Lipid nanoparticle formulations for optimal RNA-based topical delivery to murine airways.

INTRODUCTION: Lipid nanoparticles (LNP) have been successfully used as a platform technology for delivering nucleic acids to the liver. To broaden the application of LNPs in targeting non-hepatic tissues, we developed LNP-based RNA therapies (siRNA or mRNA) for the respiratory tract. Such optimized LNP systems could offer an early treatment strategy for viral respiratory tract infections such as COVID-19. METHODS: We generated a small library of six LNP formulations with varying helper lipid compositions and characterized their hydrodynamic diameter, size distribution and cargo entrapment properties. Next, we screened these LNP formulations for particle uptake and evaluated their potential for transfecting mRNA encoding green fluorescence protein (GFP) or SARS-CoV2 nucleocapsid-GFP fusion reporter gene in a human airway epithelial cell line in vitro. Following LNP-siGFP delivery, GFP protein knockdown efficiency was assessed by flow cytometry to determine %GFP+ cells and median fluorescence intensity (MFI) for GFP. Finally, lead LNP candidates were validated in Friend leukemia virus B (FVB) male mice via intranasal delivery of an mRNA encoding luciferase, using in vivo bioluminescence imaging. RESULTS: Dynamic light scattering revealed that all LNP formulations contained particles with an average diameter of <100 nm and a polydispersity index of <0.2. Human airway epithelial cell lines in culture internalized LNPs with differential GFP transfection efficiencies (73-97%). The lead formulation LNP6 entrapping GFP or Nuc-GFP mRNA demonstrated the highest transfection efficiency (97%). Administration of LNP-GFP siRNA resulted in a significant reduction of GFP protein expression. For in vivo studies, intranasal delivery of LNPs containing helper lipids (DSPC, DOPC, ESM or DOPS) with luciferase mRNA showed significant increase in luminescence expression in nasal cavity and lungs by at least 10 times above baseline control. CONCLUSION: LNP formulations enable the delivery of RNA payloads into human airway epithelial cells, and in the murine respiratory system; they can be delivered to nasal mucosa and lower respiratory tract via intranasal delivery. The composition of helper lipids in LNPs crucially modulates transfection efficiencies in airway epithelia, highlighting their importance in effective delivery of therapeutic products for airways diseases.

Tailoring the homing capacity of human Tregs for directed migration to sites of Th1-inflammation or intestinal regions.

Cell-based therapy with CD4+ FOXP3+ regulatory T cells (Tregs) is a promising strategy to limit organ rejection and graft-vs-host disease. Ongoing clinical applications have yet to consider how human Tregs could be modified to direct their migration to specific inflammation sites and/or tissues for more targeted immunosuppression. We show here that stable, homing-receptor-tailored human Tregs can be generated from thymic Tregs isolated from pediatric thymus or adult blood. To direct migration to Th1-inflammatory sites, addition of interferon-γ and IL-12 during Treg expansion produced suppressive, epigenetically stable CXCR3+ TBET+ FOXP3+ T helper (Th)1-Tregs. CXCR3 remained expressed after injection in vivo and Th1-Tregs migrated efficiently towards CXCL10 in vitro. To induce tissue-specific migration, addition of retinoic acid (RA) during Treg expansion induced expression of the gut-homing receptors α4ß7-integrin and CCR9. FOXP3+ RA-Tregs had elevated expression of the functional markers latency-associated peptide and glycoprotein A repetitions predominant, increased suppressive capacity in vitro and migrated efficiently to healthy and inflamed intestine after injection into mice. Homing-receptor-tailored Tregs were epigenetically stable even after long-term exposure to inflammatory conditions, suppressive in vivo and characterized by Th1- or gut-homing-specific transcriptomes. Tailoring human thymic Treg homing during in vitro expansion offers a new and clinically applicable approach to improving the potency and specificity of Treg therapy.

A magnetically actuated cellular strain assessment tool for quantitative analysis of strain induced cellular reorientation and actin alignment.

Commercially available cell strain tools, such as pneumatically actuated elastomer substrates, require special culture plates, pumps, and incubator setups. In this work, we present a magnetically actuated cellular strain assessment tool (MACSAT) that can be implemented using off-the-shelf components and conventional incubators. We determine the strain field on the MACSAT elastomer substrate using numerical models and experimental measurements and show that a specific region of the elastomer substrate undergoes a quasi-uniaxial 2D stretch, and that cells confined to this region of the MACSAT elastomer substrate undergo tensile, compressive, or zero axial strain depending on their angle of orientation. Using the MACSAT to apply cyclic strain on endothelial cells, we demonstrate that actin filaments within the cells reorient away from the stretching direction, towards the directions of minimum axial strain. We show that the final actin orientation angles in strained cells are spread over a region of compressive axial strain, confirming previous findings on the existence of a varied pre-tension in the actin filaments of the cytoskeleton. We also demonstrate that strained cells exhibit distinctly different values of actin alignment coherency compared to unstrained cells and therefore propose that this parameter, i.e., the coherency of actin alignment, can be used as a new readout to determine the occurrence/extent of actin alignment in cell strain experiments. The tools and methods demonstrated in this study are simple and accessible and can be easily replicated by other researchers to study the strain response of other adherent cells.

α-Integrin expression and function modulates presentation of cell surface calreticulin.

Calreticulin presentation on the cell surface is an important hallmark of immunogenic cell death (ICD), serving as the prophagocytic signal for macrophages. Cell adhesion is a physiologically relevant stimulus previously shown to increase calreticulin interaction with α-integrins via the juxtamembrane, cytosolic GFFKR motif. This study assessed whether integrin function can regulate surface calreticulin levels in ICD. We generated calreticulin-null T-lymphoblasts and confirmed the loss of surface calreticulin expression on cells treated with doxorubicin, an ICD inducer. Reconstituted expression with full-length calreticulin targeted to the endoplasmic reticulum (ER) successfully rescued doxorubicin-induced surface calreticulin. Reconstitution with a truncation mutant calreticulin targeted to the cytosol led to constitutively high surface calreticulin that was not further elevated by doxorubicin, suggesting calreticulin released from the stressed ER transits the cytosol before its translocation to the cell surface. When stimulated to engage integrin substrates, doxorubicin-treated wild-type T-lymphoblasts exhibited decreased surface calreticulin compared with cells under non-adherent conditions. The inhibitory effect on surface calreticulin was recapitulated for cells in suspension treated with a ß1-integrin-activating antibody, 9EG7. Similarly, cells expressing a truncated α-integrin cytosolic tail, bearing only the juxtamembrane GFFKR calreticulin-binding motif, exhibited low surface calreticulin with doxorubicin treatment under non-adherent conditions. Using partial permeabilization techniques to distinguish between cytosolic and ER staining, we found that ICD inducers promoted the accumulation of cytosolic calreticulin with negligible change in total calreticulin, suggesting that integrin-mediated inhibition of surface calreticulin was due to reduced cytosolic to surface translocation. T-lymphoblasts co-treated with an ICD inducer and 9EG7 exhibited reduced phagocytosis by macrophages when compared with treatment with only ICD inducer. This study reveals a previously uncharacterized function of integrins as negative regulators of ICD by suppressing presentation of cell surface calreticulin.

Magnetically actuated microstructured surfaces can actively modify cell migration behaviour.

We present a study on the application of magnetically actuated polymer micropillar surfaces in modifying the migration behaviour of cells. We show that micropillar surfaces actuated at a frequency of 1 Hz can cause more than a 5-fold decrease in cell migration rates compared to controls, whereas non-actuated micropillar surfaces cause no statistically significant alterations in cell migration rates. The effectiveness of the micropillar arrays in impeding cell migration depends on micropillar density and placement patterns, as well as the direction of micropillar actuation with respect to the direction of cell migration. Since the magnetic micropillar surfaces presented can be actuated remotely with small external magnetic fields, their integration with implants could provide new possibilities for in-vivo tissue engineering applications.

Using pictograms to assist caregivers in liquid medication administration: a systematic review.

WHAT IS KNOWN AND OBJECTIVE: It has been reported that more than 80% of out-of-hospital medication errors among the young children involve liquid formulations. The usefulness of pictorial aids to improve communication of medication instructions has not been extensively investigated for child health. The objective of this study was to determine the effectiveness of pictorial aids used to assist caregivers in the administration of liquid medications. METHODS: MEDLINE, CINAHL, PsycINFO, ScienceDirect, Scopus and the Cochrane Library were searched for articles published up to February 2015. Studies that used pictorial aids with liquid medications and measured at least one of the following outcomes were included: dosing accuracy, comprehension of medication instructions, recall of information and adherence of caregivers. Two authors independently selected studies, extracted data and assessed methodological quality of studies using the Cochrane Collaboration's tool. RESULTS AND DISCUSSION: Five experimental studies (four hospital based and one community based) with a total of 962 participants were included. A wide range of liquid formulations were studied, including both prescription and over-the-counter medications. The existing findings suggest that pictographic interventions reduced dosing errors, enhanced comprehension and recall of medication instructions and improved adherence of caregivers. Incorporating pictorial aids into verbal medication counselling or text-based instructions was more beneficial than using the single approach alone. Mixed results were identified for the relationship between health literacy of caregivers and effectiveness of pictorial aids. WHAT IS NEW AND CONCLUSION: The evidence remains limited due to the small number of studies found and variations in methodological quality. This review suggests that pictorial aids might be potential interventions, but more high-quality studies are needed to support the routine use of any pictogram-based materials with liquid medications in the clinical settings.

The orally active urotensin receptor antagonist, KR36676, attenuates cellular and cardiac hypertrophy.

BACKGROUND AND PURPOSE: Blockade of the actions of urotensin-II (U-II) mediated by the urotensin (UT) receptor should improve cardiac function and prevent cardiac remodelling in cardiovascular disease. Here, we have evaluated the pharmacological properties of the recently identified UT receptor antagonist, 2-(6,7-dichloro-3-oxo-2H-benzo[b][1,4]oxazin-4(3H)-yl)-N-methyl-N-(2-(pyrrolidin-1-yl)-1-(4-(thiophen-3-yl)phenyl) ethyl)acetamide (KR36676). EXPERIMENTAL APPROACH: Pharmacological properties of KR36676 were studied in a range of in vitro assays (receptor binding, calcium mobilization, stress fibre formation, cellular hypertrophy) and in vivo animal models such as cardiac hypertrophy induced by transverse aortic constriction (TAC) or myocardial infarction (MI). KEY RESULTS: KR36676 displayed high binding affinity for the UT receptor (Ki : 0.7 nM), similar to that of U-II (0.4 nM), and was a potent antagonist at that receptor (IC50 : 4.0 nM). U-II-induced stress fibre formation and cellular hypertrophy were significantly inhibited with low concentrations of KR36676 (≥0.01 µM). Oral administration of KR36676 (30 mg·kg(-1) ) in a TAC model in mice attenuated cardiac hypertrophy and myocardial fibrosis. Moreover, KR36676 restored cardiac function and myocyte size in rats with MI-induced cardiac hypertrophy. CONCLUSIONS AND IMPLICATIONS: A highly potent UT receptor antagonist exerted anti-hypertrophic effects not only in infarcted rat hearts but also in pressure-overloaded mouse hearts. KR36676 could be a valuable pharmacological tool in elucidating the complicated physiological role of U-II and UT receptors in cardiac hypertrophy.

First pregnancy and live birth resulting from cryopreserved embryos obtained from in vitro matured oocytes after oophorectomy in an ovarian cancer patient.

In vitro maturation (IVM) of immature oocytes retrieved from surgically resected ovaries has been proposed as a method of fertility preservation in ovarian cancer patients undergoing definitive surgery. While there had been several reports of successful derivation of mature oocytes and or embryos, there have been no reports as yet of successful pregnancies. In this case report, we present a pregnancy and live birth from a young patient, with stage IIIC ovarian cancer, who had undergone fertility sparing surgery. The immature oocytes recovered after oophorectomy were fertilized after IVM. The embryos obtained were cryopreserved and later transferred to achieve a singleton healthy pregnancy leading to a live birth.

Oxidative torrefaction of biomass residues and densification of torrefied sawdust to pellets.

Oxidative torrefaction of sawdust with a carrier gas containing 3-6% O(2) was investigated in a TG and a fluidized bed reactor, with the properties of the torrefied sawdust and pellets compared with traditional torrefaction without any O(2), as well as the dry raw material. It is found that the oxidative torrefaction process produced torrefied sawdust and pellets of similar properties as normally torrefied sawdust and corresponding pellets, especially on the density, energy consumption for pelletization, higher heating value and energy yield. For moisture absorption and hardness of the torrefied pellets, the oxidative torrefaction process showed slightly poor but negligible performance. Therefore, it is feasible to use oxygen laden combustion flue gases as the carrier gas for torrefaction of biomass. Besides, torrefied sawdust can be made into dense and strong pellets of high hydrophobicity at a higher die temperature than normally used in the production of traditional control pellets.

Torrefaction of sawdust in a fluidized bed reactor.

In the present work, stable fluidization of sawdust was achieved in a bench fluidized bed with an inclined orifice distributor without inert bed materials. A solids circulation pattern was established in the bed without the presence of slugging and channeling. The effects of treatment severity and weight loss on the solid product properties were identified. The decomposition of hemicelluloses was found to be responsible for the significant changes of chemical, physical and mechanical properties of the torrefied sawdust, including energy content, particle size distribution and moisture absorption capacity. The hydrophobicity of the torrefied sawdust was improved over the raw sawdust with a reduction of around 40 wt.% in saturated water uptake rate, and enhanced with increasing the treatment severity due to the decomposition of hemicelluloses which are rich in hydroxyl groups. The results in this study provided the basis for torrefaction in fluidized bed reactors.

The antiangiogenic and antinociceptive activities of n-propyl gallate.

n-Propyl gallate dose-dependently displayed an inhibitory effect on chick chorioallantoic membrane (CAM) angiogenesis. It markedly increased the endostatin level in both isolated CAM tissues and human umbilical vein endothelial cells (HUVECs). n-Propyl gallate was also able to enhance the endostatin mRNA level in HUVECs. Antinociceptive activity of n-propyl gallate was assessed using an acetic acid-induced writhing test in mice. In brief, n-propyl gallate possesses antiangiogenic activity via up-regulation of endostatin.

WW domain-mediated interaction with Wbp2 is important for the oncogenic property of TAZ.

The transcriptional co-activators YAP and TAZ are downstream targets inhibited by the Hippo tumor suppressor pathway. YAP and TAZ both possess WW domains, which are important protein-protein interaction modules that mediate interaction with proline-rich motifs, most commonly PPXY. The WW domains of YAP have complex regulatory roles as exemplified by recent reports showing that they can positively or negatively influence YAP activity in a cell and context-specific manner. In this study, we show that the WW domain of TAZ is important for it to transform both MCF10A and NIH3T3 cells and to activate transcription of ITGB2 but not CTGF, as introducing point mutations into the WW domain of TAZ (WWm) abolished its transforming and transcription-promoting ability. Using a proteomic approach, we discovered potential regulatory proteins that interact with TAZ WW domain and identified Wbp2. The interaction of Wbp2 with TAZ is dependent on the WW domain of TAZ and the PPXY-containing C-terminal region of Wbp2. Knockdown of endogenous Wbp2 suppresses, whereas overexpression of Wbp2 enhances, TAZ-driven transformation. Forced interaction of WWm with Wbp2 by direct C-terminal fusion of full-length Wbp2 or its TAZ-interacting C-terminal domain restored the transforming and transcription-promoting ability of TAZ. These results suggest that the WW domain-mediated interaction with Wbp2 promotes the transforming ability of TAZ.

Simple and inexpensive method of wood pellets macro-porosity measurement.

A novel simplified stereometric measurement method for determining the macro-porosity of wood pellets through geometrical approach was successfully developed and tested. The irregular ends of pellets of circular cross-section were sanded flat so that their geometry becomes cylinder and their volumes evaluated using mensuration formula. Such formed cylindrical pellets were loose or tap filled to selected volumes to evaluate the macro-porosity and the constant specific weight. The method was extended to evaluate actual wood pellets properties. Overall macro-porosity of actual wood pellets was determined as 41.0+/-2.5% and 35.5+/-2.7%, mean bulk density as 670+/-29 kg m(-3) and 731+/-31 kg m(-3), and classified as "Class-3:Medium" and "Class-3&4:Medium to Low" for loose and tapped fills, respectively. Hausner ratio and Carr's compressibility index classify wood pellets as "freely flowing." The developed stereometric method can be used as a handy inexpensive laboratory procedure to estimate the macro-porosity of different types and makes of wood pellets and other similar packaged materials.

Permeability of wood pellets in the presence of fines.

Broken pellets and fines are produced when pellets are handled. The resistance to air flow was measured for clean pellets and for pellets mixed with 1-20% broken pellets (fines). A pellet diameter was 6mm. The lengths ranged from 6 to 12 mm. Clean pellets were defined as particles that remained on a 4mm screen. A typical sieve analysis showed 30% of the mass of particles that passed through the 4mm screen was smaller than 1mm. The airflow rates used in the experiment ranged from 0.004 to 0.357 ms(-1). The corresponding pressure drop ranged from 1.9 to 271 Pam(-1) for clean pellets, from 4.8 to 1100 Pam(-1) for 10% fines content, and from 7.9 to 1800 Pam(-1) for 20% fines content. Coefficients of Hukill and Ives' equation were estimated for clean pellets and a multiplier was defined to calculate pressure drop for pellets mixed with fines.

Assessment of Physical Activity Level among Individuals with Type 2 Diabetes Mellitus at Cheras Health Clinic, Kuala Lumpur.

A cross-sectional study was carried out to assess the physical activity levels among patients with type 2 diabetes mellitus (DM) at Cheras Health Clinic in Kuala Lumpur. A total of 132 subjects (62 men and 70 women) aged 30 years and above participated in this study. Data was collected using an interview based questionnaire to obtain socio-demographic and health profile information. Physical activity was assessed using a shortened version of the International Physical Activity Questionnaire (IPAQ). Anthropometric measurements and body fat were also taken. Glycaemic status, that is, HbA1c, fasting blood sugar (FBS) and 2 hours post-prandial (2-HPP) were obtained from medical records. Results showed that the mean age of the patients was 51.9 + 5.8 years. The majority of patients had poor glycaemic control based on HbA1c (70.7%), FBS (71.9%) and 2HPP (85.4%). Patients who were unmarried and aged(60 years and above had a lower physical activity level (p< 0.05). In the older age group, low physical activity was associated with poor glycaemic control (p< 0.05). Patients in the moderate and high physical activity level were motivated to perform physical activity so as to be healthy (68.1%). Low physical activity level among patients was due to lack of time (54.5%) and lack of energy (21.2%). In conclusion, physical activity levels of the patients were unsatisfactory and associated with poor glycaemic control, especially in the elderly. There is a need to encourage diabetic patients to undertake regular physical activity in order to achieve optimal glycaemic control.

Expression of the atf1+ gene is upregulated in fission yeast under nitrosative and nutritional stresses.

This work was designed to assess regulation of the atf1+ gene in the fission yeast Schizosaccharomyces pombe under nitrosative and nutritional stresses, using the atf1+-lacZ fusion gene and RT-PCR. Nitric oxide (NO)-generating sodium nitroprusside (SNP; 10 micromol/L) and nitrogen depletion significantly enhanced synthesis of beta-galactosidase from the atf1+-lacZ fusion gene in S. pombe Pap1-positive KP1 cells, but not in S. pombe Pap1-negative TP108-3C cells. SNP (10 micromol/L) and nitrogen depletion also caused a significant increase in atf1+ mRNA levels in Pap1-positive cells, but not in Pap1-negative cells. Depletion of glucose marginally increased synthesis of beta-galactosidase from the fusion gene in S. pombe Pap1-positive cells. Taken together, the S. pombe atf1+ gene is upregulated by nitrosative and nutritional stresses on a transcriptional level, possibly via the mediation of Pap1.

Protein phosphatase 1 nuclear targeting subunit is a hypoxia inducible gene: its role in post-translational modification of p53 and MDM2.

p53, the most commonly mutated tumor suppressor gene in human cancers, is a master regulator of apoptosis in many types of cells. Recently, protein phosphatase-1 (PP1) has emerged as a key phosphatase of p53, which modulates the interaction of p53 with its regulatory protein mouse double minute 2 (MDM2) and transcriptional activity. In the present study, we demonstrate the potential role of PP1 nuclear targeting subunit (PNUTS) in regulating the phosphorylation and apoptotic activities of p53. Hypoxia significantly increased mRNA and protein expression of PNUTS in various cell lines concomitantly with increases in p53. Promoter analysis confirmed the presence of hypoxia response elements in the promoter region of the PNUTS gene, which respond to hypoxia and forced expression of hypoxia-inducible factor 1 alpha. Overexpression of PNUTS markedly increased cell death in response to hypoxia, with increased expression of Bax, an apoptosis-related gene induced by p53. Consistently, PNUTS increased the nuclear localization, phosphorylation, and transcriptional activity of p53 as well as the ubiquitin-dependent proteosomal degradation of MDM2. However, the W401A mutant form of PNUTS, which is incapable of binding to PP1, failed to induce these events. Taken together, our findings suggest that PNUTS may play an important role in controlling cell death in response to cellular stresses such as hypoxia through the post-translational modification of p53 and MDM2.

Enhancement of the sulfur capture capacity of limestones by the addition of Na2CO3 and NaCl.

The ability of Na2CO3 and NaCl to enhance the sulfur capture capacity of three limestones was evaluated via fixed-bed calcination and sulfation experiments. The tested limestones represent three different sulfation morphologies: unreacted-core, network, and uniformly sulfated. Treatment with aqueous or powdered Na2CO3 significantly increased the Ca-utilization for two stones which normally sulfate in an unreacted-core pattern (20% to 45%) and network pattern (33% to 49%). The increase was lower for the uniformly sulfated stone (44% to 48%). Na2CO3 treatment increased the number of macropores leading to uniform sulfation of all particles, nearly eliminating the normal strong dependence of utilization on limestone type and particle size. The effect of Na2CO3 is believed to be associated with formation of a eutectic melt which enhances ionic diffusion and accelerates molecular rearrangement of the CaO. Treatment with aqueous NaCl solution caused a decrease in utilization, probably due to formation of large grains and plugging of pores caused by formation of a large amount of eutectic melt. The effect of Na2CO3 is less sensitive than that of NaCl to the amount added and the combustion environment (temperature and gas composition). In addition, Na2CO3 neither promotes corrosion nor forms chlorinated byproducts, which are main concerns associated with NaCl. Thus, Na2CO3 appears to have significant advantages over NaCl for enhancement of limestone sulfur capture capacity in fluidized-bed combustors.

RasC is required for optimal activation of adenylyl cyclase and Akt/PKB during aggregation.

Disruption of Dictyostelium rasC, encoding a Ras subfamily protein, generated cells incapable of aggregation. While rasC expression is enriched in a cell type-specific manner during post-aggregative development, the defect in rasC(-) cells is restricted to aggregation and fully corrected by application of exogenous cAMP pulses. cAMP is not produced in rasC(-) cells stimulated by 2'-deoxy-cAMP, but is produced in response to GTPgammaS in cell lysates, indicating that G-protein-coupled cAMP receptor activation of adenylyl cyclase is regulated by RasC. However, cAMP-induced ERK2 phosphorylation is unaffected in rasC(-) cells, indicating that RasC is not an upstream activator of the mitogen-activated protein kinase required for cAMP relay. rasC(-) cells also exhibit reduced chemotaxis to cAMP during early development and delayed response to periodic cAMP stimuli produced by wild-type cells in chimeric mixtures. Furthermore, cAMP-induced Akt/PKB phosphorylation through a phosphatidylinositide 3-kinase (PI3K)-dependent pathway is dramatically reduced in rasC(-) cells, suggesting that G-protein-coupled serpentine receptor activation of PI3K is regulated by RasC. Cells lacking the RasGEF, AleA, exhibit similar defects as rasC(-) cells, suggesting that AleA may activate RasC.