Enzyme Release Research Articles

Polyamidoamine Dendrimers Functionalized with ZnO-Chitosan Nanoparticles as an Efficient Surface for L-asparaginase Immobilization.

In this study, the third-generation polyamidoamine dendrimer was functionalized with a 5-amino-1H-tetrazole heterocycle to load the synthesis enzyme and its surface groups. Then, chitosan was attached to the dendrimer by a suitable linker, and finally, zinc oxide nanoparticles were inserted into dendrimer cavities to increase loading. FTIR, FESEM, TEM, and DLS analysis showed that this new dendrimer has specific branches, and ZnO nanoparticles were spread between the branches and connected with the branches and chitosan biopolymer. Also proved the presence of stabilized L-asparaginase enzyme and ZnO nanoparticles in the designed system. Furthermore, the extent of L-asparaginase enzyme loading and release was investigated in the laboratory with a dialysis bag. Examining the toxicity of the new third-generation polyamidoamine (PAMAM) dendrimeric nanocarrier based on chitosan-zinc oxide biopolymer (PAMAM-G3@ZnO-Cs nanocarrier) on the Jurkat cell line (human acute lymphoblastic leukemia) at pH 7.4 showed that this nanocarrier effectively encapsulates the drug L-asparaginase and slowly releases it and also preventing the growth of cancer cells. The activity of the loaded enzyme in the nanocarrier and the free enzyme was calculated. During the investigations, it was found that the enzyme attached to the nanocarrier is more stable than the free enzyme at optimal pH and temperature and at high temperatures, acidic and basic pHs. Vmax and Km values were lower for loaded enzymes. The synthesized PAMAM-G3@ZnO-Cs nanocarrier can be a promising candidate in the pharmaceutical industry and medical science for cancer treatment due to its biocompatibility, non-toxicity, stability, and slow release of L-asparaginase.

An “On-Off” self-powered biosensor via GOD activated signal transduction for ultrasensitive detection of multiple biomarkers

A “on–off” self-powered biosensor was developed based on enzymatic biofuel cells (EBFCs) for sequential detection of microRNA-21 (miRNA-21) and miRNA-155 by capture and release of a single-bioanode enzyme, i.e., glucose oxidase. When miRNA-21 is present, it hybridizes with DNA1 and the glucose oxidase (GOD)-modified DNA2, i.e., DNA2-GOD. Then, GOD oxidizes glucose to produce a large number of electrons, and a significantly increased open-circuit voltage (E1OCV) is observed, corresponding to the “on” state. If miRNA-155 is present, it hybridizes with DNA1 and DNA3-functionalized SiO2 nanosphere@gold nanoparticles, i.e., SiO2@AuNPs-DNA3, and replaces miRNA-21 to release GOD at the bioanode, thus leading to decreased E2OCV, corresponding to the “off” state. The “on–off” self-powered biosensor shows ultra-sensitive detection of miRNA-21 and miRNA-155 with detection limits of 0.17 fM and 0.37 fM, respectively. It is believed that this study provides a feasible model for designing self-powered biosensors for multi-targets detection.

Comparison Between Terminal or Preterminal Conditioning of Donor Livers by Ex Situ Machine Perfusion.

The successful implementation of end-ischemic normothermic machine perfusion (NMP) into clinical practice comes along with unusual demands for trained personnel and technical facilities in the implantation clinic. This creates an interest to bundle expertise and professional equipment for execution of MP at regional pump centers at the disadvantage of adding a second short period of cold preservation while sending the reconditioned grafts to the actual implant clinic. Differences of liver recovery upon reperfusion either immediately after NMP or after 3 h of cold storage subsequent to NMP should therefore be evaluated. Rat livers were cold stored for 18 h, subjected to 2 h of NMP, and then either directly evaluated by ex vivo reperfusion or exposed to a second cold storage period of 3 h to simulate transport from the hub center to the implant clinic. Livers stored for 18 h by cold storage only served as controls. Both MP regimens significantly reduced hepatic enzyme release and improved bile production, clearance of lactate, and energetic recovery compared with the controls. However, no differences were seen between the 2 MP groups. The study provides first evidence that machine perfusion at regional perfusion centers may be a safe and economical alternative to the widespread individual efforts in the respective implantation clinics.

P319 MID VENTRICULAR TAKOTSUBO SYNDROME IN A PATIENT WITH COVID 19 PNEUMONIA: A CASE REPORT

Abstract Background Takotsubo Syndrome (TTS) is an acute and transient myocardial disfunction associated with chest pain, electrocardiographic changes and minimal myocardial enzymatic release mimicking acute myocardial infarction but without significant coronary artery disease. Physical or emotional stress play a central role in the pathogenesisof this cardiomyopathy. Several variants have been described including the classic type, reverse Takotsubo, midventricular type and focal type. Mid ventricular Takotsubo is characterized by circumferential mid–ventricular hypokinesia with hypercontractility of base and apex. This atypical variant is one of the most rare form that occurs in 15% of patients with Takotsubo syndrome. We report a Takotsubo case with mid–ventricular pattern in a postmenopausal woman with COVID–19 interstitial pneumonia. Clinical case A 56–year–old female patient was admitted to our intensive cardiac care unit for acute chest pain and dyspnea. In clinical history hypertension, dyslipidemia, anemia and schizophrenia were documented. COVID–19 molecular test was positive. CT scan showed multiple ground–glass opacities bilaterally. Hemodynamic parameters were normal but ECG showed ST–segment elevation and T waves inversion in the lateral precordial leads. Troponin–I was elevated and peaked at 1704 ng/mL. A transthoracic echocardiogram showed ballooning of the midventricular segments with hyperkinesis of the basal and apical segment. Coronary angiography showed normal coronaries. The patient was treated with beta–blockers and intravenous fluid infusion. After 10 days in hospital, echocardiography showed normalization of regional contractility and improvement of ejection fraction. The patient was discharged in good condition. Conclusions Recent studies have reported an increase in TTS incidence during the COVID–19 pandemic. SARS-CoV-2 infection induces physical and psychological stress in patients, which may lead to an increase in the risk of TTS development. The pathophysiology of COVID-19-induced TTS still remains unclear. The mortalityrate of TTS is higher (36.5%) in COVID–19 patients.

Multifunctional Double-Layer and Dual Drug-Loaded Microneedle Patch Promotes Diabetic Wound Healing.

Chronic nonhealing diabetic wounds are a serious complication of diabetes, with a high morbidity rate that can cause disability or death. The long period of inflammation and dysfunctional angiogenesis are the main reasons for wound-healing difficulty in diabetes. In this study, a multifunctional double-layer microneedle (DMN) is constructed to control infection and promote angiogenesis, meeting the multiple demands of the healing process of a diabetic wound. The double-layer microneedle is consisted in a hyaluronic acid substrate and a mixture of carboxymethyl chitosan and gelatin as the tip. The antibacterial drug tetracycline hydrochloride (TH) is loaded into the substrate of the microneedle to achieve rapid sterilization and promote resistance to external bacterial infections. The microneedle tip loaded with recombinant human epidermal growth factor (rh-EGF) is inserted into the skin, in response to gelatinase produced by resident microbe and disassociate to achieve the enzymatic response release. The double-layer drug-loaded microneedles (DMN@TH/rh-EGF) have antibacterial and antioxidant effects, and promote cell migration and angiogenesis in vitro. In an in vivo diabetic wound model, using rats, the DMN@TH/rh-EGF patch is able to inhibit inflammation, promote angiogenesis, collagen deposition, and tissue regeneration during the wound healing process, promoting its healing.

Cytosolic protein delivery using ultrasound-guided vaporization of perfluorocarbon nano-droplets.

Ultrasound-guided protein delivery is promising for site-specific control of cellular functions in the deep interior of the body in a noninvasive manner. Herein, we propose a method for cytosolic protein delivery based on ultrasound-guided intracellular vaporization of perfluorocarbon nano-droplets. The nano-droplets were conjugated with cargo proteins through a bio-reductively cleavable linker and introduced into living cells via antibody-mediated binding to a cell-surface receptor, which gets internalized through endocytosis. After the cells were exposed to ultrasound for endosomal escape of proteins, the ultrasound-responsive cytosolic release of a cargo enzyme was confirmed by visualizing the hydrolysis of the fluorogenic substrate using confocal microscopy. Moreover, a significant decrease in cell viability was achieved via the release of a cytotoxic protein in response to ultrasound treatment. The results of this study provide the proof of a principle that protein-conjugated nano-droplets can be used as carriers in ultrasound-guided cytosolic delivery of proteins. This article is protected by copyright. All rights reserved.

In Situ Non‐Invasive Imaging of Neutrophil Myeloperoxidase and Skin Reactive Oxygen Species in Experimental Murine Atopic Dermatitis

AbstractNeutrophils play a key role in the innate immune inflammatory response, notably through the release of the myeloperoxidase enzyme from azurophilic granules, locally generating reactive oxygen species. Although short‐lived, these reactive oxygen species are directly involved in local tissue damage in response to microbial intrusion. Neutrophil‐derived myeloperoxidase has been reported as an important factor in the elicitation of atopic dermatitis and is considered a potential target and biomarker. This study describes the use of in situ imaging techniques comprising both chemiluminescent resonance energy‐transfer and ratiometric fluorescent microtattoos to locally and non‐invasively image myeloperoxidase activity and skin reactive oxygen species in a murine model of calcipotriol‐induced atopic dermatitis. Using neutrophil depletion to assess granulocyte contribution to the observed imaging signals, the non‐invasive longitudinal data are found to correlate with endpoint biochemical activity assays for both myeloperoxidase and reactive oxygen species by‐products, as well as with immunohistochemical analysis.

A Novel Patient-Personalized Nanovector Based on Homotypic Recognition and Magnetic Hyperthermia for an Efficient Treatment of Glioblastoma Multiforme.

Glioblastoma multiforme (GBM) is the deadliest brain tumor, characterized by an extreme genotypic and phenotypic variability, besides a high infiltrative nature in healthy tissues. Apart from very invasive surgical procedures, to date, there are no effective treatments, and life expectancy is very limited. In this work, an innovative therapeutic approach based on lipid-based magnetic nanovectors is proposed, owning a dual therapeutic function: chemotherapy, thanks to an antineoplastic drug (regorafenib) loaded in the core, and localized magnetic hyperthermia, thanks to the presence of iron oxide nanoparticles, remotely activated by an alternating magnetic field. The drug is selected based on ad hoc patient-specific screenings; moreover, the nanovector is decorated with cell membranes derived from patients' cells, aiming at increasing homotypic and personalized targeting. It is demonstrated that this functionalization not only enhances the selectivity of the nanovectors toward patient-derived GBM cells, but also their blood-brain barrier in vitro crossing ability. The localized magnetic hyperthermia induces both thermal and oxidative intracellular stress that lead to lysosomal membrane permeabilization and to the release of proteolytic enzymes into the cytosol. Collected results show that hyperthermia and chemotherapy work in synergy to reduce GBM cell invasion properties, to induce intracellular damage and, eventually, to prompt cellular death.

Germinação in vitro de sementes de maracujá em função da remoção do tegumento e meios de cultura

ABSTRACT The presence of salts in the culture medium and the addition of phytohormones, necessary to break seed dormancy in the in vitro germination of passion fruit, can induce critical values of osmotic potential. This can change the membrane permeability of seeds, affecting the hydration rate, enzyme release, ion transport, pH, and inhibitor values, which may decrease germination percentage. The aim of this study was to evaluate different types of culture media, composed of other substances, combined with mechanical techniques of tegument removal to determine the most appropriate culture medium for the in vitro germination of Passiflora edulis (Sims flavicarpa Deg.) seeds obtained by open pollination. The experimental design was completely randomized in a 10 × 3 factorial scheme (10 culture media × three types of seed-intact, scarified, and cut), with eight replicates and five seeds per plot. Scarified seeds cultivated in a medium composed of a commercial substrate and gibberellic acid presented a higher percentage of germination than intact or cut seeds cultivated in different concentrations of Murashige & Skoog (MS) medium. Scarification is a satisfactory method for breaking the dormancy of passion fruit seeds, and commercial substrates Bioflora® enriched with gibberellic acid may replace in vitro germination.

Lyososome Membrane Permeabilization Induces Plasma Membrane Macropores to Activate K +-Efflux-Dependent NLRP3 Inflammasomes in Murine Dendritic Cells

Abstract NLRP3 is a cytosolic sensor protein in myeloid leukocytes that recruits the adaptor protein ASC and activates caspase-1 in functional NLRP3 inflammasomes. Caspase-1 coordinately cleaves pro-IL-1β to bioactive IL-1β and gasdermin D (GSDMD) to induce N-GSDMD macropores in the plasma membrane (PM). The latter mediate efflux of mature IL-1β and initiate progression to pyroptosis, a lytic cell death. NLRP3 activation is triggered by perturbed cellular homeostasis with decreased cytosolic [K +] being the best-characterized signal. The required K +efflux is typically induced by the ionophore nigericin or extracellular ATP as an agonist for P2X7 receptor channels. K +efflux-dependent activation of NLRP3 is also triggered by soluble or particulate agents which accumulate in lysosomes to induce lysosomal membrane permeabilization (LMP) and release of hydrolytic enzymes into the cytosol; particulates include alum widely used as a vaccine adjuvant. The mechanisms by which LMP rapidly triggers changes in plasma membrane permeability remain undefined. We used a model of murine bone marrow derived dendritic cells (BMDC) treated with the soluble LMP agent Leu-Leu-O-methyl ester (LLME) to elicit K+ efflux and NLRP3 activation. Notably, this K+ efflux was coincident with a rapidly induced influx of propidiumiodide (PI), a 668 Da organic salt routinely used to track assembly of plasma membrane macropores such as GSDMD. Equivalent LLME-induced PI influx was observed in wildtype and Gsdmd−/−BMDC and was unaffected by inhibition of NLRP3 or caspase-1. These observations indicate that LMP triggers rapid activation of an endogenous pore-forming protein distinct from GSDMD. Current studies are evaluating gasdermin E or pannexin-1 as candidates. Supported by grants from the NIH (P01-AI141350 and R01-EY014362)

Oxidative stress and inflammation: the root causes of aging

Oxygen free radicals [reactive oxygen species (ROS)] and nitrogen free radicals [reactive nitrogen species (RNS)] are generated by mitochondria during adenosine triphosphate synthesis, and catalytic activities of cytochrome P450, nicotinamide adenine dinucleotide phosphate oxidases (NOXs), cyclooxygenases, and nitric oxide synthases during drug catabolism, phagocytosis, and acute inflammation. Under normal circumstances, low levels of ROS and RNS provide redox signalings that control many essential physiological processes. As age progresses ROS and RNS increase excessively due to dysfunctional mitochondria, dysregulated NOX, and other free-radical generating sources, leading to oxidative stress, which causes oxidation and denaturation of key cellular components including DNA, proteins, and lipids, which become abnormal, constituting damage-associated molecular pattern (DAMP), recognized as ‘non-self’ by immune cells, leading to inflammation which is mediated by nuclear factor kappa B-inflammasome, p38-c-Jun N-terminal kinase and Janus kinase-signal transducer and activator of transcription pathways. DAMPs are continuously released from damaged and senescent cells, causing an otherwise normally transient inflammation turning into systemic chronic inflammation, the root cause of aging and age-associated diseases (AADs). Cells restore redox balance by activating the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway that induces the synthesis and release of antioxidation molecules and enzymes including haem oxygenase-1, which also inhibits the three inflammatory pathways. Furthermore, upregulation of autophagy (AP) can get rid of abnormal molecules, prevent the generation of DAMPs, and attenuate inflammation. Both AP and Nrf2 signalings decrease with age. The upregulations of Nrf2, AP, and downregulation of inflammation are controlled by sensors of energy and stress levels, i.e., adenosine monophosphate-activated protein kinase, silent information regulator 1, and Sestrins, as well as the extracellular matrix, while mammalian targets for rapamycin complex 1, a nutrient sensor, act in the opposite direction. If the balance of these sensor systems becomes dysregulated, aging process accelerates, and the risk of AADs increases.

Chronic intermittent oxygen deprivation alters hippocampal cholinergic and glutamatergic system via oxido-inflammatory burden and HIF-1a/Bcl-2 activity in hypothyroid mice: Ameliorative role of Ginkgo biloba supplement

BackgroundSeveral investigations in recent years have reported a relationship between hypothyroidism or ischemia and central nervous system (CNS) beginning from fetal to adult life, but the effect of ischemia and hypothyroidism comorbidity on CNS and whether phytotherapeutic approach would attenuate this pathology remains unknown. Thus, the study investigated the role of ginkgo biloba supplement (GBS), a potent anti-oxido-inflammatory and neurorestorative plant-based product on hypoxic stress-induced neurobehavioral and neurophysiological alterations in hypothyroid mice, and the underpinning molecular mechanisms MethodologyMice were orally pre-treated with Carbimazole (1.2 mg/kg) for 14 days to develop hypothyroidism. Post-hypothyroid induction, mice were treated orally with GBS (20 mg/kg) and levothyroxine (10 µg/kg) 1 hr before 20 min exposure to hypoxia (5 times daily) for 14 consecutive days. Symptoms of behavioral deficit and neuropsychiatry were evaluated in using different models. Thereafter, brain hippocampi were sectioned for biochemical assays, immunohistochemistry and histoarchitectural studies. ResultsHerein, treatment with GBS suppressed spatial memory deficit and neuropsychiatric phenotypes and attenuated hippocampal cholinergic excitotoxicity by enhancing acetylcholinesterase enzyme and glutamatergic release in the hypothyroid mice following hypoxic stress exposure. The hippocampal endogenous antioxidant system was also upregulated with concomitant downregulation of inflammatory mediators. GBS treatment consequently regulated the hypothalamic-pituitary-adrenal axis to reduce corticosterone release. Additionally, our data showed that the suppressive impact of GBS on oxido-inflammatory mainstream decreases immunoexpression of hypoxic inducible factor-1alpha (HIF-1α) and loss of hippocampal pyramidal neurons in the CA3 region with marked increase in viable neuronal cells and upregulated immunoexpression of B-cell lymphoma-2 (Bcl-2) anti-apoptotic marker. ConclusionHerein, we deduced that prolonged intermittent exposure to hypoxia in hypothyroidism may provoke further the psychological and physiological status in the hippocampal brain region. Meanwhile, reversal of these provocative effects by the GBS treatment might be playing an important effect to suppress the hypoxic/ischemic triggered neurobehavioral and neurophysiological alterations.

A novel method to estimate biofilm activity based on enzymatic oxygen release from hydrogen peroxide decomposition

Biofilm is central for biological water treatment processes in recirculating aquaculture systems (RAS). A lack of suitable methods for quantifying biofilm activity, however, makes it difficult to assess and compare the microbial status of biofilm. This type of information of the biofilm will be useful to assess the colonization status of nitrifying biocarriers or to evaluate the effect of disinfectants on the biofilm activity.Here we introduce a novel assay for rapid assessment of microbial activities in the biofilm attached on bioelements from a RAS biofilter. The assay consisted of an intermittent respirometer platform where biofilter elements were exposed to 10 mg/L hydrogen peroxide (H2O2) for 1 h, following concurrent measurements of oxygen release from the decomposition of H2O2 caused by biofilm-associated enzymes. A different number of colonized, mature bioelements from a moving bed biofilter in a freshwater RAS were tested with repeated H2O2 exposure, and compared against their autoclaved forms. A substantial increase in dissolved oxygen (DO) concentration (0.92–2.31 mg O2/L) occurred with mature bioelements during 1 h of H2O2 exposure, compared to small amounts of DO release (≤0.27 mg O2/L) with autoclaved bioelements. This substantiates that H2O2 decomposition by biofilm is mainly governed by microbial enzymatic activities. A monomolecular model fitted well with the observed oxygen release profiles of tested mature bioelements after H2O2 exposure (R2 > 0.98). The kinetic rate constant of net oxygen release (kor, h−1) was proportional (R2 for linear fit = 0.99) to the number of mature bioelements tested. Repeated exposure of H2O2 to the same bioelements did not change kor, which indicates that 10 mg/L H2O2 with an exposure time of 1 h does not suppress enzymatic activity in biofilm. Our study provides a new rapid method that allows simple quantification of microbial activity in biofilm samples from aquaculture systems, which could potentially be also applied to study biofilm from wastewater treatment plants and other industries.

Amino Acid Starvation-Induced Glutamine Accumulation Enhances Pneumococcal Survival.

Bacteria are known to cope with amino acid starvation by the stringent response signaling system, which is mediated by the accumulation of the (p)ppGpp alarmones when uncharged tRNAs stall at the ribosomal A site. While a number of metabolic processes have been shown to be regulatory targets of the stringent response in many bacteria, the global impact of amino acid starvation on bacterial metabolism remains obscure. This work reports the metabolomic profiling of the human pathogen Streptococcus pneumoniae under methionine starvation. Methionine limitation led to the massive overhaul of the pneumococcal metabolome. In particular, methionine-starved pneumococci showed a massive accumulation of many metabolites such as glutamine, glutamic acid, lactate, and cyclic AMP (cAMP). In the meantime, methionine-starved pneumococci showed a lower intracellular pH and prolonged survival. Isotope tracing revealed that pneumococci depend predominantly on amino acid uptake to replenish intracellular glutamine but cannot convert glutamine to methionine. Further genetic and biochemical analyses strongly suggested that glutamine is involved in the formation of a "prosurvival" metabolic state by maintaining an appropriate intracellular pH, which is accomplished by the enzymatic release of ammonia from glutamine. Methionine starvation-induced intracellular pH reduction and glutamine accumulation also occurred to various extents under the limitation of other amino acids. These findings have uncovered a new metabolic mechanism of bacterial adaptation to amino acid limitation and perhaps other stresses, which may be used as a potential therapeutic target for infection control. IMPORTANCE Bacteria are known to cope with amino acid starvation by halting growth and prolonging survival via the stringent response signaling system. Previous investigations have allowed us to understand how the stringent response regulates many aspects of macromolecule synthesis and catabolism, but how amino acid starvation promotes bacterial survival at the metabolic level remains largely unclear. This paper reports our systematic profiling of the methionine starvation-induced metabolome in S. pneumoniae. To the best of our knowledge, this represents the first reported bacterial metabolome under amino acid starvation. These data have revealed that the significant accumulation of glutamine and lactate enables S. pneumoniae to form a "prosurvival" metabolic state with a lower intracellular pH, which inhibits bacterial growth for prolonged survival. Our findings have provided insightful information on the metabolic mechanisms of pneumococcal adaptation to nutrient limitation during the colonization of the human upper airway.

Sclerotinia sclerotiorum (Lib.) de Bary: Insights into the Pathogenomic Features of a Global Pathogen.

Sclerotinia sclerotiorum (Lib.) de Bary is a broad host-range fungus that infects an inclusive array of plant species and afflicts significant yield losses globally. Despite being a notorious pathogen, it has an uncomplicated life cycle consisting of either basal infection from myceliogenically germinated sclerotia or aerial infection from ascospores of carpogenically germinated sclerotia. This fungus is unique among necrotrophic pathogens in that it inevitably colonizes aging tissues to initiate an infection, where a saprophytic stage follows the pathogenic phase. The release of cell wall-degrading enzymes, oxalic acid, and effector proteins are considered critical virulence factors necessary for the effective pathogenesis of S. sclerotiorum. Nevertheless, the molecular basis of S. sclerotiorum pathogenesis is still imprecise and remains a topic of continuing research. Previous comprehensive sequencing of the S. sclerotiorum genome has revealed new insights into its genome organization and provided a deeper comprehension of the sophisticated processes involved in its growth, development, and virulence. This review focuses on the genetic and genomic aspects of fungal biology and molecular pathogenicity to summarize current knowledge of the processes utilized by S. sclerotiorum to parasitize its hosts. Understanding the molecular mechanisms regulating the infection process of S. sclerotiorum will contribute to devising strategies for preventing infections caused by this destructive pathogen.

Effect of high‐pressure homogenisation on metabolic potential of Lacticaseibacillus paracasei 90: in vitro and in situ studies in fermented milk and semihard cheese

The influence of high‐pressure homogenisation (HPH) at 100 MPa on Lacticaseibacillus paracasei 90 (L90) was studied in vitro and in situ (fermented milk and cheese). Lysis and injury signs were observed in cells treated with HPH, which were linked to the release of intracellular enzymes and changes in the metabolic activity of L90. HPH treatment led to a higher lactic acid content and lower pH and pyruvic acid levels in fermented milk. The microbiological and peptide profiles of cheeses were modified by using cells or cell‐free extracts of L90. Finally, this study suggests that L90 could release bioactive peptides.

Coating of SPIONs with a Cysteine-Decorated Copolyester: A Possible Novel Nanoplatform for Enzymatic Release.

Superparamagnetic iron oxide nanoparticles (SPIONs) have their use approved for the diagnosis/treatment of malignant tumors and can be metabolized by the organism. To prevent embolism caused by these nanoparticles, they need to be coated with biocompatible and non-cytotoxic materials. Here, we synthesized an unsaturated and biocompatible copolyester, poly (globalide-co-ε-caprolactone) (PGlCL), and modified it with the amino acid cysteine (Cys) via a thiol-ene reaction (PGlCLCys). The Cys-modified copolymer presented reduced crystallinity and increased hydrophilicity in comparison to PGlCL, thus being used for the coating of SPIONS (SPION@PGlCLCys). Additionally, cysteine pendant groups at the particle's surface allowed the direct conjugation of (bio)molecules that establish specific interactions with tumor cells (MDA-MB 231). The conjugation of either folic acid (FA) or the anti-cancer drug methotrexate (MTX) was carried out directly on the amine groups of cysteine molecules present in the SPION@PGlCLCys surface (SPION@PGlCLCys_FA and SPION@PGlCLCys_MTX) by carbodiimide-mediated coupling, leading to the formation of amide bonds, with conjugation efficiencies of 62% for FA and 60% for MTX. Then, the release of MTX from the nanoparticle surface was evaluated using a protease at 37 °C in phosphate buffer pH~5.3. It was found that 45% of MTX conjugated to the SPIONs were released after 72 h. Cell viability was measured by MTT assay, and after 72 h, 25% reduction in cell viability of tumor cells was observed. Thus, after a successful conjugation and subsequent triggered release of MTX, we understand that SPION@PGlCLCys has a strong potential to be treated as a model nanoplatform for the development of treatments and diagnosis techniques (or theranostic applications) that can be less aggressive to patients.

Liraglutide Attenuates Myocardial Ischemia/Reperfusion Injury Through the Inhibition of Necroptosis by Activating GLP-1R/PI3K/Akt Pathway.

Necroptosis is a crucial programmed cell death that is tightly associated with myocardial ischemia/reperfusion injury (MI/RI). Liraglutide is an effective option for the treatment of type 2 diabetes and has recently been reported to exert cardioprotective effects on MI/RI. Researchers do not know whether the cardioprotective effect of liraglutide is involved in regulating necroptosis. This study aimed to explore the effect of liraglutide on MI/RI-induced necroptosis and its potential mechanisms. Hypoxia/reoxygenation (H/R) was performed on H9c2 cells in vitro to simulate ischemia/reperfusion (I/R) injury, and an MI/RI rat model was established in vivo by ligating the anterior descending branch of the left coronary artery. H/R or I/R damage was assessed by performing biochemical assay, Hoechst 33342/PI staining, H&E (hematoxylin and eosin) staining, and Annexin-V/PI staining. Our data revealed that liraglutide resulted in markedly increased cell viability and reduced cardiac enzyme release by protecting cardiomyocytes from a necrosis-like phenotype after H/R. The myocardial infarct size and cardiac enzyme release were reduced in the heart tissues from the liraglutide-treated group. The levels of necroptosis-associated proteins (receptor-interacting protein kinase 3 (RIPK3), p-RIPK3, and phosphorylated-mixed lineage kinase domain-like protein (p-MLKL)) were also reduced by the liraglutide treatment. Mechanistically, we revealed that liraglutide exerted cardioprotective effects through a glucagon-like peptide-1 receptor (GLP-1R) and phosphatidylinositol-3 kinase (PI3K)-dependent pathway. Both the GLP-1R inhibitor exendin (9-39) and the PI3K inhibitor LY294002 abrogated the protective effects of liraglutide in vitro. We found that liraglutide may attenuate MI/RI by inhibiting necroptosis, in part by enhancing the activity of the GLP-1R/PI3K/Akt pathway.

Characterization of the l-arabinofuranose-specific GafABCD ABC transporter essential for l-arabinose-dependent growth of the lignocellulose-degrading bacterium Shewanella sp. ANA-3.

Microbes that have evolved to live on lignocellulosic biomass face unique challenges in the effective and efficient use of this material as food. The bacterium Shewanella sp. ANA-3 has the potential to utilize arabinan and arabinoxylan, and uptake of the monosaccharide, l-arabinose, derived from these polymers, is known to be mediated by a single ABC transporter. We demonstrate that the substrate binding protein of this system, GafASw, binds specifically to l-arabinofuranose, which is the rare furanose form of l-arabinose found in lignocellulosic biomass. The structure of GafASw was resolved to 1.7 Å and comparison to Escherichia coli YtfQ (GafAEc) revealed binding site adaptations that confer specificity for furanose over pyranose forms of monosaccharides, while selecting arabinose over another related monosaccharide, galactose. The discovery of a bacterium with a natural predilection for a sugar found abundantly in certain lignocellulosic materials suggests an intimate connection in the enzymatic release and uptake of the sugar, perhaps to prevent other microbes scavenging this nutrient before it mutarotates to l-arabinopyranose. This biological discovery also provides a clear route to engineer more efficient utilization of plant biomass components in industrial biotechnology.

Lead-induced cardiomyocytes apoptosis by inhibiting gap junction intercellular communication via modulating the PKCα/Cx43 signaling pathway

ObjectiveThe aim of this study was to investigate the regulatory mechanism of Pb regulates gap junction intercellular communication to induced apoptosis in H9c2 cells. MethodsH9c2 cell line is used as the research object in this study, and treated with different concentrations of Pb acetate. Subsequently, Cell viability was measured by the Cell Counting Kit-8 (CCK-8) assay. The levels of lactate dehydrogenase (LDH), aspartate transaminase (AST) and creatine kinase-MB (CK-MB) in the supernatants were measured using respective commercial enzyme-linked immune sorbent assay (ELISA) kits. Western blot was used to detect the expression of apoptosis-related protein in H9c2 cells in each group. Quantitative RT-PCR Analysis Total RNA was extracted from frozen H9c2 cells using Trizol reagent, the PKCα and Cx43 in the supernatant of H9c2 cells was determined by the BCA protein detection kit. ResultsH9c2 cells increased release of cardiac enzymes (LDH, AST, and CK-MB) and decreased cell survival rate, and the Cx43, p-Cx43, PKCα and p-PKCα protein levels showed a dose-dependent decrease after Pb treatment. PKCα was activated with PMA, the relative expression level of Cx43 protein increased significantly, the expression of Bcl-2 increased and Bax and Cyt-c decreased compared with Pb exposure group, and the myocardial enzymes (LDH, AST, and CK-MB) in cell culture supernatant decreased compared with Pb exposure group, indicating that the degree of cell damage was alleviated. Results showed that Pb inhibited PKCα activity, decreased the expression of total Cx43 and P-Cx43 protein, and aggravated myocardial injury. ConclusionsPb decrease gap junction intercellular communication, which induce apoptosis in H9c2 cells by inhibiting the PKCα/Cx43 signaling pathway.