Primary mouse hepatocytes are an important tool in the biomedical research field for the assessment of hepatocyte function. Several methods for hepatocyte isolation have been published; however, many of these methods require extensive handling and can therefore compromise the viability and function of the isolated cells. Since one advantage of utilizing freshly isolated cells is to maintain an environment in which the cells are more comparable to their in vivo state, it is important to have robust methods that produce cells with high viability, good purity and that function in a similar manner to that in their in vivo state. Here we describe a modified two-step method for the rapid isolation and characterization of mouse primary hepatocytes that results in high yields of viable cells. The asialoglycoprotein receptor (ASGPR), which is one of the most abundant cell surface receptors on hepatocytes, was used to monitor the function of the isolated hepatocytes by demonstrating specific binding of its ligand using a newly developed flow cytometry based ligand-receptor binding assay. Also, an in vitro screening method for siRNA drug candidates was successfully developed utilizing freshly isolated hepatocytes with minimum culture time.
Hepatocytes; Two-step isolation method; Asialoglycoprotein receptor (ASGPR); GalNAc
Mediator complex is an integrative hub for transcriptional regulation. Here we show that Mediator regulates alternative mRNA processing via its Med23 subunit. Combining tandem affinity purification and mass spectrometry, we identified a number of mRNA processing factors that bind to a soluble recombinant Mediator subunit MED23 but not to several other Mediator components. One of these factors, hnRNP L, specifically interacts with MED23 in vitro and in vivo. Consistently, Mediator partially colocalizes with hnRNP L and the splicing machinery in the cell. Functionally Med23 regulates a subset of hnRNP L-targeted alternative splicing (AS) and alternative cleavage and polyadenylation (APA) events as shown by minigene reporters and exon array analysis. ChIP-seq analysis revealed that Med23 can regulate hnRNP L occupancy at their co-regulated genes. Taken together, these results demonstrate a crosstalk between Mediator and the splicing machinery, providing a molecular basis for coupling mRNA processing to transcription.
Although empathic responses to stimuli with emotional contents may occur automatically, humans are capable to intentionally empathize with other individuals. Intentional empathy for others is even possible when they do not show emotional expressions. However, little is known about the neuronal mechanisms of this intentionally controlled empathic process. To investigate the neuronal substrates underlying intentional empathy, we scanned 20 healthy Chinese subjects, using fMRI, when they tried to feel inside the emotional states of neutral or angry faces of familiar (Asian) and unfamiliar (Caucasian) models. Skin color evaluation of the same stimuli served as a control task. Compared to a baseline condition, the empathy task revealed a network of established empathy regions, including the anterior cingulate cortex, bilateral inferior frontal cortex and bilateral anterior insula. The contrast of intentional empathy vs skin color evaluation, however, revealed three regions: the bilateral inferior frontal cortex, whose hemodynamic responses were independent of perceived emotion and familiarity and the right-middle temporal gyrus, whose activity was modulated by emotion but not by familiarity. These findings extend our understanding of the role of the inferior frontal cortex and the middle temporal gyrus in empathy by demonstrating their involvement in intentional empathy.
fMRI; brain imaging; empathy
Our recent work showed that close relationships result in shared cognitive and neural representations of the self and one’s mother in collectivistic individuals (Zhu et al., 2007, Neuroimage, 34, 1310–7). However, it remains unknown whether close others, such as mother, father and best friend, are differentially represented in collectivistic brains. Here, using functional magnetic resonance imaging and a trait judgment task, we showed evidence that, while trait judgments of the self and mother generated comparable activity in the medial prefrontal cortex (MPFC) and anterior cingulate (ACC) of Chinese adults, trait judgments of mother induced greater MPFC/ACC activity than trait judgments of father and best friend. Our results suggest that, while neural representations of the self and mother overlapped in the MPFC/ACC, close others such as mother, father and best friend are unequally represented in the MPFC/ACC of collectivistic brains.
close other; self; medial prefrontal cortex; anterior cingulate; fMRI
Host genotype and gender are among the factors that influence the composition of gut microbiota. We studied the population structure of gut microbiota in two lines of chickens maintained under the same husbandry and dietary regimes. The lines, which originated from a common founder population, had undergone 54 generations of selection for high (HW) or low (LW) 56-day body weight, and now differ by more than 10-fold in body weight at selection age. Of 190 microbiome species, 68 were affected by genotype (line), gender, and genotype by gender interactions. Fifteen of the 68 species belong to Lactobacillus. Species affected by genotype, gender, and the genotype by gender interaction, were 29, 48, and 12, respectively. Species affected by gender were 30 and 17 in the HW and LW lines, respectively. Thus, under a common diet and husbandry host quantitative genotype and gender influenced gut microbiota composite.
Recent studies have shown that circulating microRNAs might be useful, novel biomarkers for the diagnosis of acute myocardial infarction. The aims of this study were to evaluate the expression of cardiac-specific miRNAs (miR-1, -133a, -208b, and -499) in patients with acute myocardial infarction and to compare the diagnostic values of these miRNAs with that of cardiac troponin T.
Sixty-seven plasma samples obtained from patients with acute myocardial infarction and 32 plasma specimens collected from healthy volunteers were analyzed in this study. The levels of cardiac-specific miRNAs (miR-1, -133a, -208b, and -499) were measured by quantitative reverse transcription-polymerase chain reaction, and the concentrations of plasma cardiac troponin T were measured using electrochemiluminescence-based methods and an Elecsys 2010 Immunoassay Analyzer.
The levels of plasma miR-1, -133a, -208b, and -499 were significantly higher in acute myocardial infarction patients (all p<0.001) than in healthy volunteers. The expression of the cardiac-specific miRNAs in acute myocardial infarction patients decreased to close to the baseline levels at the time of hospital discharge (all p>0.05). There were no correlations between the levels of the four circulating miRNAs and the clinical characteristics of the study population (all p>0.05). Furthermore, receiver operating characteristic curve analyses showed that the four plasma miRNAs were not superior to cardiac troponin T for the diagnosis of acute myocardial infarction (all p>0.05).
Our results demonstrate that circulating miR-1, -133a, -208b, and -499 may be useful biomarkers in acute myocardial infarction patients but that these miRNAs are not superior to cardiac troponin T for the diagnosis of acute myocardial infarction.
microRNAs; cardiac troponin T; Acute Myocardial Infarction; Circulating Biomarkers
A motif in a network is a connected graph that occurs significantly more frequently as an induced subgraph than would be expected in a similar randomized network. By virtue of being atypical, it is thought that motifs might play a more important role than arbitrary subgraphs. Recently, a flurry of advances in the study of network motifs has created demand for faster computational means for identifying motifs in increasingly larger networks. Motif detection is typically performed by enumerating subgraphs in an input network and in an ensemble of comparison networks; this poses a significant computational problem. Classifying the subgraphs encountered, for instance, is typically performed using a graph canonical labeling package, such as Nauty, and will typically be called billions of times. In this article, we describe an implementation of a network motif detection package, which we call NetMODE. NetMODE can only perform motif detection for -node subgraphs when , but does so without the use of Nauty. To avoid using Nauty, NetMODE has an initial pretreatment phase, where -node graph data is stored in memory (). For we take a novel approach, which relates to the Reconstruction Conjecture for directed graphs. We find that NetMODE can perform up to around times faster than its predecessors when and up to around times faster when (the exact improvement varies considerably). NetMODE also (a) includes a method for generating comparison graphs uniformly at random, (b) can interface with external packages (e.g. R), and (c) can utilize multi-core architectures. NetMODE is available from netmode.sf.net.
To explore current status and choices regarding diagnosis and treatment of Parkinson’s disease (PD) among physicians, general neurologists and movement disorders specialists in China via a national survey.
The cross-sectional questionnaire-based survey was conducted from November, 2010 to July, 2011. Six hundreds and twelve doctors from different cities in China were recruited for this study.
68.6% (n=420) and 23.9% (n=146) of doctors have read the national and international guidelines, respectively. There was a larger proportion of movement disorders specialists reading the guidelines, in contrast to physicians and general neurologists (P<0.001). Up to 76.4% (n=465) and 81.8% (n=498) of doctors would choose standard oral levodopa test and conventional MRI(with T1 and T2), respectively; Whereas susceptibility weighed imaging(SWI)(16.1%; n=98), transcranial sonography (TCS) (1.8%; n=11) and functional neuroimaging test, such as single photon emission computed tomography(SPECT) (10.2%; n=62) and positron emission tomography(PET)(13.3%; n=81) were less used for suspected patients with PD in clinical practice. Doctors at different levels or from different hospitals and cities would choose different medication for motor complications and non-motor symptoms of patients with PD, in addition to initial drug selection for newly diagnosed PD. Doctors who had read the guidelines had significantly better knowledge of medication selections for PD under specific circumstances.
Compared with commonly employed standard oral levodopa test and conventional MRI, SWI complements MRI, TCS and functional neuroimaging were less performed for diagnosis of PD in clinical practice in China. The choices of diagnostic methods and therapeutic strategy of PD vary among physicians, general neurologists and movement disorders specialists. Guideline awareness is markedly beneficial to reasonable PD medications strategy in China.
Parkinson’s disease; National survey; Clinical practice guideline; Medication
Our study aimed to investigate the prevalence of cognitive impairment(CI) and the associated risk factors among elderly people in Shanghai urban area, China.
A population-based survey was conducted among people aged 55 years or older in urban areas of Shanghai. Face-to-face interviews were carried out to collect information including demographic characteristics, medical history, and medication use, etc. The validated Chinese version of the Mini-Mental State Examination(MMSE) was used to screen subjects with CI, and the criteria of CI were adjusted for education levels.
A total of 3,176 home-living residents (≥55 years old) were included in the study. Among them, 266 people (102 men and 164 women) were identified as cognition impaired, with a prevalence of 8.38% (266/3,176, 95% CI: (8.26, 8.49)) for both genders, 9.21% (102/1,107,95% CI: (9.18, 9.33)) for men and 7.93% (164/2,069, 95% CI: (7.80, 8.09)) for women, respectively. Furthermore, we found that several significant risk factors, including social factors(education, number of children, marriage status, and family structure), physiological factors (age, blood glucose level, and obesity), factors on living styles(physical exercise, diet & chronic diseases), and genetic factor(ApoE), associated with CI onset.
This study confirms the high prevalence of CI among the elderly population in the Shanghai urban in China, similar to previous epidemiologic studies in Western countries. The putative risk factors associated with CI merit further investigated.
Cognitive impairment; Prevalence; Risk factors
Mechanistic exploration of the origins of the unparalleled soil microbial biodiversity represents a vast and uncharted scientific frontier. Quantification of candidate mechanisms that promote and sustain such diversity must be linked with microbial functions and measurable biophysical interactions at appropriate scales. We report a novel microbial coexistence index (CI) that links macroscopic soil hydration conditions with microscale aquatic habitat fragmentation that impose restrictions on cell dispersion and growth rates of competing microbial populations cohabiting soil surfaces. The index predicts a surprisingly narrow range of soil hydration conditions that suppress microbial coexistence; and for most natural conditions found in soil hydration supports coexistence. The critical hydration conditions and relative abundances of competing species are consistent with limited experimental observations and with individual-based model simulations. The proposed metric offers a means for systematic evaluation of factors that regulate microbial coexistence in an ecologically consistent fashion.
Gastric cancer is one of the most common and lethal malignant cancers worldwide, and numerous epidemiological studies have demonstrated that Helicobacter pylori (H. pylori) infection plays a key role in the development of gastric carcinomas. Our previous studies showed that aquaporin 3 (AQP3) is overexpressed in gastric carcinoma and promotes the migration and proliferation of human gastric carcinoma cells, suggesting that AQP3 may be a potentially important determinant of gastric carcinoma. However, the role of AQP3 in H. pylori carcinogenesis is unknown.
The AQP3 protein and H. pylori were detected in human gastric tissues by immunohistochemistry and modified Giemsa staining respectively. AQP3 knockdown was obtained by small interfering (si) RNA. Western blot assays and RT-PCR were used to evaluate the change of AQP3 in the human gastric cancer AGS and SGC7901 cell lines after co-culture with H. pylori. Sprague Dawley rats were orally inoculated with H. pylori to establish a rat model colonized by H. pylori.
The present study found that AQP3 expression correlated with H. pylori infection status in gastric cancer tissues and corresponding normal mucosa, and H. pylori co-culture upregulated AQP3 expression in human gastric adenocarcinoma cells in vitro via the extracellular signal-regulated kinase signaling pathway. H. pylori infection also increased AQP3 expression in gastric mucosa colonized by H. pylori in a Sprague Dawley rat model.
These findings provide further information to understand the mechanism of H. pylori carcinogenesis and a potential strategy for the treatment of H. pylori-associated gastric carcinoma.
To kill invading bacteria, neutrophils must interpret spatial cues, migrate, and reach target sites. Although initiation of chemotactic migration has been extensively studied, little is known about its termination. Here we report that two mitogen-activated protein kinases played opposing roles in neutrophil trafficking. The extracellular signal-regulated kinase (Erk) potentiated G protein-coupled receptor kinase GRK2 activity and inhibited neutrophil migration, whereas p38 MAPK acted as a non-canonical GRK that phosphorylated the formyl peptide receptor FPR1 and facilitated neutrophil migration by blocking GRK2 function. Therefore, the dynamic balance between Erk and p38 MAPK controls neutrophil “stop” and “go” behaviors, ensuring neutrophils precisely reach their final destination as the first line of host-defense.
Renal cancers are highly aggressive and clinically challenging, but a transgenic mouse model to promote pathologic studies and therapeutic advances has yet to be established. Here we report the generation of a transgenic mouse model of von Hippel-Lindau (VHL) renal cancer termed the TRACK model (transgenic cancer of the kidney). TRACK mice specifically express a mutated, constitutively active HIF1α in kidney proximal tubule (PT) cells. Kidney histologies displayed by TRACK mice are highly similar to histologies seen in patients with VHL disease, including areas of distorted tubular structure, cells with clear cytoplasm and increased glycogen and lipid deposition, multiple renal cysts, and early onset of clear cell renal cell carcinoma (ccRCC). Distorted tubules in TRACK mice exhibit higher levels of CA-IX, Glut1, and VEGF than tubules in non-transgenic control mice. Further, these tubules exhibit increased numbers of endothelial cells, increased cell proliferation, and increased expression of the human ccRCC marker CD70 (TNFSF7). Moreover, PT cells in kidney tubules from TRACK mice exhibit increased genomic instability, as monitored by elevated levels of γH2AX. Our findings establish that activated HIF1α in murine kidney PT cells is sufficient to promote cell proliferation, angiogenesis, genomic instability, and other phenotypic alterations characteristic of human VHL kidney disease, establishing the TRACK mouse as a valid preclinical model of human renal cell carcinoma.
HIF1α; DNA repair; genomic instability; mouse model; clear cell renal cell carcinoma (ccRCC)
Resting-state fMRI is a novel approach to measure spontaneous brain activity in patients with major depressive disorder (MDD). Although most resting-state fMRI studies have focused on the examination of temporal correlations between low-frequency oscillations (LFOs), few studies have explored the amplitude of these LFOs in MDD. In this study, we applied the approaches of amplitude of low-frequency fluctuation (ALFF) and fractional ALFF to examine the amplitude of LFOs in MDD.
A total of 36 subjects, 18 first-episode, treatment-naive patients with MDD matched with 18 healthy controls (HCs) completed the fMRI scans. Compared with HCs, MDD patients showed increased ALFF in the right fusiform gyrus and the right anterior and posterior lobes of the cerebellum but decreased ALFF in the left inferior temporal gyrus, bilateral inferior parietal lobule, and right lingual gyrus. The fALFF in patients was significantly increased in the right precentral gyrus, right inferior temporal gyrus, bilateral fusiform gyrus, and bilateral anterior and posterior lobes of the cerebellum but was decreased in the left dorsolateral prefrontal cortex, bilateral medial orbitofrontal cortex, bilateral middle temporal gyrus, left inferior temporal gyrus, and right inferior parietal lobule. After taking gray matter (GM) volume as a covariate, the results still remained.
These findings indicate that MDD patients have altered LFO amplitude in a number of regions distributed over the frontal, temporal, parietal, and occipital cortices and the cerebellum. These aberrant regions may be related to the disturbances of multiple emotion- and cognition-related networks observed in MDD and the apparent heterogeneity in depressive symptom domains. Such brain functional alteration of MDD may contribute to further understanding of MDD-related network imbalances demonstrated in previous fMRI studies.
Although endoplasmic reticulum (ER) stress is a pathologic mechanism in a variety of chronic diseases, it is unclear what role it plays in chronic hypertension (HTN). Dysregulation of brain mechanisms controlling arterial pressure is strongly implicated in HTN, particularly in models involving angiotensin II (Ang II). We tested the hypothesis that ER stress in the brain is causally linked to Ang II–dependent HTN. Chronic systemic infusion of low-dose Ang II in C57BL/6 mice induced slowly developing HTN, which was abolished by co-infusion of the ER stress inhibitor tauroursodeoxycholic acid (TUDCA) into the lateral cerebroventricle. Investigations of the brain regions involved revealed robust increases in ER stress biomarkers and profound ER morphological abnormalities in the circumventricular subfornical organ (SFO), a region outside the blood-brain barrier and replete with Ang II receptors. Ang II–induced HTN could be prevented in this model by selective genetic supplementation of the ER chaperone 78-kDa glucose-regulated protein (GRP78) in the SFO. These data demonstrate that Ang II–dependent HTN is mediated by ER stress in the brain, particularly the SFO. To our knowledge, this is the first report that ER stress, notably brain ER stress, plays a key role in chronic HTN. Taken together, these findings may have broad implications for the pathophysiology of this disease.
Mosquitoes are insects of the Diptera, Nematocera, and Culicidae families, some species of which are important disease vectors. Identifying mosquito species based on morphological characteristics is difficult, particularly the identification of specimens collected in the field as part of disease surveillance programs. Because of this difficulty, we constructed DNA barcodes of the cytochrome c oxidase subunit 1, the COI gene, for the more common mosquito species in China, including the major disease vectors. A total of 404 mosquito specimens were collected and assigned to 15 genera and 122 species and subspecies on the basis of morphological characteristics. Individuals of the same species grouped closely together in a Neighborhood-Joining tree based on COI sequence similarity, regardless of collection site. COI gene sequence divergence was approximately 30 times higher for species in the same genus than for members of the same species. Divergence in over 98% of congeneric species ranged from 2.3% to 21.8%, whereas divergence in conspecific individuals ranged from 0% to 1.67%. Cryptic species may be common and a few pseudogenes were detected.
A new approach of fabricating supramolecular nanoparticles generated by self-assembly polyrotaxanes for antitumor drug delivery has been reported. Cinnamic-acid-modified poly(ethylene glycol) chains were threaded in α-cyclodextrins to form polyrotaxanes. The polyrotaxanes self-assembled supramolecular nanoparticles. The morphology of the nanoparticles was changed from nanovesicle to micelle after the antitumor drug, doxorubicin, was loaded. The release profile of the drug-loaded nanoparticles was investigated, and it was found that the sustaining release time could last for 32 hours. The drug-loaded nanoparticles were co-cultured with mouse 4T1 breast cancer cells with a drug concentration of 10 μg/mL; the cell survival rate was 3.3% after a 72-hour incubation. In an in vivo study of breast cancer in a mouse model, the drug-loaded nanoparticles were injected in the tail veins of mice with a dose of 5 mg/kg body weight. The tumor inhibition rate of drug-loaded nanoparticles was 53%, which was better than that of doxorubicin hydrochloride. The cardiac toxicity of doxorubicin was decreased greatly after the encapsulation into supramolecular polyrotaxane nanoparticles.
polyrotaxane; self-assembly; nanoparticle; doxorubicin; supermolecular
Repeatability of community composition has been a critical aspect for community structure, which is closely associated with community stability, predictability, conservation biology and ecological restoration. It has been shown that both immigration and local dispersal limitation can affect the community composition in both neutral and niche model. Hence, we use a spatially explicit individual-based model to investigate the potential influence of immigration rate and strength of local dispersal limitation on repeatability in both neutral and niche models. Similarity measures are used to quantify repeatability. We examine the repeatability of community composition among replicate communities (which means the same community repeats many times), and between niche and neutral replicate communities. We find the correlation between repeatability and immigration rate is positive in the neutral model and an inverted unimodal in the niche model. The correlation between repeatability and local dispersal distance is positive in the niche model and negative in the neutral model. High repeatability between niche communities and neutral communities is observed with high immigration rates or when high local dispersal distance appears in the niche model or low local dispersal distance in the neutral model. Our results show that repeatability of community composition is not only dependent on the types of community models (niche vs. neutrality) but also strongly determined by immigration rates and local dispersal limitation.
There is evidence that drug-specific T cells are involved in inducing keratinocyte apoptosis in acute stage of Steven-Johson syndrome (SJS) and Toxic epidermal necrolysis (TEN). However, there are few studies that have attempted to examine T cell memory responses over time. We sought to determine the duration of IFN-γ and sFasL memory response to causal drugs in patients with SJS and TEN after remission. Eight patients with previous SJS and TEN were enrolled. Memory T cells were measured by 10-day cultured IFN-γ enzyme-linked immunosorbent spot-forming cell (ELISpot) assay. Effector T-cell responses were measured by ex vivo IFN-γ ELISpot assay and sFasL ELISA. The sFasL-mediated toxicities of drug-stimulated PBMC supernatants against keratinocyte line were further investigated by MTT proliferation assay and Annexin-V staining. We observed significant cultured and ex vivo IFN-γ ELISpot responses against causal drugs in all 8 patients. In addition, the sFasL levels were specifically increased in the supernatant of PBMCs cultured with causal drugs from 6 of 8 patients. Drug-stimulated PBMC supernatants were cytotoxic against keratinocyte line, which was inhibited by anti-FasL mAb in a dose-dependent manner. Our findings confirmed that drug-specific IFN-γ and sFasL memory response against causal drugs could be sustained over several years and further suggest that patients should avoid causal drug re-exposure after the recovery of TEN and SJS.
Transmissible gastroenteritis (TGE) is a highly contagious viral disease of swine, characterized by severe vomiting, diarrhea, and high mortality. Currently, the vaccines for it are only partially effective and no specific drug is available for treatment of TGE virus (TGEV) infection. RNA interference has been confirmed as a new approach for controlling viral infections. In this study, the inhibitory effect of short hairpin RNAs (shRNAs) targeting the ORF 7 gene of TGEV on virus replication was examined.
Four theoretically effective sequences of TGEV ORF 7 gene were designed and selected for construction of shRNA expression plasmids. In the reporter assays, three of four shRNA expression plasmids were able to inhibit significantly the expression of ORF 7 gene and replication of TGEV, as shown by real-time quantitative RT-PCR analysis of viral ORF 7 and N genes and detection of virus titers (TCID50/ml). Stable swine testicular (ST) cells expressing the shRNAs were established. Observation of the cytopathic effect and apoptosis, as well as a cell proliferation assay demonstrated that the three shRNAs were capable of protecting ST cells against TGEV destruction, with high specificity and efficiency.
Our results indicated that plasmid-transcribed shRNAs targeting the ORF 7 gene in the TGEV genome effectively inhibited expression of the viral target gene and viral replication in vitro. These findings provide evidence that the shRNAs have potential therapeutic application for treatment of TGE.
Transmissible gastroenteritis virus; RNA interference; Short hairpin RNA; ORF 7
Effective gene transfection without serum deprivation is a prerequisite for successful stem cell-based gene therapy. Polyethylenimine (PEI) is an efficient nonviral gene vector, but its application has been hindered by serum sensitivity and severe cytotoxicity.
To solve this problem, a new family of lipopolyplexes was developed by coating PEI/DNA polyplexes with three serum-resistant cationic lipids, namely, lysinylated, histidylated, and arginylated cholesterol. The physical properties, transfection efficiency, cellular uptake, subcellular distribution, and cytotoxicity of the lipopolyplexes was investigated.
The outer coat composed of lysinylated or histidylated cholesterol remarkably improved the transfection efficiency of the polyplex with a low PEI/DNA ratio of 2 in the presence of serum. The resulting lysinylated and histidylated cholesterol lipopolyplexes were even more efficient than the best performing polyplex with a high PEI/DNA ratio of 10. Results from cellular uptake and subcellular distribution studies suggest that their higher transfection efficiency may result from accelerated DNA nuclear localization. The superiority of the lipopolyplexes over the best performing polyplex was also confirmed by delivering the therapeutic gene, hVEGF165. Equally importantly, the lipid coating removed the necessity of introducing excess free PEI chains into the transfection solution for higher efficiency, generating lipopolyplexes with no signs of cytotoxicity.
Noncovalent modification of polyplexes with lysinylated and histidylated cholesterol lipids can simultaneously improve efficiency and reduce the toxicity of gene delivery under serum conditions, showing great promise for genetic modification of bone marrow stem cells.
gene delivery; nonviral vectors; cationic lipids; polyethylenimine; lipopolyplex; bone marrow stem cells
Therapeutics based on RNA interference (RNAi) have emerged as a potential new class of drugs for treating human disease by silencing the target messenger RNA (mRNA), thereby reducing levels of the corresponding pathogenic protein. The major challenge for RNAi therapeutics is the development of safe delivery vehicles for small interfering RNAs (siRNAs). We previously showed that cholesterol-conjugated siRNAs (chol-siRNA) associate with plasma lipoprotein particles and distribute primarily to the liver after systemic administration to mice. We further demonstrated enhancement of silencing by administration of chol-siRNA pre-associated with isolated high-density lipoprotein (HDL) or low-density lipoprotein (LDL). In this study, we investigated mimetic lipoprotein particle prepared from recombinant apolipoprotein A1 (apoA) and apolipoprotein E3 (apoE) as a delivery vehicle for chol-siRNAs. We show that apoE-containing particle (E-lip) is highly effective in functional delivery of chol-siRNA to mouse liver. E-lip delivery was found to be considerably more potent than apoA-containing particle (A-lip). Furthermore, E-lip–mediated delivery was not significantly affected by high endogenous levels of plasma LDL. These results demonstrate that E-lip has substantial potential as delivery vehicles for lipophilic conjugates of siRNAs.
To investigate the regulation of K17 expression by the pro-inflammatory cytokine IL-22 in keratinocytes and its important role in our previously hypothesized “K17/T cell/cytokine autoimmune loop” in psoriasis.
Materials and Methods
K17 expression was examined in the IL-22-treated keratinocytes by real-time quantitative PCR, ELISA, Western blot and Immunofluorescence. In addition, the signaling pathways involved in K17 regulation were investigated with related inhibitors and siRNAs. In addition, K17 expression was examined in the epidermis of IL-22-injected mouse skin.
IL-22-induced K17 expression was confirmed in keratinocytes and the epidermis of IL-22-injected mouse skin at both mRNA and protein levels, which is an important complement to the autoimmune loop. We further investigated the regulatory mechanisms and found that both STAT3 and ERK1/2 were involved in the up-regulation of K17 expression induced by IL-22.
IL-22 up-regulates K17 expression in keratinocytes in a dose-dependent manner through STAT3- and ERK1/2-dependent mechanisms. These findings indicated that IL-22 was also involved in the K17/T cell/cytokine autoimmune loop and may play an important role in the progression of psoriasis.
Prohibitin is an essential mitochondrial protein that has been implicated in a wide variety of functions in many cell types, but its role in neurons remains unclear. In a proteomic screen of rat brains in which ischemic tolerance was induced by electrical stimulation of the cerebellar fastigial nucleus, we found that prohibitin is upregulated in mitochondria. This observation prompted us to investigate the role of prohibitin in neuronal death and survival. We found that prohibitin is upregulated also in the ischemic tolerance induced by transient ischemia in vivo, or oxygen-glucose deprivation in neuronal cultures. Cell fractionation and electron microscopic immunolabeling studies demonstrated that prohibitin is localized to neuronal mitochondria. Upregulation of prohibitin in neuronal cultures or hippocampal slices was markedly neuroprotective, whereas prohibitin gene-silencing increased neuronal vulnerability, an effect associated with loss of mitochondrial membrane potential and increased mitochondrial production of reactive oxygen species. Prohibitin upregulation was associated with reduced production of reactive oxygen species in mitochondria exposed to the complex I inhibitor rotenone. In addition, prohibitin protected complex I activity from the inhibitory effects of rotenone. These observations, collectively, establish prohibitin as an endogenous neuroprotective protein involved in ischemic tolerance. Prohibitin exerts beneficial effects on neurons by reducing mitochondrial free radical production. The data with complex I activity suggest that prohibitin may stabilize the function of complex I. The protective effect of prohibitin has potential translational relevance in diseases of the nervous system associated with mitochondrial dysfunction and oxidative stress.