It is well established that activation of NMDARs plays an essential role in spinal cord synaptic plasticity (i.e., central sensitization) and pain hypersensitivity after tissue injury. Despite prominent expression of NMDARs in DRG primary sensory neurons, the unique role of peripheral NMDARs in regulating intrinsic neuronal excitability and pain sensitivity is not well understood, in part due to the lack of selective molecular tools. To address this problem, we used Advillin-Cre driver to delete the NR1 subunit of NMDARs selectively in DRG neurons. In NR1 conditional knock-out (NR1-cKO) mice, NR1 expression is absent in DRG neurons but remains normal in spinal cord neurons; NMDA-induced currents are also eliminated in DRG neurons of these mice. Surprisingly, NR1-cKO mice displayed mechanical and thermal hypersensitivity compared with wild-type littermates. NR1-deficient DRG neurons show increased excitability, as indicated by increased frequency of action potentials, and enhanced excitatory synaptic transmission in spinal cord slices, as indicated by increased frequency of miniature EPSCs. This hyperexcitability can be reproduced by the NMDAR antagonist APV and by Ca2+-activated slow conductance K+ (SK) channel blocker apamin. Furthermore, NR1-positive DRG neurons coexpress SK1/SK2 and apamin-sensitive afterhyperpolarization currents are elevated by NMDA and suppressed by APV in these neurons. Our findings reveal the hitherto unsuspected role of NMDARs in controlling the intrinsic excitability of primary sensory neurons possibly via Ca2+-activated SK channels. Our results also call attention to potential opposing effects of NMDAR antagonists as a treatment for pain and other neurological disorders.
Ethyl pyruvate (EP), a potent reactive oxygen species scavenger, has been reported to contribute to the inflammatory process. However, the protective effect of ethyl pyruvate on Concanavalin A (Con A)-induced autoimmune hepatitis have not been explored. Thus, the aims of this study are to investigate both the effects of ethyl pyruvate and its mechanism of protection on Con A-induced autoimmune hepatitis in mice.
Materials and Methods
Acute autoimmune hepatitis was induced by Con A (20 mg/kg) in Balb/C mice; ethyl pyruvate (40 mg/kg and 80 mg/kg) was administrated 1h prior to the Con A injection. At 3h, 6h and 24h post Con A injection, histological grading, proinflammatory cytokine levels and nuclear factor kappa B (NF-κB) activity were determined.
Following Con A challenge, cytokines TNF-α, IL-2, IL-1β and IL-6 were expressed at 3h and 6h, and the level of HMGB1 significantly increased by 24h. Pretreatment with ethyl pyruvate ameliorated the pathological effects of Con A-induced autoimmune hepatitis and significantly decreased the levels of TNF-α, IL-2, IL-6 and IL-1β at 3h and 6h and the level of HMGB1 at 6h and 24h post injection. Ethyl pyruvate blocked the degradation of IκB α and IκB β and decreased the expression of NF-κB at 24h.
Taken together, these results indicated that ethyl pyruvate protected against Con A-induced autoimmune hepatitis by decreasing both early (TNF-α, IL-2, IL-1β and IL-6) and late (HMGB1) cytokine expression in mice. The reduction of HMGB1 may correlate with the amelioration of NF-κB activity.
Rodents begin to use bilaterally coordinated, rhythmic sweeping of their vibrissae (“whisking”) for environmental exploration around two weeks after birth. Whether and how vibrissal control circuitry changes after birth is unknown, and relevant premotor circuitry remains poorly characterized. Using a modified rabies virus transsynaptic tracing strategy, we labeled neurons synapsing directly onto vibrissa facial motor neurons (vFMNs). Sources of potential excitatory, inhibitory, and modulatory vFMN premotor neurons, and differences between the premotor circuitry for vFMNs innervating intrinsic versus extrinsic vibrissal muscles, were systematically characterized. The emergence of whisking is accompanied by the addition of “new” sets of bilateral excitatory inputs to vFMNs from neurons in the lateral paragigantocellularis (LPGi). Furthermore, descending axons from the motor cortex directly innervate LPGi premotor neurons. Thus, neural modules well suited to facilitate the bilateral coordination and cortical control of whisking are added to premotor circuitry in parallel with the emergence of this exploratory behavior.
Purpose: Targeted radiotherapy (TRT) is an emerging approach for tumor treatment. Previously, 3PRGD2 (a dimeric RGD peptide with 3 PEG4 linkers) has been demonstrated to be of advantage for integrin αvβ3 targeting. Given the promising results of 99mTc-3PRGD2 for lung cancer detection in human beings, we are encouraged to investigate the radiotherapeutic efficacy of radiolabeled 3PRGD2. The goal of this study was to investigate and optimize the integrin αvβ3 mediated therapeutic effect of 177Lu-3PRGD2 in the animal model.
Experimental Design: Biodistribution, gamma imaging and maximum tolerated dose (MTD) studies of 177Lu-3PRGD2 were performed. The targeted radiotherapy (TRT) with single dose and repeated doses as well as the combined therapy of TRT and the anti-angiogenic therapy (AAT) with Endostar were conducted in U87MG tumor model. The hematoxylin and eosin (H&E) staining and immunochemistry (IHC) were performed post-treatment to evaluate the therapeutic effect.
Results: The U87MG tumor uptake of 177Lu-3PRGD2 was relatively high (6.03 ± 0.65 %ID/g, 4.62 ± 1.44 %ID/g, 3.55 ± 1.08 %ID/g, and 1.22 ± 0.18 %ID/g at 1 h, 4 h, 24 h, and 72 h postinjection, respectively), and the gamma imaging could visualize the tumors clearly. The MTD of 177Lu-3PRGD2 in nude mice (>111 MBq) was twice to that of 90Y-3PRGD2 (55.5 MBq). U87MG tumor growth was significantly delayed by 177Lu-3PRGD2 TRT. Significantly increased anti-tumor effects were observed in the two doses or combined treatment groups.
Conclusion: The two-dose TRT and combined therapy with Endostar potently enhanced the tumor growth inhibition, but the former does not need to inject daily for weeks, avoiding a lot of unnecessary inconvenience and suffering for patients, which could potentially be rapidly translated into clinical practice in the future.
Integrin αvβ3; Arg-Gly-Asp (RGD); 177Lu; radionuclide therapy; combination therapy.
Background. Hepatic ischemia-reperfusion (I/R) injury is a pivotal clinical problem occurring in many clinical conditions such as transplantation, trauma, and hepatic failure after hemorrhagic shock. Apoptosis and autophagy have been shown to contribute to cell death in hepatic I/R injury. Ethyl pyruvate, a stable and simple lipophilic ester, has been shown to have anti-inflammatory properties. In this study, the purpose is to explore both the effect of ethyl pyruvate on hepatic I/R injury and regulation of intrinsic pathway of apoptosis and autophagy.
Methods. Three doses of ethyl pyruvate (20 mg/kg, 40 mg/kg, and 80 mg/kg) were administered 1 h before a model of segmental (70%) hepatic warm ischemia was established in Balb/c mice. All serum and liver tissues were obtained at three different time points (4 h, 8 h, and 16 h).
Results. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and pathological features were significantly ameliorated by ethyl pyruvate (80 mg/kg). The expression of Bcl-2, Bax, Beclin-1, and LC3, which play an important role in the regulation of intrinsic pathway of apoptosis and autophagy, was also obviously decreased by ethyl pyruvate (80 mg/kg). Furthermore, ethyl pyruvate inhibited the HMGB1/TLR4/ NF-κb axis and the release of cytokines (TNF-α and IL-6).
Conclusion. Our results showed that ethyl pyruvate might attenuate to hepatic I/R injury by inhibiting intrinsic pathway of apoptosis and autophagy, mediated partly through downregulation of HMGB1/TLR4/ NF-κb axis and the competitive interaction with Beclin-1 of HMGB1.
A meta-analysis was performed of RCTs comparing therapies that combine UDCA and corticosteroids with UDCA monotherapy. In this paper, we found that the combination therapy of UDCA and corticosteroids was more effective for PBC-AIH.
Some cardiovascular risk factors have been confirmed to be positively correlated with arterial stiffness. However, it is unclear whether HDL-C, a well-established anti-risk factor, has an independent association with arterial stiffness. The aim of this study was to evaluate the relationship between HDL-C levels and arterial stiffness and the possible role of high-sensitivity C-reactive protein (hs-CRP) in this potential correlation in apparently healthy adults undergoing a general health examination in China.
Materials and Methods
This was a cross-sectional survey. In total, 15,302 participants (age range, 18–82 years; mean, 43.88±8.44 years) were recruited during routine health status examinations. A questionnaire was used and we measured the body mass index, systolic and diastolic blood pressure, and fasting glucose, and serum lipid, uric acid, hs-CRP, and serum creatinine levels of each participant. Central arterial stiffness was assessed by carotid–femoral pulse wave velocity (cf-PWV).
HDL-C levels decreased as cf-PWV increased. Pearson’s correlation analysis revealed that HDL-C levels were associated with cf-PWV (r=−0.18, P<0.001). hs-CRP levels were positively associated with cf-PWV (r=0.13). After adjustment for all confounders, HDL-C was inversely independently associated with all quartiles of cf-PWV. Furthermore, HDL-C was associated with cf-PWV in different quartiles of hs-CRP, and the correlation coefficients (r) gradually decreased with increasing hs-CRP levels (quartiles 1–4).
HDL-C is inversely independently associated with central arterial stiffness. The anti-inflammatory activity of HDL-C may mediate its relationship with cf-PWV. Further, long-term follow-up studies are needed to evaluate whether high HDL-C levels are protective against central artery stiffening through the anti-inflammatory activity of HDL-C.
A pharmacokinetic [PK]-driven screening process was implemented to select new agents for brain tumor chemotherapy from a series of low molecular weight anticancer agents [ON27x] that consisted of 141 compounds. The screening procedures involved a combination of in silico, in vitro and in vivo mouse studies that were cast into a pipeline of tier 1 and tier 2 failures that resulted in a final investigation of 2 analogues in brain tumor-bearing mice. Tier 1 failures included agents with a molecular weight of > 450 Da, a predicted log P (log P) of either < 2 or > 3.5, and a cytotoxicity IC50 value of > 2 uM. Next, 18 compounds underwent cassette dosing studies in normal mice that identified compounds with high systemic clearance, and low blood-brain barrier [BBB] penetration. These indices along with a derived parameter, referred to as the brain exposure index, comprised tier 2 failures that led to the administration of 2 compounds [ON27570, ON27740] as single agents [discrete dosing] to mice bearing intracerebral tumors. Comparison of ON27570’s resultant PK parameters to those obtained in the cassette dosing format suggested a drug-drug interaction most likely at the level of BBB transport, and prompted the use of the in vitro MDCK-MDR1 transport model to help assess the nature of the discrepancy. Overall, the approach was able to identify candidate compounds with suitable PK characteristics yet further revisions to the method, such as the use of in vitro metabolism and transport assays, may improve the PK-directed approach to identify efficacious agents for brain tumor chemotherapy.
Pharmacokinetics; Drug development; Brain tumor; CNS; Preclinical
The rodent tactile vibrissae are innervated by several different types of touch sensory neurons. The central afferents of all touch neurons from one vibrissa collectively project to a columnar structure called barrelette in brainstem. Delineating how distinct types of sensors connect to second-order neurons within each barrelette is critical for understanding tactile information coding and processing. Using genetic and viral techniques, we selectively labeled slowly-adapting (SA) mechanosensory neurons, rapidly-adapting (RA) mechanosensory neurons, afferent synapses, and second-order projection neurons with different “colors”, respectively, to examine the connectivity. We discovered that within each vibrissa column, individual sensory neurons project collaterals to multiple distributed locations; inputs from SA and RA afferents are spatially intermixed without any discernible stereotypy or topography; second-order projection neurons receive convergent SA and RA inputs. Our findings reveal a “one-to-many and many-to-one” connectivity scheme and the circuit architecture for tactile information processing at the first-order synapses.
How neural circuits associated with sexually dimorphic organs are differentially assembled during development is unclear. Here, we report a sexually dimorphic pattern of mouse mammary gland sensory innervation and the mechanism of its formation. Brain-derived neurotrophic factor (BDNF), emanating from mammary mesenchyme and signaling through its receptor TrkB on sensory axons, is required for establishing mammary gland sensory innervation of both sexes at early developmental stages. Subsequently, in males, androgens promote mammary mesenchymal expression of a truncated form of TrkB, which prevents BDNF-TrkB signaling in sensory axons and leads to a rapid loss of mammary gland innervation independent of neuronal apoptosis. Thus, sex hormone regulation of a neurotrophic factor signal directs sexually dimorphic axonal growth and maintenance, resulting in generation of a sex-specific neural circuit.
Reduced kidney function is independently associated with low high-density lipoprotein cholesterol (HDL-C) levels in patients with end-stage renal disease (ESRD), those on hemodialysis, and those with stage 3–5 chronic kidney disease (CKD). However, epidemiological data investigating the relationship between HDL-C levels and kidney function in the general population with roughly normal kidney function are limited, and the results are also inconsistent. The aim of this study was to evaluate the relationship between HDL-C levels and the estimated glomerular filtration rate (eGFR) in a community-based population in China.
This was a community-based cross-sectional survey. In total, 4925 participants (age range, 18–96 years; mean, 51.30±11.98 years) were recruited during routine health status examinations. A questionnaire was used to ascertain age, smoking status, and the history of hypertension and diabetes mellitus for each participant. We measured the body mass index, waist circumference, systolic and diastolic blood pressure, and fasting glucose, total cholesterol, triglyceride, HDL-C, low-density lipoprotein cholesterol, uric acid, and serum creatinine level of each participant. eGFR was evaluated using the Chinese modified Modification of Diet in Renal Disease equation.
The HDL-C level was higher in the first quartile (lowest quartile) of eGFR than in the fourth quartile (the highest quartile). Additionally, HDL-C levels decreased as eGFR decreased. Pearson’s correlation analysis revealed that HDL-C levels were associated with eGFR (r=0.16). After adjustment for some confounders, HDL-C was independently associated with all quartiles of eGFR in the participants.
HDL-C was independently associated with kidney function in a community-dwelling general population. The association between low HDL-C levels and a decreased eGFR gradually strengthened as eGFR declined.
Synthesis of phosphatidylinositol-3-phosphate (PI3P) by Vps34, a class III phosphatidylinositol 3-kinase (PI3K), is critical for the initial steps of autophagosome (AP) biogenesis. Although Vps34 is the sole source of PI3P in budding yeast, mammalian cells can produce PI3P through alternate pathways, including direct synthesis by the class II PI3Ks; however, the physiological relevance of these alternate pathways in the context of autophagy is unknown. Here we generated Vps34 knockout mouse embryonic fibroblasts (MEFs) and using a higher affinity 4x-FYVE finger PI3P-binding probe found a Vps34-independent pool of PI3P accounting for ~35% of the total amount of this lipid species by biochemical analysis. Importantly, WIPI-1, an autophagy-relevant PI3P probe, still formed some puncta upon starvation-induced autophagy in Vps34 knockout MEFs. Additional characterization of autophagy by electron microscopy as well as protein degradation assays showed that while Vps34 is important for starvation-induced autophagy there is a significant component of functional autophagy occurring in the absence of Vps34. Given these findings, class II PI3Ks (α and β isoforms) were examined as potential positive regulators of autophagy. Depletion of class II PI3Ks reduced recruitment of WIPI-1 and LC3 to AP nucleation sites and caused an accumulation of the autophagy substrate, p62, which was exacerbated upon the concomitant ablation of Vps34. Our studies indicate that while Vps34 is the main PI3P source during autophagy, class II PI3Ks also significantly contribute to PI3P generation and regulate AP biogenesis.
Objective. Necrostatin-1 (Nec-1) inhibits receptor-interacting protein 1 (RIP1) kinase and programmed necrosis. This study was designed to examine the protective effects and mechanisms of Nec-1 in concanavalin A- (ConA-) induced hepatitis in mice. Methods. C57BL/6 mice were exposed to ConA via tail vein injection and injected intraperitoneally with Nec-1 or vehicle. Levels of serum liver enzymes and histopathology were determined. Levels of inflammatory cytokines with ConA-induced hepatitis were determined with real-time polymerase chain reaction (real-time PCR). The expression of TNF-α, RIP1, and LC3 was detected with immunohistochemical staining. The expression of TNF-α, IFN-γ, IL2, IL6, caspase 3, RIP1, beclin-1, and LC3 protein was assessed by immunofluorescence and western blotting. Autophagosomes were observed with transmission electron microscopy (TEM). Results. Amelioration in liver functions and histopathological changes and the
suppression of inflammatory cytokine production were observed in Nec-1-injected mice. Western blotting analysis showed that the expression of TNF-α, IFN-γ, IL2, IL6, and RIP1 was significantly reduced in the Nec-1-injected mice, which was confirmed by immunofluorescence and immunohistochemistry. Autophagosome formation was significantly reduced by Nec-1 treatment, as the expression of beclin-1 and LC3, determined with immunofluorescence and western blotting. Conclusion. These results demonstrate that Nec-1 prevents ConA-induced liver injury via RIP1-related and autophagy-related pathways.
Alcohol dependence (AD) is a common neuropsychiatric disorder with high heritability. A number of studies have analyzed the association between the Taq1A polymorphism (located in the gene cluster ANKK1/DRD2) and AD. In the present study, we conducted a large-scale meta-analysis to confirm the association between the Taq1A polymorphism and the risk for AD in over 18,000 subjects included in 61 case-control studies that were published up to August 2012. Our meta-analysis demonstrated both allelic and genotypic association between the Taq1A polymorphism and AD susceptibility [allelic: P(Z)=1.1×10−5, OR=1.19; genotypic: P(Z)=3.2×10−5, OR=1.24]. The association remained significant after adjustment for publication bias using the trim and fill method. Sensitivity analysis showed that the effect size of the Taq1A polymorphism on AD risk was moderate and not influenced by any individual study. The pooled odds ratio from published studies decreased with the year of publication but stabilized after the year 2001. Subgroup analysis indicated that publication bias could be influenced by racial ancestry. In summary, this large-scale meta-analysis confirmed the association between the Taq1A polymorphism and AD. Future studies are required to investigate the functional significance of the ANKK1/DRD2 Taq1A polymorphism in AD.
Alcohol dependence; meta-analysis; ANKK1 and DRD2 gene cluster; the Taq1A polymorphism
TMEFF2 is a single-transmembrane protein containing one EGF-like and two follistatin-like domains. Some studies implicated TMEFF2 as a tumor suppressor for prostate and other cancers, whereas others reported TMEFF2 functioning as a growth factor for neurons and other cells. The gain insights into the apparently conflicting roles of TMEFF2, we generated a null allele of Tmeff2 gene by replacing its first coding exon with human placental alkaline phosphatase cDNA (Tmeff2PLAP). Tmeff2PLAP/PLAP homozygous mutant mice are born normal, but show growth retardation and die around weaning age. Tmeff2 is widely expressed in the nervous system, and the Tmeff2PLAP knock-in allele enables the visualization of neuronal innervations of skin and internal organs with a simple alkaline phosphatase staining. Tmeff2 is also highly expressed in prostate gland and white adipose tissues (WAT). However, with the exception of reduced WAT mass, extensive anatomical and molecular analyses failed to detect any structural or molecular abnormalities in the brain, the spinal cord, the enteric nervous system, or the prostate in the Tmeff2 mutants. No tumors were found in Tmeff2-mutant mice. The Tmeff2PLAP/PLAP knock-in mouse is a useful tool for studying the in vivo biological functions of TMEFF2.
Tmeff2; mutant mouse; central nervous system; peripheral nervous system; axon innervation; growth retardation
Cerebral microdialysis is used to study anti-cancer drug penetration in the central nervous system (CNS) and brain tumors in animal models. Genetically engineered mouse models (GEMMs) have been recently used to study many aspects of CNS tumors as they represent a more relevant model than orthotopic brain tumor xenograft models. However, it is challenging to implant microdialysis cannula in these animals because T2-weighted MRI imaging does not show the reference point (bregma) traditionally used to obtain stereotactic coordinates. Thus, an alternative reference point that can be visualized on MRI images is needed. In this study a novel reference point, identified as the intersection between the olfactory bulb/frontal lobe border and the midline between cerebral hemispheres on T2-weighted MRI images, was used to calculate anterior-posterior (AP) and medial-lateral (ML) coordinates of brain tumors in a GEMM. This point overlies a visible crossover between the rostral rhinal vein and the midline suture on the mouse skull, allowing for the conversion of the MRI coordinates into surgical stereotactic coordinates. Post-mortem MRI and histological examination confirmed accurate probe placement. This procedure will facilitate the accurate and precise implantation of microdialysis probes for the study of anti-cancer drug penetration in brain tumors of GEMMs.
microdialysis; MRI; spontaneous tumors; glioma
Tardive dyskinesia (TD) is a human hyperkinetic movement disorder as a result of potentially irreversible long-term chronic first-generation antipsychotic medications. Unfortunately, mechanisms involved in the development of TD have been poorly understood. Previous studies have indicated that some genetic polymorphisms of immune system and dopamine beta-hydroxylase (DBH) genes may be involved in the pathogenesis of TD. Rs1800872 and rs72393728 are located on the promoter of interleukin-10 (IL10) and DBH gene, respectively. The genetic association between the rs1800872 and TD is unclear. Previous studies have indicated that genetic variations of IL 10 and DBH are implicated in the positive and negative symptoms in schizophrenia. However, the interaction of two variations with severity of TD and symptoms of schizophrenic patients with TD has not been reported. The present study investigated whether these variations and their interaction were associated with clinical phenotypes of TD with schizophrenia in a genetically homogeneous northern Chinese Han population.
Rs1800872 and rs72393728 were genotyped in schizophrenic patients with TD (n = 372) and without TD (NTD; n = 412). The Abnormal Involuntary Movement Scale (AIMS) and Positive and Negative Syndrome Scale (PANSS) were applied to assess the severity of TD and psychopathology of schizophrenia, respectively.
The allele and genotype frequencies of rs1800872 and rs72393728 did not significantly differ between TD and NTD patients (p>0.05). No significant difference was found in the AIMS total score among the genotypes of two loci (p>0.05). Interestingly, the interaction of rs1800872 and rs72393728 showed a significant association with the PANSS general score (p = 0.011), and a trend toward to the PANSS total score (p = 0.055).
These findings suggest that the interaction of rs1800872 and rs72393728 variants may play a role in psychopathology of the general symptoms on PANSS in schizophrenic patients with TD in a northern Chinese Han population.
To observe the relationship between the perihematomal glutamate levels and the blood–brain barrier (BBB) permeability in a rabbit model of intracerebral hemorrhage (ICH).
Seventy-two rabbits were randomly divided into an intracerebral hemorrhage (ICH) model group and a normal control (NC) group, and each group of 36 rabbits was subsequently divided into 6, 12, 18, 24, 48 and 72 h groups (n = 6 each). An ICH model was induced by stereotactic injection of autologous, arterial, non-anticoagulated blood into rabbit basal ganglia. The same procedures were performed in the NC group, but blood was not injected. The rabbits were sacrificed at specific time points after the experiment began depending on their group. Perihematomal brain tissues were collected to determine glutamate levels, BBB permeability and brain water content (BWC).
All of the assessed parameters were increased 6 hour after blood infusion and continued to gradually increase, peaking at 48 hours. Differences were observed when ICH values were compared with those of the NC group (p < 0.05).
Perihematomal glutamate increased significantly after ICH. High levels of glutamate are closely associated with BBB disruption and the brain edema. Therefore, glutamate may play an important role in the pathogenesis of secondary brain injury after (ICH).
Intracerebral hemorrhage; Perihematomal glutamate; Brain water content; Blood–brain-barrier; Evans blue
Abnormal DNA methylation has been observed in promoter regions of a number of genes in human alcoholics. It is unclear whether DNA methylation changes in alcoholics result directly from alcohol consumption or predated the occurrence of alcohol abuse or dependence and whether altered DNA methylation influences gene expression.
We investigated ethanol (EtOH)-induced DNA methylation changes in mouse serotonin receptor 3a gene (Htr3a). A 5-day drinking-in-the-dark paradigm was applied to 28 male outbred CD-1 mice (15 EtOH-drinking and 13 water-drinking). The Sequenom MassARRAY approach was used to quantify methylation levels of 8 CpGs around Htr3a transcription start site in trunk blood and 9 brain regions (dorsomedial prefrontal cortex [DMPFC], ventromedial prefrontal cortex, ventral tegmental area, dorsolateral striatum, dorsomedial striatum [DMSTR], ventral striatum, amygdala, hippocampus [HIPPO], and cerebellum). DNA methylation differences between the 2 groups of mice (EtOH- and water-drinking) were analyzed using multivariate analysis of covariance with consideration of EtOH consumption amount. Expression levels of Htr3a in the DMSTR were measured by real-time PCR in 14 EtOH-drinking and 14 water-drinking male CD-1 mice.
EtOH drinking increased methylation levels of specific Htr3a promoter CpGs in mouse blood (CpG−27: p = 0.028; CpG+54: p = 0.044) and HIPPO (CpG+151: p = 0.012) but reduced methylation levels of specific Htr3a promoter CpGs in mouse DMSTR (CpG−96: p = 0.020; CpG−27: p = 0.035) and DMPFC (CpG+138: p = 0.011; CpG+151: p = 0.040). Nevertheless, methylation levels of Htr3a promoter CpGs in 6 other brain regions were not significantly altered by EtOH consumption. Additionally, the expression level of Htr3a in the DMSTR was 1.43-fold higher in alcohol-drinking mice than in water-drinking mice (p = 0.044).
Our findings indicate that alcohol consumption may induce tissue-specific DNA methylation changes and further suggest that Htr3a promoter methylation levels may be reversely correlated with Htr3a expression levels in specific brain regions such as DMSTR.
Mouse Serotonin Receptor 3a Gene; DNA Methylation; Gene Expression; Ethanol Drinking-in-the-Dark
The increased vulnerability to alcohol dependence (AD) seen in individuals with childhood adversity (CA) may result in part from CA-induced epigenetic changes. To examine CA-associated DNA methylation changes in AD patients, we examined peripheral blood DNA methylation levels of 384 CpGs in promoter regions of 82 candidate genes in 279 African Americans [AAs; 88 with CA (70.5% with AD) and 191 without CA (38.2% with AD)] and 239 European Americans [EAs; 61 with CA (86.9% with AD) and 178 without CA (46.6% with AD)] using Illumina GoldenGate Methylation Array assays. The effect of CA on methylation of individual CpGs and overall methylation in promoter regions of genes was evaluated using a linear regression analysis (with consideration of sex, age, and ancestry proportion of subjects) and a principal components-based analysis, respectively. In EAs, hypermethylation of 10 CpGs in seven genes (ALDH1A1, CART, CHRNA5, HTR1B, OPRL1, PENK, and RGS19) were cross validated in AD patients and healthy controls who were exposed to CA. P values of two CpGs survived Bonferroni correction when all EA samples were analyzed together to increase statistical power [CHRNA5_cg17108064: Padjust = 2.54×10−5; HTR1B_cg06031989: Padjust = 8.98×10−5]. Moreover, overall methylation levels in the promoter regions of three genes (ALDH1A1, OPRL1 and RGS19) were elevated in both EA case and control subjects who were exposed to CA. However, in AAs, CA-associated DNA methylation changes in AD patients were not validated in healthy controls. Our findings suggest that CA could induce population-specific methylation alterations in the promoter regions of specific genes, thus leading to changes in gene transcription and an increased risk for AD and other disorders.
A prolonged PR interval is a sign of increased risk of cardiac arrhythmia. Recent genome-wide association studies found that the single-nucleotide polymorphism (SNP) rs3825214 in T-box 5 (TBX5) was positively associated with PR interval, QRS duration, QT interval, and common arrhythmia disorders such as atrial fibrillation (AF) and advanced atrioventricular block. However, other independent replication studies are required to validate the result. This study assessed associations between rs3825214 and ECG parameters, AF, and ventricular tachycardia (VT) in a Chinese Han population.
To assess the association between rs3825214 and AF and VT, we carried out case-control association studies with 692 AF patients (including 275 lone AF patients), 235 VT patients, and 856 controls. Genotyping was performed using a Rotor-Gene TM 6000 High Resolution Melt system. Statistical analyses of associations were adjusted for potential confounding factors. A moderate association was detected between rs3825214 and AF (Padj = 0.036, OR = 0.79) and a highly significant association was detected between the G allele of rs3825214 and lone AF (Padj = 0.001, OR = 0.65; genotypic P = 3.75×10−4 with a dominant model). We also found that rs3825214 showed a significant association with atrial-ventricular block (AVB; P = 0.028; Padj = 0.035, OR = 0.494).
Our results indicate that rs3825214 conferred a significant risk of lone AF in this Chinese Han population.
Gaucher’s disease (GD) is characterized by loss of lysosomal glucocerebrosidase (GC) activity. Mutations in the gene encoding GC destabilize the protein’s native folding leading to ER-associated degradation (ERAD) of the misfolded enzyme. Enhancing the cellular folding capacity by remodeling the proteostasis network promotes native folding and lysosomal activity of mutated GC variants. However, proteostasis modulators reported so far, including ERAD inhibitors, trigger cellular stress and lead to induction of apoptosis. We show herein that lacidipine, an L-type Ca2+ channel blocker that also inhibits ryanodine receptors on the ER membrane, enhances folding, trafficking and lysosomal activity of the most severely destabilized GC variant achieved via ERAD inhibition in fibroblasts derived from patients with GD. Interestingly, reprogramming the proteostasis network by combining modulation of Ca2+ homeostasis and ERAD inhibition remodels the unfolded protein response and dramatically lowers apoptosis induction typically associated with ERAD inhibition.
Panitumumab (ABX-EGF or Vectibix), the first fully human monoclonal antibody targeting epidermal growth factor receptor (EGFR), was approved by the Food and Drug Administration for treatment of patients with metastatic colorectal cancer. Here, we report for the first time the radioimmunotherapy (RIT) of EGFR-positive human head and neck cancer in a nude mouse model using pure β− emitter 90Y-labeled panitumumab. Biodistribution and planar γ-imaging studies were carried out with 111In-DOTA-panitumumab. The RIT efficacy of 90Y-DOTA-panitumumab was evaluated in UM-SCC-22B tumor model. CD31, Ki67, terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling, and H&E staining were done on UM-SCC-22B tumor sections after treatment. The tumor uptake of 111In-DOTA-panitumumab in UM-SCC-22B tumor-bearing nude mice was 26.10 ± 4.93, 59.11 ± 7.22, 44.57 ± 9.80, 40.38 ± 7.76, and 14.86 ± 7.23 % injected dose per gram of tissue at 4, 24, 72, 120, and 168 hours after injection, respectively. Immunotherapy with cold panitumumab (four doses of 10 mg/kg) did not cause significant antitumor effect. RIT with a single dose of 100 μCi 90Y-DOTA-panitumumab caused significant tumor growth delay and improved the survival in UM-SCC-22B tumor model. A single dose of 200 μCi 90Y-DOTA-panitumumab led to almost complete tumor regression (tumor volumes were 34.83 ± 11.11 mm3 and 56.02 ± 39.95 mm3 on days 0 and 46 after treatment, respectively). Histopathologic analysis of tumors and normal organs further validated the therapeutic efficacy and limited systemic toxicity of 90Y-DOTA-panitumumab. The high tumor uptake and prolonged tumor retention, as well as effective therapy, reveal that 90Y-DOTA-panitumumab may be a promising radioimmunotherapeutic agent to treat EGFR-positive solid tumors.
Integrin αv β3 plays great roles in tumor angiogenesis, invasion, and metastasis. We report here the noninvasive visualization of tumor integrin αv β3 expression by using near-infrared fluorescence (NIRF) imaging of an IRDye800-labeled new cyclic RGD (arginine-glycine-aspartic acid) dimer with tetra(ethylene glycol) (PEG4) linkers (ie, E[PEG4-c(RGDfK)]2, PEG4 = 15-amino-4,7,10,13-tetraoxapentadecanoic acid) in a U87MG tumor model. Fluorescent dye–labeled E[PEG4-c(RGDfK)]2 were subjected to in vitro cell staining, in vivo NIRF imaging, ex vivo NIRF imaging, and histologic studies. The in vitro and in vivo characterization of dye-labeled E[PEG4-c(RGDfK)]2 were compared with dye-labeled RGD dimer without PEG4 linkers (namely, E[c(RGDfK)]2). Both Cy5.5-E[PEG4 -c(RGDfK)]2 and Cy5.5-E[c(RGDfK)]2 exhibited integrin αv β3 binding specificity in a cell-staining experiment. In vivo NIRF imaging showed higher tumor accumulation and tumor to background contrast of IRDye800-E[PEG4-c(RGDfK)]2 over IRDye800-E[c(RGDfK)]2. The tumor integrin αv β3 specificity of IRDye800-E[PEG4-c(RGDfK)]2 was confirmed by successful inhibition of tumor uptake in the presence of an excess dose of c(RGDfK). Histologic examination revealed both tumor vasculature and tumor cell integrin αv β3 binding of IRDye800-E[PEG4-c(RGDfK)]2 in vivo. In summary, NIRF imaging with IRDye800-E[PEG4-c(RGDfK)]2 offers an easy, fast, and low-cost way to detect and semiquantify tumor integrin αv β3 expression in living subjects.
HWTI is a 55-residue protein isolated from the venom of the spider Ornithoctonus huwena. It is a potent trypsin inhibitor and a moderate voltage-gated potassium channel blocker. Here, we designed and expressed two HWTI mutants, HWTI-mut1 and HWTI-mut2, in which the potassium channel inhibitory activity was reduced while the trypsin inhibitory activity of the wild type form (approximately 5 EPU/mg) was retained. Animal studies showed that these mutants were less toxic than HWTI. The effects of HWTI and HWTI-mut1 were examined in a mouse model of acute pancreatitis induced by intraperitoneal injection of a large dose of L-arginine (4 mg/kg, twice). Serum amylase and serum lipase activities were assessed, and pathological sections of the pancreas were examined. Treatment with HWTI and HWTI-mut1 significantly reduced serum amylase and lipase levels in a dose dependent manner. Compared with the control group, at 4 mg/kg, HWTI significantly reduced serum amylase level by 47% and serum lipase level by 73%, while HWTI-mut1 significantly reduced serum amylase level by 59% and serum lipase level by 72%. Moreover, HWTI and HWTI-mut1 effectively protected the pancreas from acinar cell damage and inflammatory cell infiltration. The trypsin inhibitory potency and lower neurotoxicity of HWTI-mut1 suggest that it could potentially be developed as a drug for the treatment of acute pancreatitis with few side effects.