Engineered nanoparticles promote cardiac tropism of AAV vectors.

Cardiac muscle targeting is a notoriously difficult task. Although various nanoparticle (NP) and adeno-associated viral (AAV) strategies with heart tissue tropism have been developed, their performance remains suboptimal. Significant off-target accumulation of i.v.-delivered pharmacotherapies has thwarted development of disease-modifying cardiac treatments, such as gene transfer and gene editing, that may address both rare and highly prevalent cardiomyopathies and their complications. Here, we present an intriguing discovery: cargo-less, safe poly (lactic-co-glycolic acid) particles that drastically improve heart delivery of AAVs and NPs. Our lead formulation is referred to as ePL (enhancer polymer). We show that ePL increases selectivity of AAVs and virus-like NPs (VLNPs) to the heart and de-targets them from the liver. Serotypes known to have high (AAVrh.74) and low (AAV1) heart tissue tropisms were tested with and without ePL. We demonstrate up to an order of magnitude increase in heart-to-liver accumulation ratios in ePL-injected mice. We also show that ePL exhibits AAV/NP-independent mechanisms of action, increasing glucose uptake in the heart, increasing cardiac protein glycosylation, reducing AAV neutralizing antibodies, and delaying blood clearance of AAV/NPs. Current approaches utilizing AAVs or NPs are fraught with challenges related to the low transduction of cardiomyocytes and life-threatening immune responses; our study introduces an exciting possibility to direct these modalities to the heart at reduced i.v. doses and, thus, has an unprecedented impact on drug delivery and gene therapy. Based on our current data, the ePL system is potentially compatible with any therapeutic modality, opening a possibility of cardiac targeting with numerous pharmacological approaches.

De Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation.

[Figure: see text].

A body map of somatic mutagenesis in morphologically normal human tissues.

Somatic mutations that accumulate in normal tissues are associated with ageing and disease1,2. Here we performed a comprehensive genomic analysis of 1,737 morphologically normal tissue biopsies of 9 organs from 5 donors. We found that somatic mutation accumulations and clonal expansions were widespread, although to variable extents, in morphologically normal human tissues. Somatic copy number alterations were rarely detected, except for in tissues from the oesophagus and cardia. Endogenous mutational processes with the SBS1 and SBS5 mutational signatures are ubiquitous among normal tissues, although they exhibit different relative activities. Exogenous mutational processes operate in multiple tissues from the same donor. We reconstructed the spatial somatic clonal architecture with sub-millimetre resolution. In the oesophagus and cardia, macroscopic somatic clones that expanded to hundreds of micrometres were frequently seen, whereas in tissues such as the colon, rectum and duodenum, somatic clones were microscopic in size and evolved independently, possibly restricted by local tissue microstructures. Our study depicts a body map of somatic mutations and clonal expansions from the same individual.

Straw Incorporation with Nitrogen Amendment Shapes Bacterial Community Structure in an Iron-Rich Paddy Soil by Altering Nitrogen Reserves.

Incorporation of crop straw into the soil along with inorganic fertilization is a widespread agricultural practice and is essential in nutrient-scarce soils, such as iron-rich (ferruginous) paddy soils. The responses of soil bacterial communities to straw incorporation under different nitrogen inputs in iron-rich soils remain unclear. Therefore, 6000 kg ha-1 dry wheat (Triticum aestivum L. cv. Zhengmai 12) straw was applied to a rice paddy with and without nitrogen amendment (0, 80, 300, and 450 kg ha-1 N as urea), to investigate its effects on soil fertility and bacterial community structure. Organic matter, total nitrogen, and water contents tended to decrease in straw-incorporated soils with different nitrogen inputs. Proteobacteria was the dominant bacterial phylum across all treatments (26.3-32.5% of total sequences), followed by Chloroflexi, Acidobacteria, and Nitrospirae. Up to 18.0% of all the taxa in the bacterial communities were associated with iron cycling. Straw incorporation with nitrogen amendment increased the relative abundance of iron oxidizers, Gallionellaceae, while decreasing the relative abundance of iron reducers, Geobacteraceae. Bacterial community composition shifted in different treatments, with total nitrogen, water, and Fe(III) contents being the key drivers. Straw incorporation supplemented by 300 kg ha-1 N increased bacterial richness and enhanced all the predicted bacterial functions, so that it is recommended as the optimal nitrogen dosage in practice.

Somatic MAP3K3 mutation defines a subclass of cerebral cavernous malformation.

Cerebral cavernous malformations (CCMs) are vascular disorders that affect up to 0.5% of the total population. About 20% of CCMs are inherited because of familial mutations in CCM genes, including CCM1/KRIT1, CCM2/MGC4607, and CCM3/PDCD10, whereas the etiology of a majority of simplex CCM-affected individuals remains unclear. Here, we report somatic mutations of MAP3K3, PIK3CA, MAP2K7, and CCM genes in CCM lesions. In particular, somatic hotspot mutations of PIK3CA are found in 11 of 38 individuals with CCMs, and a MAP3K3 somatic mutation (c.1323C>G [p.Ile441Met]) is detected in 37.0% (34 of 92) of the simplex CCM-affected individuals. Strikingly, the MAP3K3 c.1323C>G mutation presents in 95.7% (22 of 23) of the popcorn-like lesions but only 2.5% (1 of 40) of the subacute-bleeding or multifocal lesions that are predominantly attributed to mutations in the CCM1/2/3 signaling complex. Leveraging mini-bulk sequencing, we demonstrate the enrichment of MAP3K3 c.1323C>G mutation in CCM endothelium. Mechanistically, beyond the activation of CCM1/2/3-inhibited ERK5 signaling, MEKK3 p.Ile441Met (MAP3K3 encodes MEKK3) also activates ERK1/2, JNK, and p38 pathways because of mutation-induced MEKK3 kinase activity enhancement. Collectively, we identified several somatic activating mutations in CCM endothelium, and the MAP3K3 c.1323C>G mutation defines a primary CCM subtype with distinct characteristics in signaling activation and magnetic resonance imaging appearance.

Low-density lipoprotein nanomedicines: mechanisms of targeting, biology, and theranostic potential.

Native nanostructured lipoproteins such as low- and high-density lipoproteins (LDL and HDL) are powerful tools for the targeted delivery of drugs and imaging agents. While the cellular recognition of well-known HDL-based carriers occurs via interactions with an HDL receptor, the selective delivery and uptake of LDL particles by target cells are more complex. The most well-known mode of LDL-based delivery is via the interaction between apolipoprotein B (Apo-B) - the main protein of LDL - and the low-density lipoprotein receptor (LDLR). LDLR is expressed in the liver, adipocytes, and macrophages, and thus selectively delivers LDL carriers to these cells and tissues. Moreover, the elevated expression of LDLR in tumor cells indicates a role for LDL in the targeted delivery of chemotherapy drugs. In addition, chronic inflammation associated with hypercholesterolemia (i.e., high levels of endogenous LDL) can be abated by LDL carriers, which outcompete the deleterious oxidized LDL for uptake by macrophages. In this case, synthetic LDL nanocarriers act as 'eat-me' signals and exploit mechanisms of native LDL uptake for targeted drug delivery and imaging. Lastly, recent studies have shown that the delivery of LDL-based nanocarriers to macrophages via fluid-phase pinocytosis is a promising tool for atherosclerosis imaging. Hence, the present review summarizes the use of natural and synthetic LDL-based carriers for drug delivery and imaging and discusses various mechanisms of targeting.

Zenglv Fumai Granule protects cardiomyocytes against hypoxia/reoxygenation-induced apoptosis via inhibiting TRIM28 expression.

Myocardial ischemia/reperfusion (MIR) injury, which occurs following acute myocardial infarction, can cause secondary damage to the heart. Tripartite interaction motif (TRIM) proteins, a class of E3 ubiquitin ligases, have been recognized as critical regulators in MIR injury. Zenglv Fumai Granule (ZFG) is a clinical prescription for the treatment of sick sinus syndrome, a disease that is associated with MIR injury. The present study aimed to investigate the effect of ZFG on MIR injury and to determine whether ZFG exerts its effects via regulation of TRIM proteins. In order to establish an in vitro MIR model, human cardiomyocyte cell line AC16 was cultured under hypoxia for 5 h and then under normal conditions for 1 h. Following hypoxia/reoxygenation (H/R) treatment, these cells were cultured with different ZFG concentrations. ZFG notably inhibited H/R-induced cardiomyocyte apoptosis. The expression levels of four TRIM proteins, TRIM7, TRIM14, TRIM22 and TRIM28, were also detected. These four proteins were significantly upregulated in H/R-injured cardiomyocytes, whereas their expression was inhibited following ZFG treatment. Moreover, TRIM28 knockdown inhibited H/R-induced cardiomyocyte apoptosis, whereas TRIM28 overexpression promoted apoptosis and generation of reactive oxygen species (ROS) in cardiomyocytes. However, the effects of TRIM28 overexpression were limited by the action of ROS inhibitor N-acetyl-L-cysteine. In addition, the mRNA and protein levels of antioxidant enzyme glutathione peroxidase (GPX)1 were significantly downregulated in H/R-injured cardiomyocytes. TRIM28 knockdown restored GPX1 protein levels but had no effect on mRNA expression levels. Co-immunoprecipitation and ubiquitination assays demonstrated that TRIM28 negatively regulated GPX1 via ubiquitination. In sum, the present study revealed that ZFG attenuated H/R-induced cardiomyocyte apoptosis by regulating the TRIM28/GPX1/ROS pathway. ZFG and TRIM28 offer potential therapeutic options for the treatment of MIR injury.

Bioaccessibility and health risk assessment of trace metals in soils of greenhouse vegetable production near the industrial areas of the Yangtze River Delta, China.

As a common environmental problem in China, trace metal accumulation and contamination in soils of greenhouse vegetable production (GVP) may pose significant health risk via oral ingestion, inhalation, and dermal contact to vegetable farmers and children playing in greenhouse fields. Thus, bioaccessibility and health risk of Cr, Ni, Cu, Zn, Cd, and Pb in GVP soils collected from 13 GVP farms or bases near industrial areas of the Yangtze River Delta, China, were investigated as a case study. The results suggested that both GVP and industrial discharges contributed a lot to accumulation or contamination especially of Zn and Cd in soil, which subsequently increased their bioaccessible concentrations. In addition, soil acidification caused by GVP also increased bioaccessible Cr and Ni concentrations in soil of the Anthrosols study area. However, the health risk assessment of metals in GVP soil through inhalation and oral ingestion considering metal bioaccessibility suggested no non-carcinogenic and carcinogenic risks to both farmers and children. In contrast, there was potential carcinogenic risk within acceptable level posed by Cr in GVP soil through dermal contact to farmers and children. This indicates that both GVP and industrial activities had limited effect on health risk of trace metals in GVP soil via ingestion, inhalation, and dermal contact. However, the carcinogenic risk posed by Cr, which mainly originated from natural sources, still cannot be negligible. Overall, the results will provide valuable information for decision-makers to develop reasonable strategies and guidelines for risk management of trace metals in GVP soil.

Aggregation of Human Mesenchymal Stromal Cells Eliminates Their Ability to Suppress Human T Cells.

Mesenchymal stromal cells (MSCs) are administered locally to treat sites of inflammation. Local delivery is known to cause MSCs to aggregate into "spheroids," which alters gene expression and phenotype. While adherent MSCs are highly efficient in their inhibition of T cells, whether or not this property is altered upon MSC aggregation has not been thoroughly determined. In this study, we discovered that aggregation of MSCs into spheroids causes them to lose their T cell-suppressive abilities. Interestingly, adding budesonide, a topical glucocorticoid steroid, alongside spheroids partially restored MSC suppression of T cell proliferation. Through a series of inhibition and add-back studies, we determined budesonide acts synergistically with spheroid MSC-produced PGE2 to suppress T cell proliferation through the PGE2 receptors EP2 and EP4. These findings highlight critical differences between adherent and spheroid MSC interactions with human immune cells that have significant translational consequences. In addition, we uncovered a mechanism through which spheroid MSC suppression of T cells can be partly restored. By understanding the phenotypic changes that occur upon MSC aggregation and the impact of MSC drug interactions, improved immunosuppressive MSC therapies for localized delivery can be designed.

Nature vs. Nurture: Defining the Effects of Mesenchymal Stromal Cell Isolation and Culture Conditions on Resiliency to Palmitate Challenge.

As MSC products move from early development to clinical translation, culture conditions shift from xeno- to xeno-free systems. However, the impact of isolation and culture-expansion methods on the long-term resiliency of MSCs within challenging transplant environments is not fully understood. Recent work in our lab has shown that palmitate, a saturated fatty acid elevated in the serum of patients with obesity, causes MSCs to convert from an immunosuppressive to an immunostimulatory state at moderate to high physiological levels. This demonstrated that metabolically-diseased environments, like obesity, alter the immunomodulatory efficacy of healthy donor MSCs. In addition, it highlighted the need to test MSC efficacy not only in ideal conditions, but within challenging metabolic environments. To determine how the choice of xeno- vs. xeno-free media during isolation and expansion would affect future immunosuppressive function, umbilical cord explants from seven donors were subdivided and cultured within xeno- (fetal bovine serum, FBS) or xeno-free (human platelet lysate, PLT) medias, creating 14 distinct MSC preparations. After isolation and primary expansion, umbilical cord MSCs (ucMSC) were evaluated according to the ISCT minimal criteria for MSCs. Following baseline characterization, ucMSC were exposed to physiological doses of palmitate and analyzed for metabolic health, apoptotic induction, and immunomodulatory potency in co-cultures with stimulated human peripheral blood mononuclear cells. The paired experimental design (each ucMSC donor grown in two distinct culture environments) allowed us to delineate the contribution of inherent (nature) vs. environmentally-driven (nurture) donor characteristics to the phenotypic response of ucMSC during palmitate exposure. Culturing MSCs in PLT-media led to more consistent growth characteristics during the isolation and expansion for all donors, resulting in faster doubling times and higher cell yields compared to FBS. Upon palmitate challenge, PLT-ucMSCs showed a higher susceptibility to palmitate-induced metabolic disturbance, but less susceptibility to palmitate-induced apoptosis. Most striking however, was that the PLT-ucMSCs resisted the conversion to an immunostimulatory phenotype better than their FBS counterparts. Interestingly, examining MSC suppression of PBMC proliferation at physiologic doses of palmitate magnified the differences between donors, highlighting the utility of evaluating MSC products in stress-based assays that reflect the challenges MSCs may encounter post-transplantation.

Area disparity in children's perceptions of access to tobacco and cigarette purchasing experiences in Taiwan.

BACKGROUND: Adolescents who perceive easy access to tobacco are more likely to acquire cigarettes and experience smoking. This study assesses area disparities in perceptions of access to tobacco and cigarette purchasing experiences among schoolchildren. METHODS: Data on children's tobacco-related variables were obtained from the Control of School-Aged Children Smoking Study Survey in Taiwan. A stratified random sample of 65 primary schools was included. Polytomous logistic regression analyzed factors associated with tobacco accessibility and purchasing experiences. RESULTS: More than half of the children reported that tobacco retailers often or always sold cigarettes to them. Rural and mountainous children were more likely to have access to cigarettes (adjusted odds ratio [AOR] = 2.01 and 3.01, respectively) and have cigarette purchasing experiences (AOR = 3.06 and 13.76, respectively). Cigarette purchasing from retailers (AOR = 1.84) was significantly associated with children's perceptions of access to tobacco. The factors associated with cigarette purchasing experiences were families smoking (AOR = 8.90), peers smoking (AOR = 2.22), frequent exposure to entertainer smoking on TV and in films (AOR = 2.15), and perceived access to tobacco (AOR = 1.51). CONCLUSIONS: The health department should strictly enforce laws regarding retailers selling tobacco to underage, particularly in remote areas. Schools can reinforce tobacco-use prevention messages.

[Comparison of diagnostic value between DR and MSCT in fracture and dislocation of foot and ankle].

OBJECTIVE: To compare the diagnostic value between digital photography (DR) and multi-slice spiral CT (MSCT) in fracture and dislocation of foot and ankle. METHODS: From August 2010 to August 2012, the DR and MSCT data of 52 patients with fracture and dislocation of foot and ankle were compared according to results of surgery or discharge diagnosis. There were 37 males and 15 females, aged from 15 to 49 years old. Wilcoxon signed rank test was used for statistical analysis. RESULTS: The results of 52 cases of MSCT were matched with the postoperative or discharge diagnosis. A total of 172 fractures were found on MSCT and 98 fractures were found on DR, the results had significant difference in detecting fracture (V=1 081, P<0.05); A total of 24 dislocations were found on MSCT and 16 dislocations were found on DR,the results also had significant difference in detecting dislocation (V=21, P<0.05). Fractures of 6 cases with DR diagnosis were corrected and located by MSCT. CONCLUSION: MSCT is significantly better than DR in diagnosis of fracture and dislocation of foot and ankle. The examination of two parts should be performed in DR. MSCT and multi-planar reconstruction (MPR) examination should be further performed if DR results are unclear or do not match with clinical symptoms, missed diagnosis and misdiagnosis can be avoided.

Tumour necrosis factor alpha down-regulates the expression of peroxisome proliferator activated receptor alpha (PPARα) in human hepatocarcinoma HepG2 cells by activation of NF-κB pathway.

Peroxisome proliferator activated receptor-alpha (PPARα) plays a major role in the regulation of lipid and glucose homeostasis, and inflammatory responses. The objectives of the study were to systematically investigate the effects of TNF-α and its regulatory pathway on PPARα expression in HepG2 cells using Real-Time RT-PCR and western blot analysis. Here, TNF-α suppressed PPARα mRNA expression in a dose- and time-dependent manner at the level of gene transcription. Pre-treatment of cells with 10µM of Wedelolactone for 2h was sufficient to restore PPARα expression to basal levels and also affected the expression of PPARα-regulated genes. This study also demonstrated that TNF-α represses PPARα expression by augmenting the activity of canonical NF-κB signalling pathway. This was shown by the abrogation of TNF-α-mediated PPARα down-regulation, after both p65 and p50 were knocked down via siRNA. The IKK contributes to IκBα degradation and mediates inducible phosphorylation of p105 at Ser933. Surprisingly, phosphorylation of p65 at Ser468 and Ser536 were severely abrogated with Wedelolactone inhibition, suggesting that Ser468 and Ser536, but not Ser276, may mediate the TNF-α inhibitory action on PPARα gene expression. These results suggest that TNF-α might, at least in part, suppress PPARα expression through activation of IKK/p50/p105/p65 pathway. Furthermore, phosphorylation of p65 at Ser468 and Ser536 may play a crucial role in the mechanism that limits PPARα production in the human HepG2 cells.

The analgesic and anti-rheumatic effects of Thladiantha dubia fruit crude polysaccharide fraction in mice and rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The fruit of Thladiantha dubia has been used in Chinese manchu nationality folk medicine for treatment of various pains, such as rheumatic pain, lumbocrural pain, and dysmenorrhea. The aim of the present study was to evaluate the anti-rheumatic effect of Thladiantha dubia fruit crude polysaccharide (TF-P) fraction in rats. MATERIALS AND METHODS: TF-P was evaluated in mice for analgesic activity using acetic acid-induced abdominal contractions test and for anti-inflammatory activity using xylene-induced ear oedema model. Moreover, rheumatoid arthritis (RA) was induced by injection of CFA into the subplantar surface of the hind paw of the male Wistar rats. Joint swelling was measured. In order to evaluate the effect of TF-P on disease progression, proinflammatory mediators (TNF-α and IL-2), and antioxidant indicators (SOD, MDA, and NO) were determined in rats induced by CFA. Pathologic changes of RA in rats were also observed under light microscope. RESULTS AND CONCLUSIONS: In the present study, TF-P at the dose of 200mg/kg (according to the clinical traditional dosage) significantly reduced writhings and stretchings induced by the acetic acid in mice. TF-P significantly inhibited xylene-induced ear oedema in mice, compared with control group. TF-P significantly inhibited inoculated and non-inoculated joint swellings in rats induced by CFA. TF-P had no effect of body weight in rats. The histopathological analysis suggested that TF-P obviously alleviated the degree of RA rats. TF-P treated rats preserved a nearly normal histological architecture of the joint. Results of the present study confirm the use of Thladiantha dubia traditionally for the treatment of painful and inflammatory conditions. Those results suggest TF-P has protective and therapeutic effects on RA rats induced by CFA.

Rapamycin pre-treatment abrogates Tumour Necrosis Factor-alpha down-regulatory effects on LXR-alpha and PXR mRNA expression via inhibition of c-Jun N-terminal kinase 1 activation in HepG2 cells

The Liver X Receptor (LXR) and Pregnane X Receptor (PXR) are members of the nuclear receptor superfamily. Previously, they have been classified as important regulators of lipid homeostasis. However, recent studies have shown that they may be implicated in anti-inflammatory responses as well. This study shows that Tumour Necrosis Factor-alpha (TNF-alpha) treatment reduces both LXR-alpha and PXR mRNA expression. However, pre-treatment with rapamycin, an mTOR inhibitor, followed by TNF-alpha stimulation, significantly induces LXR-alpha and PXR mRNA expression to ~17- and ~2-fold, respectively. This suggests that mTORC1, a multi-molecular complex of which mTOR is a member, may act as a negative regulator that inhibits the induction of LXR-alpha and PXR as anti-inflammatory genes. It is also shown here that inhibition of JNK1 via the mTOR/Akt pathway coincides with the up-regulation of LXR-alpha and PXR mRNA, after TNF-alpha treatment. Together, these observations suggest that JNK1 possibly act downstream of mTORC1 as an LXR-alpha and PXR inhibitor. From the results gleaned in this study, rapamycin (and its analogues) may be used to reduce acute inflammation by promoting the induction of LXR-alpha and PXR as anti-inflammatory genes.

The anti-hyperplasia of mammary gland effect of Thladiantha dubia root ethanol extract in rats reduced by estrogen and progestogen.

AIM OF THE STUDY: Thladiantha dubia (Cucurbitaceae) has been used in Chinese manchu nationality folk medicine for treatment of various diseases, such as scrofula, carbuncle, lactation deficiency, etc. Its effects include softening hard masses, heat-clearing and detoxifying, reducing swelling, eliminating carbuncle, and promoting lactation. It has a history of use in the treatment of breast disorders and as an agent to increase lactation. The aim of the present study was to evaluate the anti-hyperplasia of mammary (HMG) effect of T. dubia root ethanol extract (TD-E) in estrogen combined with progestogen induced HMG rats. MATERIALS AND METHODS: Forty virgin female Wistar rats were randomly divided into blank control, HMG model, Rupixiao Tablet (RPXT, positive control), and TD-E groups, 10 in each. Injection of estrogen and progestogen were given to establish rat models of HMG, RPXT and TD-E were given at the same time. Changes of nipple heights were measured; uterus and ovary index were calculated; sex hormone levels (LH, E(2), and P) in serum were estimated. Pathologic changes of MG in rats were also observed under light microscope. RESULTS AND CONCLUSION: TD-E could decrease the increased nipple height, reduce the numbers of MG lobules and relieve the pathologic changes of HMG in rats. It could also decrease E(2) and LH levels in serum compared with the control group, nipple height, uterus index, and the levels of E(2) and LH in the HMG model group increased significantly, the level of P decreased significantly. Compared with the HMG model group, nipple height, uterus index, and the levels of E(2) and LH in the TD-E group decreased, and no obvious effect on P content. The histopathological analysis suggested that TD-E obviously alleviated the degree of HMG. Those results suggest TD-E has protective and therapeutic effects on HMG rats induced by estrogen and progestogen.