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Diabetic foot ulcers (DFU) are chronic, refractory wounds caused by diabetic neuropathy, vascular disease, and bacterial infection, and have become one of the most serious and persistent complications of diabetes mellitus because of their high incidence and difficulty in healing. Its malignancy results from a complex microenvironment that includes a series of unfriendly physiological states secondary to hyperglycemia, such as recurrent infections, excessive oxidative stress, persistent inflammation, and ischemia and hypoxia. However, current common clinical treatments, such as antibiotic therapy, insulin therapy, surgical debridement, and conventional wound dressings all have drawbacks, and suboptimal outcomes exacerbate the financial and physical burdens of diabetic patients. Therefore, development of new, effective and affordable treatments for DFU represents a top priority to improve the quality of life of diabetic patients. In recent years, nanozymes-based diabetic wound therapy systems have been attracting extensive interest by integrating the unique advantages of nanomaterials and natural enzymes. Compared with natural enzymes, nanozymes possess more stable catalytic activity, lower production cost and greater maneuverability. Remarkably, many nanozymes possess multienzyme activities that can cascade multiple enzyme-catalyzed reactions simultaneously throughout the recovery process of DFU. Additionally, their favorable photothermal-acoustic properties can be exploited for further enhancement of the therapeutic effects. In this review we first describe the characteristic pathological microenvironment of DFU, then discuss the therapeutic mechanisms and applications of nanozymes in DFU healing, and finally, highlight the challenges and perspectives of nanozyme development for DFU treatment.

The relevance of the research is due to the spread of protozoal diseases of animals, which, in the absence of the necessary veterinary care, lead to death. In this regard, the aim of the study was to investigate changes in clinical and haematological parameters in dogs under complex treatment of babesiosis with simultaneous red blood cell transfusion. Transfusions were performed in five animals infected with Babesia canis by intravenous injection of red blood cells, the volume of which was calculated using the Sackmen formula. The diagnosis of babesiosis was made based on anamnesis, clinical symptoms (weakness, refusal to food, pallor of visible mucous membranes, fever up to 40°C), and confirmed by microscopic detection of babesiae in blood smears. Before transfusion, rapid tests were performed to exclude heartworm disease in donor dogs. The compatibility of the blood of the donor and recipient animal was determined using a large cross-test. The condition of the abdominal organs of the patients was assessed using ultrasound diagnostics. Clinical examination of the dogs’ blood before treatment indicated the development of anaemia. The results of biochemical blood tests indicated an increase in the activity of alanine aminotransferase and gamma-glutamyl transpeptidase, which indicates a load on the hepatobiliary system. There were no contraindications to blood transfusion. After haemotransfusion, the clinical condition of the dogs was monitored based on the results of the examination of the animals, taking into account the anamnesis and haematological studies on the second, fifth, and tenth days of treatment. During the second and fifth days, a gradual increase in blood haemoglobin content and haematocrit value was noted. On the tenth day, there was a significant positive trend in the number of red blood cells, haemoglobin content and haematocrit to the reference values. It was established that the use of red blood cell transfusion in dogs with babesiosis as part of therapy contributed to the fastest recovery of the general condition of the animal. The material of the article is of practical value for the use of red blood cell transfusion in the treatment of dogs with babesiosis [ABSTRACT FROM AUTHOR]

Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

Quantitative systems pharmacology (QSP) has emerged as a transformative science in drug discovery and development. It is now time to fully rethink the biological functions of drug metabolizing enzymes (DMEs) and transporters within the framework of QSP models. The large set of DME and transporter genes are generally considered from the perspective of the absorption, distribution, metabolism, and excretion (ADME) of drugs. However, there is a growing amount of data on the endogenous physiology of DMEs and transporters. Recent studies-including systems biology analyses of "omics" data as well as metabolomics studies-indicate that these enzymes and transporters, which are often among the most highly expressed genes in tissues like liver, kidney, and intestine, have coordinated roles in fundamental biological processes. Multispecific DMEs and transporters work together with oligospecific and monospecific ADME proteins in a large multiorgan remote sensing and signaling network. We use the Remote Sensing and Signaling Theory (RSST) to examine the roles of DMEs and transporters in intratissue, interorgan, and interorganismal communication via metabolites and signaling molecules. This RSST-based view is applicable to bile acids, uric acid, eicosanoids, fatty acids, uremic toxins, and gut microbiome products, among other small organic molecules of physiological interest. Rooting this broader perspective of DMEs and transporters within QSP may facilitate an improved understanding of fundamental biology, physiologically based pharmacokinetics, and the prediction of drug toxicities based upon the interplay of these ADME proteins with key pathways in metabolism and signaling. The RSST-based view should also enable more tailored pharmacotherapy in the setting of kidney disease, liver disease, metabolic syndrome, and diabetes. We further discuss the pharmaceutical and regulatory implications of this revised view through the lens of systems physiology.

Therapeutic enzymes used for genetic disorders or metabolic diseases oftentimes suffer from suboptimal pharmacokinetics and stability. Nanodelivery systems have shown considerable promise for improving the performance of enzyme therapies. Here, we develop a cell membrane-camouflaged metal-organic framework (MOF) system with enhanced biocompatibility and functionality. The MOF core can efficiently encapsulate enzymes while maintaining their bioactivity. After the introduction of natural cell membrane coatings, the resulting nanoformulations can be safely administered in vivo. The surface receptors on the membrane can also provide additional functionalities that synergize with the encapsulated enzyme to target disease pathology from multiple dimensions. Employing uricase as a model enzyme, we demonstrate the utility of this approach in multiple animal disease models. The results support the use of cell membrane-coated MOFs for enzyme delivery, and this strategy could be leveraged to improve the usefulness of enzyme-based therapies for managing a wide range of important human health conditions.

Chitin is an abundant polysaccharide used by many organisms for structural rigidity and water repulsion. As such, the insoluble crystalline structure of chitin poses significant challenges for enzymatic degradation. Acidic mammalian chitinase, a processive glycosyl hydrolase, is the primary enzyme involved in the degradation of environmental chitin in mammalian lungs. Mutations to acidic mammalian chitinase have been associated with asthma, and genetic deletion in mice increases morbidity and mortality with age. We initially set out to reverse this phenotype by engineering hyperactive acidic mammalian chitinase variants. Using a screening approach with commercial fluorogenic substrates, we identified mutations with consistent increases in activity. To determine whether the activity increases observed were consistent with more biologically relevant chitin substrates, we developed new assays to quantify chitinase activity with insoluble chitin, and identified a one-pot fluorogenic assay that is sufficiently sensitive to quantify changes to activity due to the addition or removal of a carbohydrate-binding domain. We show that the activity increases from our directed evolution screen were lost when insoluble substrates were used. In contrast, naturally occurring gain-of-function mutations gave similar results with oligomeric and insoluble substrates. We also show that activity differences between acidic mammalian chitinase and chitotriosidase are reduced with insoluble substrate, suggesting that previously reported activity differences with oligomeric substrates may have been driven by differential substrate specificity. These results highlight the need for assays against physiological substrates when engineering metabolic enzymes, and provide a new one-pot assay that may prove to be broadly applicable to engineering glycosyl hydrolases.

Our objectives were to characterize responses in the field to a mix of fibrolytic enzymes using large commercial dairy herds and sufficient study power to evaluate milk production and reproductive responses to an enzyme treatment started during the precalving period. We hypothesized that the use of the enzyme treatment would increase milk production when provided to dairy cows precalving and for approximately 200 d of lactation. The study was conducted on 7,507 cows, in 8 replicates and 16 pens, at 3 dairies in the United States. Eight pens were randomly allocated as control pens and received no enzyme, and another 8 pens received enzyme treatment at a dose of 750 mL/t of dry matter feed. Milk production and energy-corrected milk yield were increased with the enzyme treatment by 0.70 and 0.80 kg/d, respectively, across a 5-month period. Milk fat percentage was not significantly increased by enzyme treatment, but milk fat yield was significantly increased by 0.040 kg/d, compared with controls. Milk protein yield increased 0.010 kg/d with enzyme treatment despite a small reduction of 0.020 percentage units in milk protein percentage. We found no evidence of an increase in the ln somatic cell count for the enzyme-treated cows. Body weight overall was not increased for enzyme-treated cows, but we did observe a numerical increase in dry matter intake (0.20 kg/head per day) for enzyme-treated cows. Most production responses to the enzyme treatment were influenced by dairy. Compared with controls, milk yield in enzyme-treated cows was significantly higher by 3.6 kg/d in dairy 2 and numerically higher by 0.60 and 0.20 kg/d in dairies 1 and 3, respectively. Reproduction, health, and risk of removal or death were not significantly influenced by treatment, apart from a reduced time to first breeding. Production responses to the enzyme treatment varied by dairy from substantial to minor increases, but variation among dairies was not evident in differences in dry matter intake or in partitioning of body weight among enzyme-treated and control pens and cows. It appears likely that the increase in production reflected increased digestibility of feed; however, further work is needed to identify factors influencing the variation in production responses to enzymes.

We review the standard model for de novo computational design of enzymes, which primarily focuses on the development of an active-site geometry, composed of protein functional groups in orientations optimized to stabilize the transition state, for a novel chemical reaction not found in nature. Its emphasis is placed on the structure and energetics of the active site embedded in an accommodating protein that serves as a physical support that shields the reaction chemistry from solvent, which is typically improved upon by laboratory-directed evolution. We also provide a review of design strategies that move beyond the standard model, by placing more emphasis on the designed enzyme as a whole catalytic construct. Starting with complete de novo enzyme design examples, we consider additional design factors such as entropy of individual residues, correlated motion between side chains (mutual information), dynamical correlations of the enzyme motions that could aid the reaction, reorganization energy, and electric fields as ways to exploit the entire protein scaffold to improve upon the catalytic rate, thereby providing directed evolution with better starting sequences for increasing biocatalytic performance.

KRAS mutations are the earliest events found in approximately 90% of pancreatic ductal adenocarcinomas (PDACs). However, little is known as to why KRAS mutations preferentially occur in PDACs and what processes/factors generate these mutations. While abnormal carbohydrate metabolism is associated with a high risk of pancreatic cancer, it remains elusive whether a direct relationship between KRAS mutations and sugar metabolism exists. Here, we show that under high-glucose conditions, cellular O-GlcNAcylation is significantly elevated in pancreatic cells that exhibit lower phosphofructokinase (PFK) activity than other cell types. This post-translational modification specifically compromises the ribonucleotide reductase (RNR) activity, leading to deficiency in dNTP pools, genomic DNA alterations with KRAS mutations, and cellular transformation. These results establish a mechanistic link between a perturbed sugar metabolism and genomic instability that induces de novo oncogenic KRAS mutations preferentially in pancreatic cells.

Many insect species display daily variation of sensitivity to insecticides when they are exposed to the same concentration at different times during the day. To date, this has not been investigated in bed bugs. To address this, we explored circadian rhythms in insecticide susceptibility, xenobiotic metabolizing (XM) gene expressions, and metabolic detoxification in the common bed bug, Cimex lectularius. An insecticide susceptible Monheim strain of C. lectularius was most tolerant of deltamethrin during the late photophase at ZT9 (i.e. nine hours after light is present in the light-dark cycle (LD) cycle) and similarly repeated at CT9 (i.e. nine hours into the subjective day in constant darkness (DD)) suggesting endogenous circadian involvement in susceptibility to deltamethrin. No diel rhythm was observed against imidacloprid insecticide despite significant daily susceptibility in both LD and DD conditions. Rhythmic expressions of metabolic detoxification genes, GSTs1 and CYP397A1 displayed similar expression patterns with total GST and P450 enzyme activities in LD and DD conditions, respectively. The oscillation of mRNA levels of GSTs1 and CYP397A1 was found consistent with peak phases of deltamethrin susceptibility in C. lectularius. This study demonstrates that circadian patterns of metabolic detoxification gene expression occur within C. lectularius. As a consequence, insecticide efficacy can vary dramatically throughout a 24 hour period.

To elucidate D-methionine's (D-met) dose and time rescue parameters from steady-state or impulse noise-induced permanent threshold shift (PTS) and determine D-met rescue's influence on serum and cochlear antioxidant levels. Five D-met doses at 0, 50, 100, or 200 mg/kg/dose administered starting at 1, 24, or 36 hours post steady-state or impulse noise exposure. Auditory brainstem responses at baseline and 21 days post-noise measured PTS. Serum (superoxide dismutase [SOD], catalase [CAT],, glutathione reductaseand glutathione peroxidase [GPx]) and cochlear (Glutathione [GSH] and glutathione disulphide [GSSG]) antioxidant levels measured physiological impact. Chinchillas (10/study group; 6–8/confirmatory groups). D-met significantly reduced PTS for impulse noise (100 mg [2, 6, 14 and 20 kHz]; 200 mg [2, 14 and 20 kHz]) and steady-state noise (all dosing groups, time parameters and tested frequencies). PTS reduction did not significantly vary by rescue time. D-met significantly increased serum SOD (100 and 200 mg for 24 hour rescue) and GPx (50 mg/kg at 24 hour rescue) at 21 days post-noise. Cochlear GSH and GSSG levels were unaffected relative to control. D-met rescues from steady-state and impulse noise-induced PTS even when administered up to 36 hours post-noise and dose-dependently influences serum antioxidant levels even 21 days post-noise. D-met's broad and effective dose/time window renders it a promising antioxidant rescue agent. [ABSTRACT FROM AUTHOR]

CBL-B is an E3 ubiquitin ligase that ubiquitinates proteins downstream of immune receptors to downregulate positive signaling cascades. Distinct homozygous mutations in CBLB were identified in 3 unrelated children with early-onset autoimmunity, one of whom also had chronic urticaria. Patient T cells exhibited hyperproliferation in response to anti-CD3 cross-linking. One of the mutations, p.R496X, abolished CBL-B expression, and a second mutation, p.C464W, resulted in preserved CBL-B expression. The third mutation, p.H285L in the SH2 domain of CBL-B, was expressed at half the normal level in the patient's cells. Mice homozygous for the CBL-B p.H257L mutation, which corresponds to the patient's p.H285L mutation, had T and B cell hyperproliferation in response to antigen receptor cross-linking. CblbH257L mice had increased percentages of T regulatory cells (Tregs) that had normal in vitro suppressive function. However, T effector cells from the patient with the p.H285L mutation and CblbH257L mice were resistant to suppression by WT Tregs. Bone marrow-derived mast cells from CblbH257L mice were hyperactivated after FcεRI cross-linking, and CblbH257L mice demonstrated exaggerated IgE-mediated passive anaphylaxis. This study establishes CBL-B deficiency as a cause of immune dysregulation. [ABSTRACT FROM AUTHOR]

An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure. [ABSTRACT FROM AUTHOR]

Objective: The intersection between immunology and metabolism contributes to the pathogenesis of obesity-associated metabolic diseases as well as molecular control of inflammatory responses. The metabolite itaconate and the cell-permeable derivatives have robust anti-inflammatory effects; therefore, it is hypothesized that cis-aconitate decarboxylase (Acod1)-produced itaconate has a protective, anti-inflammatory effect during diet-induced obesity and metabolic disease.Methods: Wild-type and Acod1-/- mice were subjected to diet-induced obesity. Glucose metabolism was analyzed by glucose tolerance tests, insulin tolerance tests, and indirect calorimetry. Gene expression and transcriptome analysis was performed using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RNA sequencing.Results: Wild-type and Acod1-/- mice on high-fat diet had equivalent weight gain, but Acod1-/- mice had impaired glucose metabolism. Insulin tolerance tests and glucose tolerance tests after 12 weeks on high-fat diet revealed significantly higher blood glucose levels in Acod1-/- mice. This was associated with significant enrichment of inflammatory gene sets and a reduction in genes related to adipogenesis and fatty acid metabolism. Analysis of naive Acod1-/- mice showed a significant increase in fat deposition at 3 and 6 months of age and obesity and insulin resistance by 12 months.Conclusions: The data show that Acod1 has an important role in the regulation of glucose homeostasis and obesity under normal and high-fat diet conditions. [ABSTRACT FROM AUTHOR]

Background: The role of club cells in the pathology of idiopathic pulmonary fibrosis (IPF) is not well understood. Protein disulfide isomerase A3 (PDIA3), an endoplasmic reticulum-based redox chaperone required for the functions of various fibrosis-related proteins; however, the mechanisms of action of PDIA3 in pulmonary fibrosis are not fully elucidated.Objectives: To examine the role of club cells and PDIA3 in the pathology of pulmonary fibrosis and the therapeutic potential of inhibition of PDIA3 in lung fibrosis.Methods: Role of PDIA3 and aberrant club cells in lung fibrosis was studied by analyses of human transcriptome dataset from Lung Genomics Research Consortium, other public resources, the specific deletion or inhibition of PDIA3 in club cells and blocking SPP1 downstream of PDIA3 in mice.Results: PDIA3 and club cell secretory protein (SCGB1A1) signatures are upregulated in IPF compared with control patients. PDIA3 or SCGB1A1 increases also correlate with a decrease in lung function in patients with IPF. The bleomycin (BLM) model of lung fibrosis showed increases in PDIA3 in SCGB1A1 cells in the lung parenchyma. Ablation of Pdia3, specifically in SCGB1A1 cells, decreases parenchymal SCGB1A1 cells along with fibrosis in mice. The administration of a PDI inhibitor LOC14 reversed the BLM-induced parenchymal SCGB1A1 cells and fibrosis in mice. Evaluation of PDIA3 partners revealed that SPP1 is a major interactor in fibrosis. Blocking SPP1 attenuated the development of lung fibrosis in mice.Conclusions: Our study reveals a new relationship with distally localised club cells, PDIA3 and SPP1 in lung fibrosis and inhibition of PDIA3 or SPP1 attenuates lung fibrosis. [ABSTRACT FROM AUTHOR]

Background: Inflammation is a potential risk factor of mental disturbance. FKBP5 that encodes FK506-binding protein 51 (FKBP51), a negative cochaperone of glucocorticoid receptor (GR), is a stress-inducible gene and has been linked to psychiatric disorders. Yet, the role of FKBP51 in the inflammatory stress-associated mental disturbance remained unclear.Methods: Fkbp5-deficient (Fkbp5-KO) mice were used to study inflammatory stress by a single intraperitoneal injection of lipopolysaccharide (LPS). The anxiety-like behaviors, neuroimaging, immunofluorescence staining, immunohistochemistry, protein and mRNA expression analysis of inflammation- and neurotransmission-related mediators were evaluated. A dexamethasone drinking model was also applied to examine the effect of Fkbp5-KO in glucocorticoid-induced stress.Results: LPS administration induced FKBP51 elevation in the liver and hippocampus accompanied with transient sickness. Notably, Fkbp5-KO but not wild-type (WT) mice showed anxiety-like behaviors 7 days after LPS injection (LPS-D7). LPS challenge rapidly increased peripheral and central immune responses and hippocampal microglial activation followed by a delayed GR upregulation on LPS-D7, and these effects were attenuated in Fkbp5-KO mice. Whole-brain [18F]-FEPPA neuroimaging, which target translocator protein (TSPO) to indicate neuroinflammation, showed that Fkbp5-KO reduced LPS-induced neuroinflammation in various brain regions including hippocampus. Interestingly, LPS elevated glutamic acid decarboxylase 65 (GAD65), the membrane-associated GABA-synthesizing enzyme, in the hippocampus of WT but not Fkbp5-KO mice on LPS-D7. This FKBP51-dependent GAD65 upregulation was observed in the ventral hippocampal CA1 accompanied by the reduction of c-Fos-indicated neuronal activity, whereas both GAD65 and neuronal activity were reduced in dorsal CA1 in a FKBP51-independent manner. GC-induced anxiety was also examined, which was attenuated in Fkbp5-KO and hippocampal GAD65 expression was unaffected.Conclusions: These results suggest that FKBP51/FKBP5 is involved in the systemic inflammation-induced neuroinflammation and hippocampal GR activation, which may contribute to the enhancement of GAD65 expression for GABA synthesis in the ventral hippocampus, thereby facilitating resilience to inflammation-induced anxiety. [ABSTRACT FROM AUTHOR]

Background: The bone marrow (BM) is the most common site of dissemination in patients with aggressive, metastatic neuroblastoma (NB). However, the molecular mechanisms underlying the aggressive behavior of NB cells in the BM niche are still greatly unknown. In the present study, we explored biological mechanisms that play a critical role in NB cell survival and progression in the BM and investigated potential therapeutic targets.Methods: Patient-derived bone marrow (BM) primary cultures were generated using fresh BM aspirates obtained from NB patients. NB cell lines were cultured in the presence of BM conditioned media containing cell-secreted factors, and under low oxygen levels (1% O2) to mimic specific features of the BM microenvironment of high-risk NB patients. The BM niche was explored using cytokine profiling assays, cell migration-invasion and viability assays, flow cytometry and analysis of RNA-sequencing data. Selective pharmacological inhibition of factors identified as potential mediators of NB progression within the BM niche was performed in vitro and in vivo.Results: We identified macrophage migration inhibitory factor (MIF) as a key inflammatory cytokine involved in BM infiltration. Cytokine profiling and RNA-sequencing data analysis revealed NB cells as the main source of MIF in the BM, suggesting a potential role of MIF in tumor invasion. Exposure of NB cells to BM-conditions increased NB cell-surface expression of the MIF receptor CXCR4, which was associated with increased cell viability, enhanced migration-invasion, and activation of PI3K/AKT and MAPK/ERK signaling pathways. Moreover, subcutaneous co-injection of NB and BM cells enhanced tumor engraftment in mice. MIF inhibition with 4-IPP impaired in vitro NB aggressiveness, and improved drug response while delayed NB growth, improving survival of the NB xenograft model.Conclusions: Our findings suggest that BM infiltration by NB cells may be mediated, in part, by MIF-CXCR4 signaling. We demonstrate the antitumor efficacy of MIF targeting in vitro and in vivo that could represent a novel therapeutic target for patients with disseminated high-risk NB. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation with aberrant epigenetic alterations, eventually leading to joint destruction. However, the epigenetic regulatory mechanisms underlying RA pathogenesis remain largely unknown. Here, we showed that ubiquitin-like containing PHD and RING finger domains 1 (UHRF1) is a central epigenetic regulator that orchestrates multiple pathogeneses in RA in a suppressive manner. UHRF1 expression was remarkably upregulated in synovial fibroblasts (SFs) from arthritis model mice and patients with RA. Mice with SF-specific Uhrf1 conditional knockout showed more severe arthritic phenotypes than littermate controls. Uhrf1-deficient SFs also exhibited enhanced apoptosis resistance and upregulated expression of several cytokines, including Ccl20. In patients with RA, DAS28, CRP, and Th17 accumulation and apoptosis resistance were negatively correlated with UHRF1 expression in synovium. Finally, Ryuvidine administration stabilized UHRF1 ameliorated arthritis pathogeneses in a mouse model of RA. This study demonstrated that UHRF1 expressed in RA SFs can contribute to negative feedback mechanisms that suppress multiple pathogenic events in arthritis, suggesting that targeting UHRF1 could be one of the therapeutic strategies for RA. [ABSTRACT FROM AUTHOR]

Activation-induced cytidine deaminase (AID) is a genome-mutating enzyme that initiates class switch recombination and somatic hypermutation of antibodies in jawed vertebrates. We previously described the biochemical properties of human AID and found that it is an unusual enzyme in that it exhibits binding affinities for its substrate DNA and catalytic rates several orders of magnitude higher and lower, respectively, than a typical enzyme. Recently, we solved the functional structure of AID and demonstrated that these properties are due to nonspecific DNA binding on its surface, along with a catalytic pocket that predominantly assumes a closed conformation. Here we investigated the biochemical properties of AID from a sea lamprey, nurse shark, tetraodon, and coelacanth: representative species chosen because their lineages diverged at the earliest critical junctures in evolution of adaptive immunity. We found that these earliest-diverged AID orthologs are active cytidine deaminases that exhibit unique substrate specificities and thermosensitivities. Significant amino acid sequence divergence among these AID orthologs is predicted to manifest as notable structural differences. However, despite major differences in sequence specificities, thermosensitivities, and structural features, all orthologs share the unusually high DNA binding affinities and low catalytic rates. This absolute conservation is evidence for biological significance of these unique biochemical properties.

The present work describes an attractive skin-worn microneedle sensing device for the minimally invasive electrochemical monitoring of subcutaneous alcohol. The device consists of an assembly of pyramidal microneedle structures integrated with Pt and Ag wires, each with a microcavity opening. The microneedle aperture was modified by electropolymerizing o-phenylene diamine onto the Pt wire microtransducer, followed by the immobilization of alcohol oxidase (AOx) in an intermediate chitosan layer, along with an outer Nafion layer. The resulting microneedle-based enzyme electrode displays an interference-free ethanol detection in artificial interstitial fluid without compromising its sensitivity, stability and response time. The skin penetration ability and the efficaciousness of the biosensor performance towards subcutaneous alcohol monitoring was substantiated by the ex vivo mice skin model analysis. Our results reveal that the new microneedle sensor holds considerable promise for continuous non-invasive alcohol monitoring in real-life situations.

Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.

The oviductal microenvironment is a site for key events that involve gamete maturation, fertilization and early embryo development. Secretions into the oviductal lumen by either the lining epithelium or by transudation of plasma constituents are known to contain elements conducive for reproductive success. Although previous studies have identified some of these factors involved in reproduction, knowledge of secreted proteins in the oviductal fluid remains rudimentary with limited definition of function even in extensively studied species like cattle. In this study, we used a shotgun proteomics approach followed by bioinformatics sequence prediction to identify secreted proteins present in the bovine oviductal fluid (ex vivo) and secretions from the bovine oviductal epithelial cells (in vitro). From a total of 2087 proteins identified, 266 proteins could be classified as secreted, 109 (41%) of which were common for both in vivo and in vitro conditions. Pathway analysis indicated different classes of proteins that included growth factors, metabolic regulators, immune modulators, enzymes, and extracellular matrix components. Functional analysis revealed mechanisms in the oviductal lumen linked to immune homeostasis, gamete maturation, fertilization and early embryo development. These results point to several novel components that work together with known elements mediating functional homeostasis, and highlight the diversity of machinery associated with oviductal physiology and early events in cattle fertility.

The Gene Ontology (GO) (Ashburner et al., Nat Genet 25(1):25-29, 2000) is a powerful tool in the informatics arsenal of methods for evaluating annotations in a protein dataset. From identifying the nearest well annotated homologue of a protein of interest to predicting where misannotation has occurred to knowing how confident you can be in the annotations assigned to those proteins is critical. In this chapter we explore what makes an enzyme unique and how we can use GO to infer aspects of protein function based on sequence similarity. These can range from identification of misannotation or other errors in a predicted function to accurate function prediction for an enzyme of entirely unknown function. Although GO annotation applies to any gene products, we focus here a describing our approach for hierarchical classification of enzymes in the Structure-Function Linkage Database (SFLD) (Akiva et al., Nucleic Acids Res 42(Database issue):D521-530, 2014) as a guide for informed utilisation of annotation transfer based on GO terms.

Obesity is a serious health problem in the US and is associated with increased risks of various human diseases. To date, the mechanisms by which obesity increases the risks of a wide range of human diseases are not well understood. Here we used a LC-MS/MS-based lipidomics, which can analyze >100 bioactive lipid mediators produced by cyclooxygenase, lipoxygenase, and cytochrome P450 enzymes, to analyze plasma profiles of lipid mediators in high-fat diet induced obesity in C57BL/6 mice. Our results show that the plasma concentrations of epoxyoctadecenoic acids (EpOMEs, also termed as leukotoxins) are significantly increased in plasma of high-fat diet-fed mice, in addition, EpOMEs are among the most abundant lipid mediators detected in mouse plasma. Since substantial studies have shown that EpOMEs and their metabolites have a large array of detrimental effects on health, enhanced levels of EpOMEs could contribute to the pathology of obesity.

Background: The interferon response can influence the primary and metastatic activity of breast cancers and can interact with checkpoint immunotherapy to modulate its effects. Using N-ethyl-N-nitrosourea mutagenesis, we found a mouse with an activating mutation in oligoadenylate synthetase 2 (Oas2), a sensor of viral double stranded RNA, that resulted in an interferon response and prevented lactation in otherwise healthy mice.Methods: To determine if sole activation of Oas2 could alter the course of mammary cancer, we combined the Oas2 mutation with the MMTV-PyMT oncogene model of breast cancer and examined disease progression and the effects of checkpoint immunotherapy using Kaplan-Meier survival analysis with immunohistochemistry and flow cytometry.Results: Oas2 mutation prevented pregnancy from increasing metastases to lung. Checkpoint immunotherapy with antibodies against programmed death-ligand 1 was more effective when the Oas2 mutation was present.Conclusions: These data establish OAS2 as a therapeutic target for agents designed to reduce metastases and increase the effectiveness of checkpoint immunotherapy. [ABSTRACT FROM AUTHOR]

In metabolism, available free energy is limited and must be divided across pathway steps to maintain a negative ΔG throughout. For each reaction, ΔG is log proportional both to a concentration ratio (reaction quotient to equilibrium constant) and to a flux ratio (backward to forward flux). Here we use isotope labeling to measure absolute metabolite concentrations and fluxes in Escherichia coli, yeast and a mammalian cell line. We then integrate this information to obtain a unified set of concentrations and ΔG for each organism. In glycolysis, we find that free energy is partitioned so as to mitigate unproductive backward fluxes associated with ΔG near zero. Across metabolism, we observe that absolute metabolite concentrations and ΔG are substantially conserved and that most substrate (but not inhibitor) concentrations exceed the associated enzyme binding site dissociation constant (Km or Ki). The observed conservation of metabolite concentrations is consistent with an evolutionary drive to utilize enzymes efficiently given thermodynamic and osmotic constraints.

There is currently no effective treatment for multiorgan failure following shock other than supportive care. A better understanding of the pathogenesis of these sequelae to shock is required. The intestine plays a central role in multiorgan failure. It was previously suggested that bacteria and their toxins are responsible for the organ failure seen in circulatory shock, but clinical trials in septic patients have not confirmed this hypothesis. Instead, we review here evidence that the digestive enzymes, synthesized in the pancreas and discharged into the small intestine as requirement for normal digestion, may play a role in multiorgan failure. These powerful enzymes are nonspecific, highly concentrated, and fully activated in the lumen of the intestine. During normal digestion they are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. However, if this barrier becomes permeable, e.g. in an ischemic state, the digestive enzymes escape into the wall of the intestine. They digest tissues in the mucosa and generate small molecular weight cytotoxic fragments such as unbound free fatty acids. Digestive enzymes may also escape into the systemic circulation and activate other degrading proteases. These proteases have the ability to clip the ectodomain of surface receptors and compromise their function, for example cleaving the insulin receptor causing insulin resistance. The combination of digestive enzymes and cytotoxic fragments leaking into the central circulation causes cell and organ dysfunction, and ultimately may lead to complete organ failure and death. We summarize current evidence suggesting that enteral blockade of digestive enzymes inside the lumen of the intestine may serve to reduce acute cell and organ damage and improve survival in experimental shock.

There is currently no effective treatment for multiorgan failure following shock other than supportive care. A better understanding of the pathogenesis of these sequelae to shock is required. The intestine plays a central role in multiorgan failure. It was previously suggested that bacteria and their toxins are responsible for the organ failure seen in circulatory shock, but clinical trials in septic patients have not confirmed this hypothesis. Instead, we review here evidence that the digestive enzymes, synthesized in the pancreas and discharged into the small intestine as requirement for normal digestion, may play a role in multiorgan failure. These powerful enzymes are nonspecific, highly concentrated, and fully activated in the lumen of the intestine. During normal digestion they are compartmentalized in the lumen of the intestine by the mucosal epithelial barrier. However, if this barrier becomes permeable, e.g. in an ischemic state, the digestive enzymes escape into the wall of the intestine. They digest tissues in the mucosa and generate small molecular weight cytotoxic fragments such as unbound free fatty acids. Digestive enzymes may also escape into the systemic circulation and activate other degrading proteases. These proteases have the ability to clip the ectodomain of surface receptors and compromise their function, for example cleaving the insulin receptor causing insulin resistance. The combination of digestive enzymes and cytotoxic fragments leaking into the central circulation causes cell and organ dysfunction, and ultimately may lead to complete organ failure and death. We summarize current evidence suggesting that enteral blockade of digestive enzymes inside the lumen of the intestine may serve to reduce acute cell and organ damage and improve survival in experimental shock.

Enzymes are complex biomolecules capable of performing unique catalysis under physiological conditions at neutral temperature and pH. However, the architecture of enzymatic catalysis is often a combination of the quantum influence of the immediate active site, as well as the electrostatic and configurational influences of amino acids surrounding the active site. As a result of this cooperation between baseline chemical reactivity and electrostatic assistance, it has become important to model enzymes using multiscale methods that take advantage of treating the active site with quantum mechanical methods, while approximately treating the surrounding protein using cheaper, classically driven force-field molecular mechanics methods. Here we describe the use of a multiscale engine which utilizes a combination of density functional theory with discrete molecular dynamics (dubbed QM/DMD) to aid in the characterization of metalloenzymes.

Enzymes are complex biomolecules capable of performing unique catalysis under physiological conditions at neutral temperature and pH. However, the architecture of enzymatic catalysis is often a combination of the quantum influence of the immediate active site, as well as the electrostatic and configurational influences of amino acids surrounding the active site. As a result of this cooperation between baseline chemical reactivity and electrostatic assistance, it has become important to model enzymes using multiscale methods that take advantage of treating the active site with quantum mechanical methods, while approximately treating the surrounding protein using cheaper, classically driven force-field molecular mechanics methods. Here we describe the use of a multiscale engine which utilizes a combination of density functional theory with discrete molecular dynamics (dubbed QM/DMD) to aid in the characterization of metalloenzymes.

The Concise Guide to PHARMACOLOGY 2015/16 provides concise overviews of the key properties of over 1750 human drug targets with their pharmacology, plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. The full contents can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13354/full. G protein-coupled receptors are one of the eight major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The Concise Guide is published in landscape format in order to facilitate comparison of related targets. It is a condensed version of material contemporary to late 2015, which is presented in greater detail and constantly updated on the website www.guidetopharmacology.org, superseding data presented in the previous Guides to Receptors & Channels and the Concise Guide to PHARMACOLOGY 2013/14. It is produced in conjunction with NC-IUPHAR and provides the official IUPHAR classification and nomenclature for human drug targets, where appropriate. It consolidates information previously curated and displayed separately in IUPHAR-DB and GRAC and provides a permanent, citable, point-in-time record that will survive database updates.

Sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor in lipogenesis and lipid metabolism, is critical for disease progression and associated with poor outcomes in prostate cancer (PCa) patients. However, the mechanism of SREBP-1 regulation in PCa remains elusive. Here, we report that SREBP-1 is transcriptionally regulated by microRNA-21 (miR-21) in vitro in cultured cells and in vivo in mouse models. We observed aberrant upregulation of SREBP-1, fatty acid synthase (FASN) and acetyl-CoA carboxylase (ACC) in Pten/Trp53 double-null mouse embryonic fibroblasts (MEFs) and Pten/Trp53 double-null mutant mice. Strikingly, miR-21 loss significantly reduced cell proliferation and suppressed the prostate tumorigenesis of Pten/Trp53 mutant mice. Mechanistically, miR-21 inactivation decreased the levels of SREBP-1, FASN, and ACC in human PCa cells through downregulation of insulin receptor substrate 1 (IRS1)-mediated transcription and induction of cellular senescence. Conversely, miR-21 overexpression increased cell proliferation and migration; as well as the levels of IRS1, SREBP-1, FASN, and ACC in human PCa cells. Our findings reveal that miR-21 promotes PCa progression by activating the IRS1/SREBP-1 axis, and targeting miR-21/SREBP-1 signaling pathway can be a novel strategy for controlling PCa malignancy. [ABSTRACT FROM AUTHOR]

Mitochondrial defects are a hallmark of early pathophysiology in Alzheimer's disease, with pathologically phosphorylated tau reported to induce mitochondrial toxicity. Mitophagy constitutes a key pathway in mitochondrial quality control by which damaged mitochondria are targeted for autophagy. However, few details are known regarding the intersection of mitophagy and pathologies in tauopathy. Here, by applying biochemical and cell biological approaches including time-lapse confocal imaging in live tauopathy neurons, combined with gene rescue experiments via stereotactic injections of adeno-associated virus particles into tauopathy mouse brains, electrophysiological recordings and behavioural tests, we demonstrate for the first time that mitochondrial distribution deficits at presynaptic terminals are an early pathological feature in tauopathy brains. Furthermore, Parkin-mediated mitophagy is extensively activated in tauopathy neurons, which accelerates mitochondrial Rho GTPase 1 (Miro1) turnover and consequently halts Miro1-mediated mitochondrial anterograde movement towards synaptic terminals. As a result, mitochondrial supply at tauopathy synapses is disrupted, impairing synaptic function. Strikingly, increasing Miro1 levels restores the synaptic mitochondrial population by enhancing mitochondrial anterograde movement and thus reverses tauopathy-associated synaptic failure. In tauopathy mouse brains, overexpression of Miro1 markedly elevates synaptic distribution of mitochondria and protects against synaptic damage and neurodegeneration, thereby counteracting impairments in learning and memory as well as synaptic plasticity. Taken together, our study reveals that activation of the Parkin pathway triggers an unexpected effect-depletion of mitochondria from synaptic terminals, a characteristic feature of early tauopathy. We further provide new mechanistic insights into how parkin activation-enhanced Miro1 degradation and impaired mitochondrial anterograde transport drive tauopathy-linked synaptic pathogenesis and establish a foundation for future investigations into new therapeutic strategies to prevent synaptic deterioration in Alzheimer's disease and other tauopathies. [ABSTRACT FROM AUTHOR]

Adult Weddell seals (Leptonychotes weddellii) haul-out on the ice in October/November (austral spring) for the breeding season and reduce foraging activities for ~4 months until their molt in the austral fall (January/February). After these periods, animals are at their leanest and resume actively foraging for the austral winter. In mammals, decreased exercise and hypoxia exposure typically lead to decreased production of O2-carrying proteins and muscle wasting, while endurance training increases aerobic potential. To test whether similar effects were present in marine mammals, this study compared the physiology of 53 post-molt female Weddell seals in the austral fall to 47 pre-breeding females during the spring in McMurdo Sound, Antarctica. Once body mass and condition (lipid) were controlled for, there were no seasonal changes in total body oxygen (TBO2) stores. Within each season, hematocrit and hemoglobin values were negatively correlated with animal size, and larger animals had lower mass-specific TBO2 stores. But because larger seals had lower mass-specific metabolic rates, their calculated aerobic dive limit was similar to smaller seals. Indicators of muscular efficiency, myosin heavy chain composition, myoglobin concentrations, and aerobic enzyme activities (citrate synthase and β-hydroxyacyl CoA dehydrogenase) were likewise maintained across the year. The preservation of aerobic capacity is likely critical to foraging capabilities, so that following the molt Weddell seals can rapidly regain body mass at the start of winter foraging. In contrast, muscle lactate dehydrogenase activity, a marker of anaerobic metabolism, exhibited seasonal plasticity in this diving top predator and was lowest after the summer period of reduced activity.

Novel ibuprofen-containing monomers comprising naturally occurring and biocompatible compounds were synthesized and subsequently polymerized via enzymatic methods. Through the use of a malic acid sugar backbone, ibuprofen was attached as a pendant group, and then subsequently polymerized with a linear aliphatic diol (1,3-propanediol, 1,5-pentanediol, or 1,8-octanediol) as comonomer using lipase B from Candida antarctica, a greener alternative to traditional metal catalysts. Polymer structures were elucidated by nuclear magnetic resonance and infrared spectroscopies, and thermal properties and molecular weights were determined. All polymers exhibited sustained ibuprofen release, with the longer chain, more hydrophobic diols exhibiting the slowest release over the 30 d study. Polymers were deemed cytocompatible using mouse fibroblasts, when evaluated at relevant therapeutic concentrations. Additionally, ibuprofen retained its chemical integrity throughout the polymerization and in vitro hydrolytic degradation processes. This methodology of enzymatic polymerization of a drug presents a more environmentally friendly synthesis and a novel approach to bioactive polymer conjugates.

The coffee berry borer, Hypothenemus hampei, is the most economically important insect pest of coffee worldwide. We present an analysis of the draft genome of the coffee berry borer, the third genome for a Coleopteran species. The genome size is ca. 163 Mb with 19,222 predicted protein-coding genes. Analysis was focused on genes involved in primary digestion as well as gene families involved in detoxification of plant defense molecules and insecticides, such as carboxylesterases, cytochrome P450, gluthathione S-transferases, ATP-binding cassette transporters, and a gene that confers resistance to the insecticide dieldrin. A broad range of enzymes capable of degrading complex polysaccharides were identified. We also evaluated the pathogen defense system and found homologs to antimicrobial genes reported in the Drosophila genome. Ten cases of horizontal gene transfer were identified with evidence for expression, integration into the H. hampei genome, and phylogenetic evidence that the sequences are more closely related to bacterial rather than eukaryotic genes. The draft genome analysis broadly expands our knowledge on the biology of a devastating tropical insect pest and suggests new pest management strategies.

Orchestrated biological materials such as enzymes and growth factors regulate the growth of tissues and organs. A chirality-controlled, single-protein technology is devised to tailor the spatiotemporally defined delivery of therapeutic proteins in response to natural enzymes present at wound sites. Sustained delivery of one protein and sequential delivery of two proteins are demonstrated for stroke and skin wound healing.

The recent identification of the interplay between metabolizing enzymes and BCRP has drawn more and more attention from people. BCRP, a transporter belonging to ATP-binding cassette (ABC) family, has been hypothesized to play roles in many aspects including protecting the human body against therapeutics because it is expressed in the tissues that function as barriers in vivo. Efficient coupling of BCRP and metabolizing enzymes enables rapid elimination of foreign compounds from the body because BCRP could facilitate the excretion of metabolites catalyzed by phase I and II enzymes into bile, urine and feces. Without BCRP coupling, pass through the cell membrane may be difficult for them by passive diffusion because of the increment of the molecular weight and water solubility. Thus the metabolism-efflux alliance has extraordinary importance to drug metabolism, distribution, pharmacological effect, toxicity and elimination. In this manuscript, a brief discussion about the interplays of BCRP and metabolizing enzymes in liver, intestine, kidney, lung and other organs were presented and summarized. Many endogenous and exogenous compounds belong to different chemical groups, for instance, the dietary flavonoids and the steroidal hormones were involved. Clarifying the cooperation mechanisms of BCRP and enzymes could lead to a better prediction of drug clearance in vitro.

Thirty-five isolates of Staphylococcus were isolated out of 100 clinical specimens were collected from different infections of human and animals. These isolates were identified as Staphylococcus aureus, 15 of them isolated from human, 15 from cows and only 5 isolates from poultry. Twenty-five (71.4%) isolates of different sources revealed positive results for the detection of protein A, capsule formation and enzyme production of phosphatase, DNase and haemolysin. Seven (20 %) isolates showed negative results for both protein A and capsule as well as for the production of enzymes while only three (8.5%) isolates were not capsulated but able to produce protein A and the enzymes. During the detection of protein A, capsule structure and production of some enzymes, we found that the best value of pH used was the range of 7-7.5. The present study indicated that the presence of protein A and capsule structure is highly associated with the virulence of isolates, and loss of protein A doesn't necessary mean loss of capsule as well. The ability of the isolates to produce their essential enzymes to induct the infection is highly correlated with the ability to produce protein A, without considering the source of the isolates.

Objective: The fetal alcohol spectrum disorder (FASD) is a group of clinical conditions associated with the in utero exposure to ethanol (EtOH). We have recently examined the effects of a moderate maternal exposure to EtOH on crucial brain enzyme activities in offspring rats, and discussed the translational challenges arising when attempting to simulate any of the clinical conditions associated with FASD.Materials and Methods: In this current study, we: (i) address the need for a more consistent and reliable in vivo experimental platform that could simulate milder cases of FASD complicated by simultaneous thiamine-deprivation during gestation and (ii) explore the effects of such a moderate maternal exposure pattern to EtOH and a thiamine-deficient diet (TDD) on crucial enzyme activities in the offspring rat brains.Results: We demonstrate a significant decrease in the newborn and 21-day-old offspring body and brain weight due to maternal dietary thiamine-deprivation, as well as evidence of crucial brain enzyme activity alterations that in some cases are present in the offspring rat brains long after birth (and the end of the maternal exposure to both EtOH and TDD).Conclusions: Our findings provide a preliminary characterization of important neurochemical effects due to maternal exposure to EtOH and TDD during gestation that might affect the offspring rat neurodevelopment, and that characterization should be further explored in a brain region-specific manner level as well as through the parallel examination of changes in the offspring rat brain lipid composition. [ABSTRACT FROM AUTHOR]

BackgroundStick and leaf insects (Phasmatodea) are an exclusively leaf-feeding order of insects with no record of omnivory, unlike other "herbivorous" Polyneoptera. They represent an ideal system for investigating the adaptations necessary for obligate folivory, including plant cell wall degrading enzymes (PCWDEs). However, their physiology and internal anatomy is poorly understood, with limited genomic resources available.ResultsWe de novo assembled transcriptomes for the anterior and posterior midguts of six diverse Phasmatodea species, with RNA-Seq on one exemplar species, Peruphasma schultei. The latter's assembly yielded >100,000 transcripts, with over 4000 transcripts uniquely or more highly expressed in specific midgut sections. Two to three dozen PCWDE encoding gene families, including cellulases and pectinases, were differentially expressed in the anterior midgut. These genes were also found in genomic DNA from phasmid brain tissue, suggesting endogenous production. Sequence alignments revealed catalytic sites on most PCWDE transcripts. While most phasmid PCWDE genes showed homology with those of other insects, the pectinases were homologous to bacterial genes.ConclusionsWe identified a large and diverse PCWDE repertoire endogenous to the phasmids. If these expressed genes are translated into active enzymes, then phasmids can theoretically break plant cell walls into their monomer components independently of microbial symbionts. The differential gene expression between the two midgut sections provides the first molecular hints as to their function in living phasmids. Our work expands the resources available for industrial applications of animal-derived PCWDEs, and facilitates evolutionary analysis of lower Polyneopteran digestive enzymes, including the pectinases whose origin in Phasmatodea may have been a horizontal transfer event from bacteria.

There are an increasing number of human pathologies that have been associated with altered metabolism, including obesity, diabetes, atherosclerosis, cancer, and neurodegenerative diseases. Most attention on metabolism has been focused on well-understood metabolic pathways and has largely ignored most of the biochemical pathways that operate in (patho)physiological settings, in part because of the vast landscape of uncharacterized and undiscovered metabolic pathways. One technology that has arisen to meet this challenge is activity-based protein profiling (ABPP) that uses activity-based chemical probes to broadly assess the functional states of both characterized and uncharacterized enzymes. This review will focus on how ABPP, coupled with inhibitor discovery platforms and functional metabolomic technologies, have led to discoveries that have expanded our knowledge of metabolism in health and disease.

Two novel synthetic α2-6-linked disialyl hexasaccharides, disialyllacto-N-neotetraose (DSLNnT) and α2-6-linked disialyllacto-N-tetraose (DS'LNT), were readily obtained by highly efficient one-pot multienzyme (OPME) reactions. The sequential OPME systems described herein allowed the use of an inexpensive disaccharide and simple monosaccharides to synthesize the desired complex oligosaccharides with high efficiency and selectivity. DSLNnT and DS'LNT were shown to protect neonatal rats from necrotizing enterocolitis (NEC) and are good therapeutic candidates for preclinical experiments and clinical application in treating NEC in preterm infants.

Alkaline phosphatase (AP) isozymes are present in a wide range of species from bacteria to man and are capable of dephosphorylation and transphosphorylation of a wide spectrum of substrates in vitro. In humans, four AP isozymes have been identified-one tissue-nonspecific (TNAP) and three tissue-specific-named according to the tissue of their predominant expression: intestinal (IAP), placental (PLAP) and germ cell (GCAP) APs. Modulation of activity of the different AP isozymes may have therapeutic implications in distinct diseases and cellular processes. For instance, changes in the level of IAP activity can affect gut mucosa tolerance to microbial invasion due to the ability of IAP to detoxify bacterial endotoxins, alter the absorption of fatty acids and affect ectopurinergic regulation of duodenal bicarbonate secretion. To identify isozyme selective modulators of the human and mouse IAPs, we developed a series of murine duodenal IAP (Akp3-encoded dIAP isozyme), human IAP (hIAP), PLAP, and TNAP assays. High throughput screening and subsequent SAR efforts generated a potent inhibitor of dIAP, ML260, with specificity for the Akp3-, compared to the Akp5- and Akp6-encoded mouse isozymes.

Matrix metalloproteinase enzymes, overexpressed in HT-1080 human fibrocarcinoma tumors, were used to guide the accumulation and retention of an enzyme-responsive nanoparticle in a xenograft mouse model. The nanoparticles were prepared as micelles from amphiphilic block copolymers bearing a simple hydrophobic block and a hydrophilic peptide brush. The polymers were end-labeled with Alexa Fluor 647 dyes leading to the formation of labeled micelles upon dialysis of the polymers from DMSO/DMF to aqueous buffer. This dye-labeling strategy allowed the presence of the retained material to be visualized via whole animal imaging in vivo and in ex vivo organ analysis following intratumoral injection into HT-1080 xenograft tumors. We propose that the material is retained by virtue of an enzyme-induced accumulation process whereby particles change morphology from 20 nm spherical micelles to micrometer-scale aggregates, kinetically trapping them within the tumor. This hypothesis is tested here via an unprecedented super-resolution fluorescence analysis of ex vivo tissue slices confirming a particle size increase occurs concomitantly with extended retention of responsive particles compared to unresponsive controls.

The involvement of nitric oxide (NO) in preventing bone loss has long been hypothesized, but despite decades of research the mechanisms remain obscure. In this issue of the JCI, Jin et al. explored NO deficiency using human cell and mouse models that lacked argininosuccinate lyase (ASL), the enzyme involved in synthesizing arginine and NO production. Osteoblasts that did not express ASL produced less NO and failed to differentiate. Notably, in the context of Asl deficiency, heterozygous deletion of caveolin 1, which normally inhibits NO synthesis, restored NO production, osteoblast differentiation, glycolysis, and bone mass. These experiments suggest that ASL regulates arginine synthesis in osteoblasts, which leads to enhanced NO production and increased glucose metabolism. After a period when research slowed, these studies, like the legendary phoenix, renew the exploration of NO in bone biology, and provide exciting translational potential. [ABSTRACT FROM AUTHOR]

New neuroprotective treatments of natural origin are being investigated. Both, plant extracts and isolated compounds have shown bioactive effects. Hempseed is known for its composition of fatty acids, proteins, fibre, vitamins, as well as a large number of phytochemical compounds. After a defatting process of the seeds, hydroxycinnamic acids and its amine derivatives are the majoritarian compounds in an ethyl acetate fraction (EAF). In the present study, we investigated in vitro effect on neuronal enzymes: MAO-A, MAO-B, tyrosinase and acetylcholinesterase. Besides, the effect of EAF on striatal biogenic amines in mice was evaluated. Both, EAF and isolated compounds (N-trans-caffeoyltyramine and N-trans-coumaroyltyramine), showed inhibitory action on MAO-A, MAO-B and tyrosinase. Furthermore, an increasing of biogenic amines was observed in the corpus striatum of the mice, after administration of EAF. These findings show that EAF and the hydroxycinnamic acid derivatives may represent a potential treatment in degenerative neuronal diseases. [ABSTRACT FROM AUTHOR]