Preparation of highly adsorptive biochar by sequential iron impregnation under refluxing and pyrolysis at low temperature for removal of tetracycline.

Iron-doping modification is a prevailing approach for improving adsorption capability of biochar with environmental friendliness, but usually requires high temperature and suffers from iron aggregation. Herein, a highly adsorptive biochar was manufactured via sequential disperse impregnation of iron by refluxing and pyrolysis at low temperature for eliminating tetracycline (TC) from aqueous solution. Iron oxides and hydroxides were impregnated and stably dispersed on the carbon matrix as pyrolyzed at 200 °C, meanwhile abundant oxygen and nitrogen functional groups were generated on surface. The iron-doped biochar exhibited up to 891.37 mg/g adsorption capacity at pH 5, and could be recycled with high adsorption capability. The adsorption of TC should be mostly contributed to the hydrogen bonding of N/O functional groups and the hydrogen bonding/coordination of iron oxides/hydroxides. This would provide a valuable guide for dispersedly doping iron and conserving functional groups on biochar, and a super iron-doped biochar was prepared with superior recyclability.

Facile production of cellulose nanofibers from raw elephant grass by an aluminum chloride-enhanced acidic deep eutectic solvent.

To develop a greener and more efficient method for producing cellulose nanofibers (CNFs) from raw plants, an AlCl3-enhanced ternary deep eutectic solvent, DES2 (consisting of choline chloride, citric acid, and AlCl3·6H2O in a molar ratio of 1:0.4:0.08), was synthesized. Raw elephant grass (EG) was pretreated with DES2, followed by sodium chlorite (NaClO2) bleaching and ultrasonic disruption to extract high-performance CNFs. The DES2 and NaClO2 treatments effectively removed hemicellulose and lignin, achieving removal rates of 99.23 % and 99.62 %, respectively, while maintaining a cellulose content of 78.3 %. DES2 demonstrated easy recyclability and maintained excellent biomass pretreatment performance even after multiple cycles. Following a brief 30-min intermittent ultrasound treatment, the resulting CNFs demonstrated superior crystallinity, increased carboxyl content, and a narrower width distribution compared to CNFs obtained from AlCl3-free DES1. Optimized conditions at 110 °C yielded CNFs with 85.3 % crystallinity, 0.64 mmol/g carboxyl content, 5.15 nm width distribution, and excellent dispersion in water for at least six months. Additionally, CNFs enhanced the tensile strength of chia seed mucilage (CM) composite films, showing a significant improvement to 26.6 MPa, representing a 231.3 % increase over the control film. This study offers a promising approach for efficiently producing CNFs from raw plants.

New function of a well-known promoter: Enhancer activity of minimal CMV promoter enables efficient dual-cassette transgene expression.

BACKGROUND: Co-expression of multiple genes in single vectors has achieved varying degrees of success by employing two promoters and/or application of viral 2A-peptide or the internal ribosome entry-site (IRES). However, promoter interference, potential functional-interruption of expressed-proteins by 2A-generated residual peptides or weaker translation of IRES-mediated downstream genes has curtailed their utilization. Thus, there is the need for single vectors that robustly express multiple proteins for enhanced gene therapy applications. METHODS: We engineered lentiviral-vectors for dual-cassette expression of green fluorescent protein and mCherry in uni- or bidirectional architectures using the short-version (Es) of elongation factor 1α (EF) promoter and simian virus 40 promoter (Sv). The regulatory function of a core fragment (cC) from human cytomegalovirus promoter was investigated with cell-lineage specificity in NIH3T3 (fibroblast) and hematopoietic cell lines U937 (monocyte/macrophage), LCL (lymphoid), DAMI (megakaryocyte) and MEL (erythroid). RESULTS: The cC element in reverse-orientation not only boosted upstream Es promoter to levels comparable to full-length EF in DAMI, U937 and 3T3 cells, but also blocked the suppression of downstream Sv promoter by Es in U937 and 3T3 cells with further improved Sv activity in DAMI cells. Such lineage-restricted up-regulation is likely attributed to two protein-binding domains of cC and diverse expression of related factors in different cell types for enhancer and terminator activities, but not spacing function. CONCLUSIONS: Such a newly developed dual-cassette vector could be advantageous, particularly in hematopoietic cell-mediated gene/cancer therapy, by allowing for independent and robust co-expression of therapeutic gene(s) and/or a selectable gene or imaging marker in the same cells.

A comprehensive review on molecular mechanism of defective dry-cured ham with excessive pastiness, adhesiveness, and bitterness by proteomics insights.

Excessive bitterness, pastiness, and adhesiveness are the main organoleptic and textural defects of dry-cured ham, which often cause a lot of financial losses to manufacturers and seriously damage the quality of the product. These sensory and textural defects are related to the protein degradation of dry-cured ham. Proteomics shows great potential to improve our understanding of the molecular mechanism of sensory and textural defects and identify biomarkers for monitoring their quality traits. This review presents some of the major achievements and considerations in organoleptic and textural defects of dry-cured ham by proteomics analysis in the recent decades and gives an overview about how to correct sensory and textural defects of dry-cured ham. Proteomics reveals that muscle proteins derived from myofibril and cytoskeleton and involved in metabolic enzymes and oxygen transport have been identified as potential biomarkers in defective dry-cured ham. Relatively high residual activities of cathepsin B and L are responsible for the excessive degradation of these protein biomarkers in defective dry-cured ham. Ultrasound-assisted mild thermal or high-pressure treatment shows a good correction for the organoleptic and textural defects of dry-cured ham by changing microstructure and conformation of muscle proteins by accelerating degradation of proteins and polypeptides into free amino acids.

Oral Pregabalin in Cardiac Surgery: A Systematic Review and Meta-Analysis of Randomized Controlled Trials.

BACKGROUND: Pregabalin has received wide clinical attention as a new type of analgesic. We undertake a systematic review and meta-analysis to evaluate the effect of pregabalin on postoperative pain in patients undergoing cardiac surgery. METHODS: We searched PubMed, Embase, and Cochrane Library (from inception to July 2020) for eligible studies. The primary outcomes were the total morphine consumption at 24 h. A secondary outcome was intraoperative fentanyl consumption, extubation time postoperative, and length of stay in hospital. We calculated pooled weighted mean difference (WMD) or odds ratio (OR) and 95% CIs using random- or fixed-effects models. RESULTS: Seven trials involving 463 patients were listed. Meta-analysis showed that the total morphine consumption at 24 h in the pregabalin group was significantly less than the control group (WMD: -5.44, 95% CI: -10.42-0.46, P = 0.03). We found that there is no significant difference between the two groups in intraoperative fentanyl consumption. Compared with the control group, the length of stay in hospital in the pregabalin group was significantly shorter (WMD = -0.87, 95% CI: -1.42-0.32, P = 0.002). And we found that there were no significant differences between the two groups in extubation time (WMD: 17.24, 95% CI: -24.36-58.84, P = 0.42). CONCLUSIONS: Oral pregabalin for cardiac surgery patients can effectively reduce the patient's 24-hour morphine consumption after surgery, shorten the patient's hospital stay, and is more conducive to early postoperative recovery.

miR-143 Regulates Lysosomal Enzyme Transport across the Blood-Brain Barrier and Transforms CNS Treatment for Mucopolysaccharidosis Type I.

During brain maturation, cation-independent mannose-6-phosphate receptor (CI-MPR), a key transporter for lysosomal hydrolases, decreases significantly on the blood-brain barrier (BBB). Such a phenomenon leads to poor brain penetration of therapeutic enzymes and subsequent failure in reversing neurological complications in patients with neuropathic lysosomal storage diseases (nLSDs), such as Hurler syndrome (severe form of mucopolysaccharidosis type I [MPS I]). In this study, we discover that upregulation of microRNA-143 (miR-143) contributes to the decline of CI-MPR on the BBB during development. Gain- and loss-of-function studies showed that miR-143 inhibits CI-MPR expression and its transport function in human endothelial cells in vitro. Genetic removal of miR-143 in MPS I mice enhances CI-MPR expression and improves enzyme transport across the BBB, leading to brain metabolic correction, pathology normalization, and correction of neurological functional deficits 5 months after peripheral protein delivery at clinically relevant levels that derived from erythroid/megakaryocytic cells via hematopoietic stem cell-mediated gene therapy, when otherwise no improvement was observed in MPS I mice at a parallel setting. These studies not only uncover a novel role of miR-143 as an important modulator for the developmental decline of CI-MPR on the BBB, but they also demonstrate the functional significance of depleting miR-143 for "rescuing" BBB-anchored CI-MPR on advancing CNS treatment for nLSDs.

Loss of BRUCE reduces cellular energy level and induces autophagy by driving activation of the AMPK-ULK1 autophagic initiating axis.

Autophagy is an intracellular catabolic system. It delivers cellular components to lysosomes for degradation and supplies nutrients that promote cell survival under stress conditions. Although much is known regarding starvation-induced autophagy, the regulation of autophagy by cellular energy level is less clear. BRUCE is an ubiquitin conjugase and ligase with multi-functionality. It has been reported that depletion of BRUCE inhibits starvation-induced autophagy by blockage of the fusion step. Herein we report a new function for BRUCE in the dual regulation of autophagy and cellular energy. Depletion of BRUCE alone (without starvation) in human osteosarcoma U2OS cells elevated autophagic activity as indicted by the increased LC3B-II protein and its autophagic puncta as well as further increase of both by chloroquine treatment. Such elevation results from enhanced induction of autophagy since the numbers of both autophagosomes and autolysosomes were increased, and recruitment of ATG16L onto the initiating membrane structure phagophores was increased. This concept is further supported by elevated lysosomal enzyme activities. In contrast to starvation-induced autophagy, BRUCE depletion did not block fusion of autophagosomes with lysosomes as indicated by increased lysosomal cleavage of the GFP-LC3 fusion protein. Mechanistically, BRUCE depletion lowered the cellular energy level as indicated by both a higher ratio of AMP/ATP and the subsequent activation of the cellular energy sensor AMPK (pThr-172). The lower energy status co-occurred with AMPK-specific phosphorylation and activation of the autophagy initiating kinase ULK1 (pSer-555). Interestingly, the higher autophagic activity by BRUCE depletion is coupled with enhanced cisplatin resistance in human ovarian cancer PEO4 cells. Taken together, BRUCE depletion promotes induction of autophagy by lowering cellular energy and activating the AMPK-ULK1-autophagy axis, which could contribute to ovarian cancer chemo-resistance. This study establishes a BRUCE-AMPK-ULK1 axis in the regulation of energy metabolism and autophagy, as well as provides insights into cancer chemo-resistance.

RUNX represses Pmp22 to drive neurofibromagenesis.

Patients with neurofibromatosis type 1 (NF1) are predisposed to develop neurofibromas, but the underlying molecular mechanisms of neurofibromagenesis are not fully understood. We showed dual genetic deletion of Runx1 and Runx3 in Schwann cells (SCs) and SC precursors delayed neurofibromagenesis and prolonged mouse survival. We identified peripheral myelin protein 22 (Pmp22/Gas3) related to neurofibroma initiation. Knockdown of Pmp22 with short hairpin RNAs increased Runx1fl/fl;Runx3fl/fl;Nf1fl/fl;DhhCre tumor-derived sphere numbers and enabled significantly more neurofibroma-like microlesions on transplantation. Conversely, overexpression of Pmp22 in mouse neurofibroma SCs decreased cell proliferation. Mechanistically, RUNX1/3 regulated alternative promoter usage and induced levels of protein expression of Pmp22 to control SC growth. Last, pharmacological inhibition of RUNX/core-binding factor ß (CBFB) activity significantly reduced neurofibroma volume in vivo. Thus, we identified a signaling pathway involving RUNX1/3 suppression of Pmp22 in neurofibroma initiation and/or maintenance. Targeting disruption of RUNX/CBFB interaction might provide a novel therapy for patients with neurofibroma.

Penicillin­binding protein 1A mutation­positive Helicobacter pylori promotes epithelial­mesenchymal transition in gastric cancer via the suppression of microRNA­134.

Evidence suggests that Helicobacter pylori (H. pylori) is not only the main cause of gastric cancer (GC), but is also closely associated with its metastasis. One of the major virulence factors in H. pylori is the cytotoxin­associated gene A (CagA). With the growing proportion of amoxicillin­resistant H. pylori strains, the present study aimed to explore the effects of CagA­ and penicillin­binding protein 1A (PBP1A) mutation­positive H. pylori (H. pyloriCagA+/P+) on GC cells, and its clinical significance. The clinical significance of H. pyloriCagA+/P+ infection was analyzed in patients with GC. In vitro, GC cells were infected with H. pyloriCagA+/P+ to investigate whether it was involved in the epithelial­mesenchymal transition (EMT) of SGC­7901 cells using immunofluorescence and western blot analysis. The results of clinical analysis demonstrated that, although CagA­negative H. pylori infection had no significant association with the characteristics of patients with GC, H. pyloriCagA+/P+ infection was significantly associated with various clinicopathological parameters, including invasion depth, lymphatic metastasis and distant metastasis. In vitro, the results indicated that H. pyloriCagA+/P+ promoted proliferation, invasion and EMT of SGC­7901 cells. MicroRNA (miR)­134 was downregulated in H. pyloriCagA+/P+ infected tissues compared with in those with H. pyloriCagA+/P­ infection. miR­134 overexpression significantly reversed H. pyloriCagA+/P+ infection­associated cell proliferation, invasion and EMT. Furthermore, the results revealed that Forkhead box protein M1 (FoxM1) was a direct target of miR­134, and FoxM1 knockdown impeded H. pyloriCagA+/P+­induced EMT. In conclusion, the present study demonstrated that miR­134 may suppress the proliferation, invasion and EMT of SGC­7901 cells by targeting FoxM1, and may serve a protective role in the process of H. pyloriCagA+/P+­induced GC. These findings may lead to an improved understanding of H. pyloriCagA+/P+­associated poor clinical characteristics in patients with GC.

Getting the Most: Enhancing Efficacy by Promoting Erythropoiesis and Thrombopoiesis after Gene Therapy in Mice with Hurler Syndrome.

Novel strategies are needed to solve the conundrum of achieving clinical efficacy with high vector copy numbers (VCNs) in hematopoietic stem cells (HSCs) while attempting to minimize the potential risk of oncogenesis in lentiviral vector (LV)-mediated gene therapy clinical trials. We previously reported the benefits of reprogramming erythroid-megakaryocytic (EMK) cells for high-level lysosomal enzyme production with less risk of activating oncogenes in HSCs. Herein, using a murine model of mucopolysaccharidosis type I (MPS I) with a deficiency of α-L-iduronidase (IDUA), we sought to determine the transgene minimum effective doses (MEDs) in major organs, and if a transient increase of IDUA-containing red blood cells and platelets by repeated phlebotomy would provide further therapeutic benefits in diseased mice after EMK-restricted LV-mediated gene therapy. The MEDs for complete metabolic correction ranged from 0.1 to 2 VCNs in major visceral organs, which were dramatically reduced to 0.005-0.1 VCN by one cycle of stress induction and were associated with a further reduction of pathological deficits in mice with 0.005 VCN. This work provides a proof of concept that transiently stimulating erythropoiesis and thrombopoiesis can further improve therapeutic benefits in HSC-mediated gene therapy for MPS I, a repeatable and reversible approach to enhance clinical efficacy in the treatment of lysosomal storage diseases.

The effect of ATP marination on the depolymerization of actin filament in goose muscles during postmortem conditioning.

In order to study the tenderization mechanism of ATP treatments by depolymerizing actin filaments, breast muscles of Eastern Zhejiang White Geese were randomly divided into 3 groups: control, 10 and 20 mM groups. Shear force (SF), sarcomere length (SL) and myofibrillar fraction index (MFI), the content of F-actin and G-actin, the expression of actin associated proteins (cofilins and tropomodulins) were investigated during conditioning. In 20 mM group, cofilins content increased from 48 to 168 h, while tropomodulins decreased; the content of F-actin decreased from 24 to 168 h, while the increased G-actin was observed upto 48 h. In the control, the degraded tropomodulins were observed at 168 h, and the increased cofilins and G-actin were detected at the same time; the increase of MFI and decrease of F-actin content were shown at 96 and 168 h. Compared to control group, 20 mM group accelerated the transformation of F-actin into G-actin; it showed higher SL and MFI, and lower SF at 48, 96 and 168 h, respectively. We concluded that depolymerization of actin filaments, which was regulated by cofilins and tropomodulins, contributed to myofibrillar fraction and low SF during conditioning.

Preoperative Antihypertensive Medication in Relation to Postoperative Atrial Fibrillation in Patients Undergoing Cardiac Surgery: A Meta-Analysis.

Background. We undertake a systematic review and meta-analysis to evaluate the effect of preoperative hypertension and preoperative antihypertensive medication to postoperative atrial fibrillation (POAF) in patients undergoing cardiac surgery. Methods. We searched PubMed, Embase, and Cochrane Library (from inception to March 2016) for eligible studies. The outcomes were the effects of preoperative hypertension, preoperative calcium antagonists regimen, preoperative ACE inhibitors regimen, and preoperative beta blocking agents regimen with POAF. We calculated pooled risk ratios (OR) and 95% CIs using random- or fixed-effects models. Results. Twenty-five trials involving 130087 patients were listed. Meta-analysis showed that the number of preoperative hypertension patients in POAF group was significantly higher (P < 0.05), while we found that there are no significant differences between two groups in Asia patients by subgroup analysis, which is in contrast to other outcomes. Compared with the Non-POAF group, the number of patients who used calcium antagonists and ACE inhibitors preoperatively in POAF group was significantly higher (P < 0.05). And we found that there were no significant differences between two groups of preoperative beta blocking agents used (P = 0.08). Conclusions. Preoperative hypertension and preoperative antihypertensive medication in patients undergoing cardiac operations seem to be associated with higher risk of POAF.

Hematopoietic Stem cell transplantation and lentiviral vector-based gene therapy for Krabbe's disease: Present convictions and future prospects.

Currently, presymtomatic hematopoietic stem and progenitor cell transplantation (HSPCT) is the only therapeutic modality that alleviates Krabbe's disease (KD)-induced central nervous system damage. However, all HSPCT-treated patients exhibit severe deterioration in peripheral nervous system function characterized by major motor and expressive language pathologies. We hypothesize that a combination of several mechanisms contribute to this phenomenon, including 1) nonoptimal conditioning protocols with consequent inefficient engraftment and biodistribution of donor-derived cells and 2) insufficient uptake of donor cell-secreted galactocerebrosidease (GALC) secondary to a naturally low expression level of the cation-independent mannose 6-phosphate-receptor (CI-MPR). We have characterized the effects of a busulfan (Bu) based conditioning regimen on the efficacy of HSPCT in prolonging twi mouse average life span. There was no correlation between the efficiency of bone marrow engraftment of donor cells and twi mouse average life span. HSPCT prolonged the average life span of twi mice, which directly correlated with the aggressiveness of the Bu-mediated conditioning protocols. HSPC transduced with lentiviral vectors carrying the GALC cDNA under control of cell-specific promoters were efficiently engrafted in twi mouse bone marrow. To facilitate HSPCT-mediated correction of GALC deficiency in target cells expressing low levels of CI-MPR, a novel GALC fusion protein including the ApoE1 receptor was developed. Efficient cellular uptake of the novel fusion protein was mediated by a mannose-6-phosphate-independent mechanism. The novel findings described here elucidate some of the cellular mechanisms that impede the cure of KD patients by HSPCT and concomitantly open new directions to enhance the therapeutic efficacy of HSPCT protocols for KD. © 2016 The Authors. Journal of Neuroscience Research Published by Wiley Periodicals, Inc.

Effect of black pepper essential oil on the quality of fresh pork during storage.

The effect of different concentrations (0, 0.1 and 0.5%, v/v) of black pepper essential oil (BPEO) on thiobarbituric acid reactive substances (TBARS), meat color, the percentage of metmyoglobin (MetMb%), microbiological parameters and total volatile basic nitrogen (TVB-N) of pork loins stored at 4°C for 9days was evaluated. BPEO treatments showed lower TBARS, MetMb%, yellowness (b*) values, Pseudomonas spp. and Enterobacteriaceae count and TVB-N values and higher lightness (L*) and redness (a*) values than the control during storage; the effectiveness of BPEO was dose-dependent. The retardation of the formation of MetMb by adding BPEO ensured higher L* and a* values and lower b* values than the control at 6 and 9days; the MetMb content has a similar trend to the lipid oxidation. The lower TVB-N value of BPEO treatments than the control could be attributed to the inhibition of Pseudomonas spp. and Enterobacteriaceae. Gram-negative bacteria were more sensitive than Gram-positive bacteria to BPEO.

Antibacterial Activity and Mechanism of Action of Black Pepper Essential Oil on Meat-Borne Escherichia coli.

The aim of this study was to investigate the antibacterial activity of black pepper essential oil (BPEO) on Escherichia coli, further evaluate the potential mechanism of action. Results showed that the minimum inhibition concentration (MIC) of BPEO was 1.0 µL/mL. The diameter of inhibition zone values were with range from 17.12 to 26.13 mm. 2 × MIC treatments had lower membrane potential and shorter kill-time than 1 × MIC, while control had the highest values. E. coli treated with BPEO became deformed, pitted, shriveled, adhesive, and broken. 2 × MIC exhibited the greatest electric conductivity at 1, 3, 5, 7, 9, 11, and 13 h, leaked DNA materials at 4, 8, 12, 16, 20, 24, and 28 h, proteins at 4, 6, 8, 10, 12, 14, and 16 h, potassium ion at 0, 0.5, 1, 1.5, and 2 h, phosphate ion at 0.5, 1, 1.5, and 2 h and ATP (P < 0.05); 1 × MIC had higher values than control. BPEO led to the leakage, disorder and death by breaking cell membrane. This study suggested that the BPEO has potential as the natural antibacterial agent in meat industry.

Dexamethasone versus ondansetron in the prevention of postoperative nausea and vomiting in patients undergoing laparoscopic surgery: a meta-analysis of randomized controlled trials.

BACKGROUND: Dexamethasone is an antiemetic alternative to ondansetron. We aimed to compare the effects of dexamethasone and ondansetron in preventing postoperative nausea and vomiting (PONV) in patients undergoing laparoscopic surgery. METHODS: We searched PubMed, Embase, Medline and Cochrane Library (from inception to July 2014) for eligible studies. The primary outcome was the incidence of PONV during the first 24 h after surgery. The secondary outcomes included PONV in the early postoperative stage (0-6 h), PONV in the late postoperative stage (6-24 h), and the postoperative anti-emetics used at both stages. We calculated pooled risk ratios (RR) and 95 % CIs using random- and fixed-effects models. RESULTS: Seven trials involving 608 patients were included in this meta-analysis, which found that dexamethasone had a comparable effectiveness in preventing PONV (RR, 0.91; 95 % CI, 0.73-1.13; P = 0.39) with that of ondansetron within 24 h of laparoscopic surgery, with no evidence of heterogeneity among the studies (I(2) = 0 %; P = 0.71). In the early postoperative stage (0-6 h), ondansetron was better at decreasing PONV than dexamethasone (RR, 1.71; 95 % CI, 1.05-2.77; P = 0.03), while in the late postoperative stage (6-24 h), dexamethasone was more effective in preventing PONV than ondansetron (RR, 0.51; 95 % CI, 0.27-0.93; P = 0.03). There was no significant difference in the postoperative anti-emetics used (RR, 0.90; 95 % CI, 0.67-1.19; P = 0.45). CONCLUSIONS: Dexamethasone was as effective and as safe as ondansetron in preventing PONV. Dexamethasone should be encouraged as an alternative to ondansetron for preventing PONV in patients undergoing laparoscopic surgery.

Intraosseous delivery of lentiviral vectors targeting factor VIII expression in platelets corrects murine hemophilia A.

Intraosseous (IO) infusion of lentiviral vectors (LVs) for in situ gene transfer into bone marrow may avoid specific challenges posed by ex vivo gene delivery, including, in particular, the requirement of preconditioning. We utilized IO delivery of LVs encoding a GFP or factor VIII (FVIII) transgene directed by ubiquitous promoters (a MND or EF-1α-short element; M-GFP-LV, E-F8-LV) or a platelet-specific, glycoprotein-1bα promoter (G-GFP-LV, G-F8-LV). A single IO infusion of M-GFP-LV or G-GFP-LV achieved long-term and efficient GFP expression in Lineage(-)Sca1(+)c-Kit(+) hematopoietic stem cells and platelets, respectively. While E-F8-LV produced initially high-level FVIII expression, robust anti-FVIII immune responses eliminated functional FVIII in circulation. In contrast, IO delivery of G-F8-LV achieved long-term platelet-specific expression of FVIII, resulting in partial correction of hemophilia A. Furthermore, similar clinical benefit with G-F8-LV was achieved in animals with pre-existing anti-FVIII inhibitors. These findings further support platelets as an ideal FVIII delivery vehicle, as FVIII, stored in α-granules, is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. Overall, a single IO infusion of G-F8-LV was sufficient to correct hemophilia phenotype for long term, indicating that this approach may provide an effective means to permanently treat FVIII deficiency.

Normalization and improvement of CNS deficits in mice with Hurler syndrome after long-term peripheral delivery of BBB-targeted iduronidase.

Mucopolysaccharidosis type I (MPS I) is a progressive lysosomal storage disorder with systemic and central nervous system (CNS) involvement due to deficiency of α-L-iduronidase (IDUA). We previously identified a receptor-binding peptide from apolipoprotein E (e) that facilitated a widespread delivery of IDUAe fusion protein into CNS. In this study, we evaluated the long-term CNS biodistribution, dose-correlation, and therapeutic benefits of IDUAe after systemic, sustained delivery via hematopoietic stem cell (HSC)-mediated gene therapy with expression restricted to erythroid/megakaryocyte lineages. Compared to the highest dosage group treated by nontargeted control IDUAc (165 U/ml), physiological levels of IDUAe in the circulation (12 U/ml) led to better CNS benefits in MPS I mice as demonstrated in glycosaminoglycan accumulation, histopathology analysis, and neurological behavior. Long-term brain metabolic correction and normalization of exploratory behavior deficits in MPS I mice were observed by peripheral enzyme therapy with physiological levels of IDUAe derived from clinically attainable levels of HSC transduction efficiency (0.1). Importantly, these levels of IDUAe proved to be more beneficial on correction of cerebrum pathology and behavioral deficits in MPS I mice than wild-type HSCs fully engrafted in MPS I chimeras. These results provide compelling evidence for CNS efficacy of IDUAe and its prospective translation to clinical application.

Platelets are efficient and protective depots for storage, distribution, and delivery of lysosomal enzyme in mice with Hurler syndrome.

Use of megakaryocytes/platelets for transgene expression may take advantage of their rapid turnover and protective storage in platelets and reduce the risk of activating oncogenes in hematopoietic stem and progenitor cells (HSCs). Here, we show that human megakaryocytic cells could overexpress the lysosomal enzyme, α-l-iduronidase (IDUA), which is deficient in patients with mucopolysaccharidosis type I (MPS I). Upon megakaryocytic differentiation, the amount of released enzyme increased rapidly and steadily by 30-fold. Using a murine MPS I model, we demonstrated that megakaryocyte/platelets were capable of producing, packaging, and storing large amounts of IDUA with proper catalytic activity, lysosomal trafficking, and receptor-mediated uptake. IDUA can be released directly into extracellular space or within microparticles during megakaryocyte maturation or platelet activation, while retaining the capacity for cross-correction in patient's cells. Gene transfer into 1.7% of HSCs led to long-term normalization of plasma IDUA and preferential distribution of enzyme in liver and spleen with complete metabolic correction in MPS I mice. Detection of GFP (coexpressed with IDUA) in Kupffer cells and hepatocytes suggested liver delivery of platelet-derived IDUA possibly via the clearance pathway for senile platelets. These findings provide proof of concept that cells from megakaryocytic lineage and platelets are capable of generating and storing fully functional lysosomal enzymes and can also lead to efficient delivery of both the enzymes released into the circulation and those protected within platelets/microparticles. This study opens a door for use of the megakaryocytes/platelets as a depot for efficient production, delivery, and effective tissue distribution of lysosomal enzymes.