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.

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.

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.

The effect of cooking temperature on the aggregation and digestion rate of myofibrillar proteins in Jinhua ham.

BACKGROUND: In order to evaluate the effect of cooking temperature on the nutrition quality of dry-cured hams, 60 biceps femoris samples from 16 Jinhua hams were divided into four groups (control, 70, 100 and 120 °C) and cooked for 30 min. Carbonyl content, sulfhydryl groups, surface hydrophobicity, microstructure, protein aggregation and digestibility of myofibrillar proteins were investigated. RESULTS: Cooking promoted carbonylation and decreased sulfhydryl groups in a temperature-dependent way. Scanning electron microscopy and Nile Red revealed that protein aggregation became a main phenomenon at 120 °C; it coincided with surface hydrophobicity. The increased carbonyl content and decreased sulfhydryl groups contributed to the formation of aggregates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles showed the initial difference in proteolysis rate among four groups. The in vitro digestibility of pepsin and of trypsin and α-chymotrypsin increased from the control to 100 °C and decreased from 100 to 120 °C. CONCLUSION: The increased digestibility could be attributed to the oxidation of proteins and exposing recognition sites of digestive enzymes, while the decreased digestibility was due to the formation of aggregates. Cooking was a main factor that affected the digestibility of Jinhua ham, and cooking at 100 °C could be an ideal way to gain the highest digestibility of Jinhua ham. © 2018 Society of Chemical Industry.

A convenient approach to facilitate monitoring Gaucher disease progression and therapeutic response.

Gaucher disease (GD) is caused by mutations on the GBA1 gene leading to deficiency in acid ß-glucosidase (GCase) and subsequent accumulation of its substrates, glucosylceramide (GlcC) and glucosylsphingosine (GlcS). GlcS in plasma has been proposed as a highly sensitive and specific biomarker for the diagnosis of GD and for monitoring disease progression and response to therapy. Here we report a novel robust and accurate hydrophilic interaction liquid chromatography tandem mass spectrometric method (HILIC-MS/MS) for the direct measurement of glucosylsphingosine (GlcS) in dried plasma spots (DPS). The method was also capable of resolving the isomeric pair, glucosylsphingosine and galactosylsphingosine, the latter of which was proposed as a promising biomarker for Krabbe disease. The method was fully validated and applied to the analysis of 19 GD patients and carriers. The GlcS levels in 9 GD type I patients who have been on enzyme replacement therapy (ERT) were reduced to a mean of 31.0 nM, much lower compared to a pre-treated specimen at a level of 85.8 nM, but still significantly elevated compared to healthy controls. GlcS concentrations in three treated type III GD patients were much lower compared to an untreated patient. In our preclinical GD studies, 4L;C* mice (subacute nGD model) exhibited comparable levels of plasma GlcS, but had much higher GlcS accumulation in the brain than those of 9V/null mice (chronic neuropathic GD model). Our method for the measurement of GlcS in DPS proved to be a very convenient approach for sample collection, storage and shipping nationwide and internationally.

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.

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.

Characterization of purple acid phosphatases involved in extracellular dNTP utilization in Stylosanthes.

Stylo (Stylosanthes spp.) is a pasture legume predominant in tropical and subtropical areas, where low phosphorus (P) availability is a major constraint for plant growth. Therefore, stylo might exhibit superior utilization of the P pool on acid soils, particularly organic P. However, little is known about mechanisms of inorganic phosphate (Pi) acquisition employed by stylo. In this study, the utilization of extracellular deoxy-ribonucleotide triphosphate (dNTP) and the underlying physiological and molecular mechanisms were examined for two stylo genotypes with contrasting P efficiency. Results showed that the P-efficient genotype, TPRC2001-1, was superior to the P-inefficient genotype, Fine-stem, when using dNTP as the sole P source. This was reflected by a higher dry weight and total P content for TPRC2001-1 than for Fine-stem, which was correlated with higher root-associated acid phosphatase (APase) activities in TPRC2001-1 under low P conditions. Subsequently, three PAP members were cloned from TPRC2001-1: SgPAP7, SgPAP10, and SgPAP26 Expression levels of these three SgPAPs were up-regulated by Pi starvation in stylo roots. Furthermore, there was a higher abundance of transcripts of SgPAP7 and SgPAP10 in TPRC2001-1 than in Fine-stem. Subcellular localization analysis demonstrated that these three SgPAPs were localized on the plasma membrane. Overexpression of these three SgPAPs could result in significantly increased root-associated APase activities, and thus extracellular dNTP utilization in bean hairy roots. Taken together, the results herein suggest that SgPAP7, SgPAP10, and SgPAP26 may differentially contribute to root-associated APase activities, and thus control extracellular dNTP utilization in stylo.

Comparison between two-dimensional and three-dimensional quantitative coronary angiography for the prediction of functional severity in true bifurcation lesions: Insights from the randomized DK-CRUSH II, III, and IV trials.

OBJECTIVE: This study investigated the diagnostic accuracy of three-dimensional quantitative coronary angiography (3D-QCA) compared with conventional 2D-QCA for predicting functional severity assessed by fractional flow reserve (FFR) for true bifurcation lesions. METHODS: Based on pooled data from the randomized DK-CRUSH II, III, and IV trials, we evaluated the patients with true bifurcation lesions who underwent coronary angiography together with functional evaluations using FFR in both the main vessel and the side branch. Off-line 2D- and 3D-QCA analyses were conducted using dedicated bifurcation QCA analysis software. Measurements of minimum lumen diameter (MLD), percentage diameter stenosis (% DS), and minimum lumen area (MLA) were compared between 2D- and 3D-QCA, and we evaluated their predictive values of functionally significant FFR. RESULTS: Ninety patients were eligible for enrollment in the present study. In the main vessel, MLA measured by 3D-QCA was the most accurate predictor of FFR <0.75 (C statistic 0.85, P < 0.001), while MLD measured by 2D-QCA was a similarly accurate predictor (C statistic 0.85, P < 0.001). In the side branch, the best metrics for predicting FFR <0.75 were % DS measured by 2D-QCA with a C statistic value of 0.91 (P < 0.001) and MLA measured by 3D-QCA with a C statistic value of 0.81 (P < 0.001). However, both 2D- and 3D-QCA metrics exhibited low accuracies for predicting FFR <0.75 in intermediate bifurcation lesions. CONCLUSIONS: 3D-QCA analysis for true bifurcation lesions did not improve the predictive accuracy of functionally significant FFR compared with 2D-QCA analysis. In lesions with intermediate stenosis, the diagnostic performance of both 2D- and 3D-QCA-derived measurements in differentiating functional severity is limited.

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.

Engineering a lysosomal enzyme with a derivative of receptor-binding domain of apoE enables delivery across the blood-brain barrier.

To realize the potential of large molecular weight substances to treat neurological disorders, novel approaches are required to surmount the blood-brain barrier (BBB). We investigated whether fusion of a receptor-binding peptide from apolipoprotein E (apoE) with a potentially therapeutic protein can bind to LDL receptors on the BBB and be transcytosed into the CNS. A lysosomal enzyme, α-L-iduronidase (IDUA), was used for biological and therapeutic evaluation in a mouse model of mucopolysaccharidosis (MPS) type I, one of the most common lysosomal storage disorders with CNS deficits. We identified two fusion candidates, IDUAe1 and IDUAe2, by in vitro screening, that exhibited desirable receptor-mediated binding, endocytosis, and transendothelial transport as well as appropriate lysosomal enzyme trafficking and biological function. Robust peripheral IDUAe1 or IDUAe2 generated by transient hepatic expression led to elevated enzyme levels in capillary-depleted, enzyme-deficient brain tissues and protein delivery into nonendothelium perivascular cells, neurons, and astrocytes within 2 d of treatment. Moreover, 5 mo after long-term delivery of moderate levels of IDUAe1 derived from maturing red blood cells, 2% to 3% of normal brain IDUA activities were obtained in MPS I mice, and IDUAe1 protein was detected in neurons and astrocytes throughout the brain. The therapeutic potential was demonstrated by normalization of brain glycosaminoglycan and ß-hexosaminidase in MPS I mice 5 mo after moderate yet sustained delivery of IDUAe1. These findings provide a noninvasive and BBB-targeted procedure for the delivery of large-molecule therapeutic agents to treat neurological lysosomal storage disorders and potentially other diseases that involve the brain.

Comparison of intravascular ultrasound guided versus angiography guided drug eluting stent implantation: a systematic review and meta-analysis.

BACKGROUND: Intravascular ultrasound (IVUS) can be a useful tool during drug-eluting stents (DES) implantation as it allows accurate assessment of lesion severity and optimal treatment planning. However, numerous reports have shown that IVUS guided percutaneous coronary intervention is not associated with improved clinical outcomes, especially in non-complex patients and lesions. METHODS: We searched the literature in Medline, the Cochrane Library, and other internet sources to identify studies that compare clinical outcomes between IVUS-guided and angiography-guided DES implantation. Random-effects model was used to assess treatment effect. RESULTS: Twenty eligible studies with a total of 29,068 patients were included in this meta-analysis. The use of IVUS was associated with significant reductions in major adverse cardiovascular events (MACE, odds ratios [OR] 0.77, 95 % confidence intervals [CI] 0.71-0.83, P < 0.001), death (OR 0.62, 95 % CI 0.54-0.71, p < 0.001), and stent thrombosis (OR 0.59, 95 % CI: 0.47-0.73, P < 0.001). The benefit was also seen in the repeated analysis of matched and randomized studies. In stratified analysis, IVUS guidance appeared to be beneficial not only in patients with complex lesions or acute coronary syndromes (ACS) but also patients with mixed lesions or presentations (MACE: OR 0.69, 95 % CI: 0.60-0.79, p < 0.001, OR 0.81, 95 % CI 0.74-0.90, p < 0.001, respectively). By employing meta-regression analysis, the benefit of IVUS is significantly pronounced in patients with complex lesions or ACS with respect to death (p = 0.048). CONCLUSIONS: IVUS guidance was associated with improved clinical outcomes, especially in patients with complex lesions admitted with ACS. Large, randomized clinical trials are warranted to identify populations and lesion characteristics where IVUS guidance would be associated with better outcomes.

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.

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.

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.

Gut microbiota impacts bone via Bacteroides vulgatus-valeric acid-related pathways.

Although the gut microbiota has been reported to influence osteoporosis risk, the individual species involved, and underlying mechanisms, remain largely unknown. We performed integrative analyses in a Chinese cohort of peri-/post-menopausal women with metagenomics/targeted metabolomics/whole-genome sequencing to identify novel microbiome-related biomarkers for bone health. Bacteroides vulgatus was found to be negatively associated with bone mineral density (BMD), which was validated in US white people. Serum valeric acid (VA), a microbiota derived metabolite, was positively associated with BMD and causally downregulated by B. vulgatus. Ovariectomized mice fed B. vulgatus demonstrated increased bone resorption and poorer bone micro-structure, while those fed VA demonstrated reduced bone resorption and better bone micro-structure. VA suppressed RELA protein production (pro-inflammatory), and enhanced IL10 mRNA expression (anti-inflammatory), leading to suppressed maturation of osteoclast-like cells and enhanced maturation of osteoblasts in vitro. The findings suggest that B. vulgatus and VA may represent promising targets for osteoporosis prevention/treatment.

K-Cl cotransporter gene expression during human and murine erythroid differentiation.

The K-Cl cotransporter (KCC) regulates red blood cell (RBC) volume, especially in reticulocytes. Western blot analysis of RBC membranes revealed KCC1, KCC3, and KCC4 proteins in mouse and human cells, with higher levels in reticulocytes. KCC content was higher in sickle versus normal RBC, but the correlation with reticulocyte count was poor, with inter-individual variability in KCC isoform ratios. Messenger RNA for each isoform was measured by real time RT-quantitative PCR. In human reticulocytes, KCC3a mRNA levels were consistently the highest, 1-7-fold higher than KCC4, the second most abundant species. Message levels for KCC1 and KCC3b were low. The ratios of KCC RNA levels varied among individuals but were similar in sickle and normal RBC. During in vivo maturation of human erythroblasts, KCC3a RNA was expressed consistently, whereas KCC1 and KCC3b levels declined, and KCC4 message first increased and then decreased. In mouse erythroblasts, a similar pattern for KCC3 and KCC1 expression during in vivo differentiation was observed, with low KCC4 RNA throughout despite the presence of KCC4 protein in mature RBC. During differentiation of mouse erythroleukemia cells, protein levels of KCCs paralleled increasing mRNA levels. Functional properties of KCCs expressed in HEK293 cells were similar to each other and to those in human RBC. However, the anion dependence of KCC in RBC resembled most closely that of KCC3. The results suggest that KCC3 is the dominant isoform in erythrocytes, with variable expression of KCC1 and KCC4 among individuals that could result in modulation of KCC activity.

Reprogramming erythroid cells for lysosomal enzyme production leads to visceral and CNS cross-correction in mice with Hurler syndrome.

Restricting transgene expression to maturing erythroid cells can reduce the risk for activating oncogenes in hematopoietic stem cells (HSCs) and their progeny, yet take advantage of their robust protein synthesis machinery for high-level protein production. This study sought to evaluate the feasibility and efficacy of reprogramming erythroid cells for production of a lysosomal enzyme, alpha-L-iduronidase (IDUA). An erythroid-specific hybrid promoter provided inducible IDUA expression and release during in vitro erythroid differentiation in murine erythroleukemia cells, resulting in phenotypical cross-correction in an enzyme-deficient lymphoblastoid cell line derived from patients with mucopolysaccharidosis type I (MPS I). Stable and higher than normal plasma IDUA levels were achieved in vivo in primary and secondary MPS I chimeras for at least 9 months after transplantation of HSCs transduced with the erythroid-specific IDUA-containing lentiviral vector (LV). Moreover, long-term metabolic correction was demonstrated by normalized urinary glycosaminoglycan accumulation in all treated MPS I mice. Complete normalization of tissue pathology was observed in heart, liver, and spleen. Notably, neurological function and brain pathology were significantly improved in MPS I mice by erythroid-derived, higher than normal peripheral IDUA protein. These data demonstrate that late-stage erythroid cells, transduced with a tissue-specific LV, can deliver a lysosomal enzyme continuously at supraphysiological levels to the bloodstream and can correct the disease phenotype in both viscera and CNS of MPS I mice. This approach provides a paradigm for the utilization of RBC precursors as a depot for efficient and potentially safer systemic delivery of nonsecreted proteins by ex vivo HSC gene transfer.

A multifaceted evaluation of microgliosis and differential cellular dysregulation of mammalian target of rapamycin signaling in neuronopathic Gaucher disease.

Neuronopathic Gaucher disease (nGD) is an inherited neurodegenerative disease caused by mutations in GBA1 gene and is associated with premature death. Neuroinflammation plays a critical role in disease pathogenesis which is characterized by microgliosis, reactive astrocytosis, and neuron loss, although molecular mechanisms leading to neuroinflammation are not well-understood. In this report, we developed a convenient tool to quantify microglia proliferation and activation independently and uncovered abnormal proliferation of microglia (∼2-fold) in an adult genetic nGD model. The nGD-associated pattern of inflammatory mediators pertinent to microglia phenotypes was determined, showing a unique signature favoring pro-inflammatory chemokines and cytokines. Moreover, highly polarized (up or down) dysregulations of mTORC1 signaling with varying lysosome dysfunctions (numbers and volume) were observed among three major cell types of nGD brain. Specifically, hyperactive mTORC1 signaling was detected in all disease-associated microglia (Iba1high) with concurrent increase in lysosome function. Conversely, the reduction of neurons presenting high mTORC1 activity was implicated (including Purkinje-like cells) which was accompanied by inconsistent changes of lysosome function in nGD mice. Undetectable levels of mTORC1 activity and low Lamp1 puncta were noticed in astrocytes of both diseased and normal mice, suggesting a minor involvement of mTORC1 pathway and lysosome function in disease-associated astrocytes. These findings highlight the differences and complexity of molecular mechanisms that are involved within various cell types of the brain. The quantifiable parameters established and nGD-associated pattern of neuroinflammatory mediators identified would facilitate the efficacy evaluation on microgliosis and further discovery of novel therapeutic target(s) in treating neuronopathic Gaucher disease.