood Disorders.

Early-life infections and associated neuroinflammation is incriminated in the pathogenesis of various mood disorders. Infection with human roseoloviruses, HHV-6A and HHV-6B, allows viral latency in the central nervous system and other tissues, which can later be activated causing cognitive and behavioral disturbances. Hence, this study was designed to evaluate possible association of HHV-6A and HHV-6B activation with three different groups of psychiatric patients. DNA qPCR, immunofluorescence and FISH studies were carried out in post-mortem posterior cerebellum from 50 cases each of bipolar disorder (BPD), schizophrenia, 15 major depressive disorder (MDD) and 50 appropriate control samples obtained from two well-known brain collections (Stanley Medical Research Institute). HHV-6A and HHV-6B late proteins (indicating active infection) and viral DNA were detected more frequently (p < 0.001 for each virus) in human cerebellum in MDD and BPD relative to controls. These roseolovirus proteins and DNA were found less frequently in schizophrenia cases. Active HHV-6A and HHV-6B infection in cerebellar Purkinje cells were detected frequently in BPD and MDD cases. Furthermore, we found a significant association of HHV-6A infection with reduced Purkinje cell size, suggesting virus-mediated abnormal Purkinje cell function in these disorders. Finally, gene expression analysis of cerebellar tissue revealed changes in pathways reflecting an inflammatory response possibly to HHV-6A infection. Our results provide molecular evidence to support a role for active HHV-6A and HHV-6B infection in BPD and MDD.

Human herpesvirus 6-induced inflammatory cardiomyopathy in immunocompetent children.

Over the last decade, human herpesvirus 6 (HHV-6) has been implicated in the etiology of pediatric myocarditis and subsequent dilated cardiomyopathy (DCM). This review provides an overview of recent literature investigating the pathophysiological relevance of HHV-6 in inflammatory cardiomyopathy. We examined 11 cases of previously published pediatric myocarditis and/or DCM associated with HHV-6 and also our experience of detection of virus particles in vascular endothelium of HHV-6 positive endomyocardial biopsy tissue by electron microscopy. The exact role of the presence of HHV-6 and its load remains controversial as the virus is also found in the heart of healthy controls. Therefore, the question remains open whether and how cardiac HHV-6 may be of pathogenetic importance. Quantitative polymerase chain reaction or mRNA testing allows differentiation between low-level latent virus found in asymptomatic myocardium and active HHV-6 infection. Although only a small number of pediatric cases have been reported in literature, HHV-6 should be considered as a causative agent of inflammatory cardiomyopathy, especially in children under three who might be experiencing a primary infection. Future studies are needed to establish a threshold for determining active infection in biopsy samples and the role of coinfections other cardiotropic viruses.

The NF-κB1 is a key regulator of acute but not chronic renal injury.

The NF-κB family of transcription factors is important for many cellular functions, in particular initiation and propagation of inflammatory and immune responses. However, recent data has suggested that different subunits of the NF-κB family can suppress the inflammatory response. NF-κB1, from the locus nfκb1, can inhibit transcription, acting as a brake to the recognised pro-inflammatory activity of other NF-κB subunits. We tested the function of NF-κB1 in an acute (nephrotoxic serum (NTS) nephritis) and a chronic (unilateral ureteric obstruction (UUO)) model of renal injury using NF-κB1 (nfκb1-/-) knockout mice. Deficiency in NF-κB1 increased the severity of glomerular injury in NTS-induced nephritis and was associated with greater proteinuria and persistent pro-inflammatory gene expression. Induction of disease in bone marrow chimeric mice demonstrated that the absence of NF-κB1 in either bone marrow or glomerular cells increased the severity of injury. Early after UUO (day 3) there was more severe histological injury in the nfκb1-/- mice but by day 10, disease severity was equivalent in wild type and nfκb1-/- mice. In conclusion, NF-κB1 modifies acute inflammatory renal injury but does not influence chronic fibrotic injury.

Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle.

Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc2Man1), while cardiac calsequestrin had five additional mannoses (GlcNAc2Man6). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca(2+)-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic "guiderail"-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca(2+) concentrations, and proper glycosylation, in turn, guarantees both effective Ca(2+) storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site.

Differential Tandem Mass Spectrometry-Based Cross-Linker: A New Approach for High Confidence in Identifying Protein Cross-Linking.

Chemical cross-linking and mass spectrometry are now widely used to analyze large-scale protein-protein interactions. The major challenge in cross-linking approaches is the complexity of the mass spectrometric data. New approaches are required that can identify cross-linked peptides with high-confidence and establish a user-friendly analysis protocol for the biomedical scientific community. Here, we introduce a novel cross-linker that can be selectively cleaved in the gas phase using two differential tandem mass-spectrometric fragmentation methods, such as collision-induced or electron transfer dissociation (CID and ETD). This technique produces two signature mass spectra of the same cross-linked peptide, thereby producing high confidence in identifying the sites of interaction. Further tandem mass spectrometry can also give additional confidence on the peptide sequences. We demonstrate a proof-of-concept for this method using standard peptides and proteins. Peptides and proteins were cross-linked and their fragmentation characteristics were analyzed using CID and ETD tandem mass spectrometry. Two sequential cleavages unambiguously identified cross-linked peptides. In addition, the labeling efficiency of the new cross-linker was evaluated in macrophage immune cells after stimulation with the microbial ligand lipopolysaccharide and subsequent pulldown experiments with biotin-avidin affinity chromatography. We believe this strategy will help advance insights into the structural biology and systems biology of cell signaling.

Lysosomal protease cathepsin D; a new driver of apoptosis during acute kidney injury.

Acute kidney injury (AKI) is an abrupt reduction in kidney function caused by different pathological processes. It is associated with a significant morbidity and mortality in the acute phase and an increased risk of developing End Stage Renal Disease. Despite the progress in the management of the disease, mortality rates in the last five decades remain unchanged at around 50%. Therefore there is an urgent need to find new therapeutic strategies to treat AKI. Lysosomal proteases, particularly Cathepsin D (CtsD), play multiple roles in apoptosis however, their role in AKI is still unknown. Here we describe a novel role for CtsD in AKI. CtsD expression was upregulated in damaged tubular cells in nephrotoxic and ischemia reperfusion (IRI) induced AKI. CtsD inhibition using Pepstatin A led to an improvement in kidney function, a reduction in apoptosis and a decrease in tubular cell damage in kidneys with nephrotoxic or IRI induced AKI. Pepstatin A treatment slowed interstitial fibrosis progression following IRI induced AKI. Renal transplant biopsies with acute tubular necrosis demonstrated high levels of CtsD in damaged tubular cells. These results support a role for CtsD in apoptosis during AKI opening new avenues for the treatment of AKI by targeting lysosomal proteases.

Anti-herpesviral effects of a novel broad range anti-microbial quaternary ammonium silane, K21.

We have created a novel quaternary ammonium silane, K21 through sol-gel chemistry, using an ethoxylated version of an organosilane quaternary ammonium compound and TetraEthyl Ortho Silicate (TEOS) as precursors. Previous studies using the precursor molecule quaternary ammonium compounds (QACs) and a methacryloxy version of K21, primarily designed for use in dental healthcare, have shown inhibited growth properties against several types of gram-positive and gram-negative bacteria including Escherichia coli, Streptococcus mutans, Actinomyces naeslundii and Candida albicans etc. Here we tested the effect of K21 on HSV-1, HHV-6A and HHV-7 in in vitro cell culture infection models. Our results show growth inhibitory effect of K21 on HSV-1, HHV-6A and HHV-7 infection.

Altered Micro-RNA Degradation Promotes Tumor Heterogeneity: A Result from Boolean Network Modeling.

Cancer heterogeneity may reflect differential dynamical outcomes of the regulatory network encompassing biomolecules at both transcriptional and post-transcriptional levels. In other words, differential gene-expression profiles may correspond to different stable steady states of a mathematical model for simulation of biomolecular networks. To test this hypothesis, we simplified a regulatory network that is important for soft-tissue sarcoma metastasis and heterogeneity, comprising of transcription factors, micro-RNAs, and signaling components of the NOTCH pathway. We then used a Boolean network model to simulate the dynamics of this network, and particularly investigated the consequences of differential miRNA degradation modes. We found that efficient miRNA degradation is crucial for sustaining a homogenous and healthy phenotype, while defective miRNA degradation may lead to multiple stable steady states and ultimately to carcinogenesis and heterogeneity.