Combined metabolome and transcriptome reveal HmF6'H1 regulating simple coumarin accumulation against powdery mildew infection in Heracleum moellendorffii Hance.

BACKGROUND: Powdery mildew, caused by Eeysiphe heraclei, seriously threatens Heracleum moellendorffii Hance. Plant secondary metabolites are essential to many activities and are necessary for defense against biotic stress. In order to clarify the functions of these metabolites in response to the pathogen, our work concentrated on the variations in the accumulation of secondary metabolites in H. moellendorffii during E. heraclei infection. RESULTS: Following E. heraclei infection, a significant upregulation of coumarin metabolites-particularly simple coumarins and associated genes was detected by RNA-seq and UPLC-MS/MS association analysis. Identifying HmF6'H1, a Feruloyl CoA 6'-hydroxylase pivotal in the biosynthesis of the coumarin basic skeleton through ortho-hydroxylation, was a significant outcome. The cytoplasmic HmF6'H1 protein was shown to be able to catalyze the ortho-hydroxylation of p-coumaroyl-CoA and caffeoyl-CoA, resulting in the formation of umbelliferone and esculetin, respectively. Over-expression of the HmF6'H1 gene resulted in increased levels of simple coumarins, inhibiting the biosynthesis of furanocoumarins and pyranocoumarins by suppressing PT gene expression, enhancing H. moellendorffii resistance to powdery mildew. CONCLUSIONS: These results established HmF6'H1 as a resistance gene aiding H. moellendorffii in combatting E. heraclei infection, offering additional evidence of feruloyl-CoA 6'-hydroxylase role in catalyzing various types of simple coumarins. Therefore, this work contributes to our understanding of the function of simple coumarins in plants' defense against powdery mildew infection.

Impulse oscillometry in the diagnosis of cough variant asthma in children.

BACKGROUND: Cough variant asthma (CVA) is one of the most common causes of chronic cough in children worldwide. The diagnosis of CVA in children remains challenging. This study aimed to assess the diagnostic utility of impulse oscillometry (IOS) pulmonary function in children with CVA. METHODS: This study included children aged 4 to 12 years diagnosed with CVA who underwent IOS pulmonary function and bronchodilation (BD) tests. A control group of healthy children was matched. Pre- and post-BD IOS parameters were recorded and presented as mean ± standard deviation or median. Receiver operating characteristic (ROC) curves were plotted, and the area under the curve (AUC) was calculated to evaluate the discriminatory potential of the IOS parameters for diagnosing CVA. RESULTS: A total of 180 patients with CVA and 65 control subjects were included. The baseline IOS parameters in the CVA group, except X5%pred, were significantly greater compared to the control group. After inhalation of salbutamol sulfate, all IOS parameters improved significantly in the CVA group. However, Z5%pred, R5%pred, and R20%pred remained greater in the CVA group compared to the control group. The improvement rates of IOS parameters in the CVA group significantly surpassed those in the control group. The ROC curve results for pre-BD IOS parameters and the improvement rate during the BD test showed that the combinations of pre-Z5%pred+△Z5% and pre-R5%pred+△R5% achieved the highest AUC value of 0.920 and 0.898, respectively. The AUC values of these combined parameters surpassed those of individual ones. CONCLUSIONS: This study highlights that children with CVA exhibit greater IOS parameters compared to healthy children. The changes in IOS parameters during the BD test provided valuable diagnostic information for CVA, and the combination of various parameters can help pediatricians accurately identify CVA in children.

Analysis of Erect-Panicle Japonica Rice in Northern China: Yield, Quality Status, and Quality Improvement Directions.

China is the only country that extensively cultivates the indica and japonica rice varieties, with the largest japonica rice production area being in northeast China. A study of the relationship between the yield and quality of japonica rice and the effect of nitrogen fertilizer application on this relationship is important. In this paper, we aimed to assess the current yield and quality of japonica rice in northeast China. We selected erect-panicle varieties as the test materials. Field experiments were conducted using different nitrogen fertilizer levels for two consecutive years to analyze the rice varieties' yield, quality, interrelationship, and nitrogen fertilizer response. The average yield following high- and low-nitrogen treatments exceeded 10,000.00 kg/hm2, with a maximum of 12,285.63 kg/hm2. The high-yield-high-nitrogen treatment group had more panicles, a higher seed-setting rate, and a higher 1000-grain weight than the other groups. The high-yield-low-nitrogen group had a higher number of panicles and seed-setting rate than the other groups. The low-yield-high-nitrogen group had a lower number of whole grains, grain length-to-width ratio, and taste value than the other groups. The low-yield-low-nitrogen group had fewer primary branches than the other groups; excluding the primary branch-setting rate and 1000-grain weight, the values of the other panicle traits of the group were significantly higher than those of the other groups. The high-nitrogen-high-flavor group had lower panicle and spikelet numbers and higher spikelet fertility rates than the other groups. The low-nitrogen-high-flavor group had higher spikelet fertility rates and 1000-grain weight than the other groups. Compared to the other groups, the low-nitrogen-high-flavor group had a higher head rice yield, and the high-nitrogen-high-flavor group had a lower chalkiness rate. The main goal of the breeding and cultivation of high-yield and high-quality erect-panicle japonica rice in northern China is to achieve "dual high, dual low, and one high and one low" conditions, signifying a high yield with high or low nitrogen levels, low protein and amylose contents, high head rice rates, and low chalkiness. This study provides a new technique for enhancing the taste of northern erect-panicle japonica rice to promote the sustainable, high-yield, and high-quality development of japonica rice in northern China.

Spirometry in the diagnosis of cough variant asthma in children.

OBJECTIVE: This study aimed to assess the diagnostic utility of spirometry, particularly focusing on small airway parameters, in children with cough variant asthma (CVA). METHODS: This study included children aged 5-12 years with a diagnosis of CVA. Pre- and postbronchodilation spirometry parameters, including FEV1 %pred, FVC%pred, FEV1 /FVC%pred, PEF%pred, FEF25 %pred, FEF50 %pred, FEF75 %pred, MMEF%pred, were recorded. Receiver operating characteristic curves were plotted, and the area under the curve (AUC) was calculated to assess the discriminatory potential of these spirometry parameters for CVA. A prediction model based on logistic regression (LR) was performed. RESULTS: A total of 200 patients with CVA and 73 control subjects were included. Baseline spirometry parameters in the CVA group, except for FVC%pred, were significantly lower compared to the control group. After inhalation of salbutamol sulfate, all parameters showed significant improvement in the CVA group. However, these parameters, except for FEV1 %pred and FVC%pred, remained lower in the CVA group compared to the control group. The improvement rate of each parameter in the CVA group, except for ∆ FVC%, was significantly higher than that in the control group. △ MMEF% achieved the highest AUC of 0.797 with a threshold value of 16.09%, followed by △ FEF75 % (0.792), △ FEV1 % (0.756), and △ FEF50 % (0.747) with threshold values of 19.01%, 4.48%, and 19.4%, respectively. The clinical prediction model included four variables (age, △ FEF25 %, △ FEF75 %, and △ MMEF%) and demonstrated excellent performance distinguishing patients with and without CVA (AUC = 0.850). In the CVA group, the △ FEV1 % showed a positive correlation with small airway parameters. CONCLUSIONS: This study highlights that children with CVA exhibit lower pulmonary function parameters compared to healthy children. Changes in small airway parameters during bronchodilator tests can be valuable in diagnosing CVA, and the LR prediction model incorporating age and several pulmonary parameters can assist physicians in accurately identifying CVA in clinical practice.

Functionalization of cellulosic hydrogels with Cu2O@CuO nanospheres: Toward antifouling applications.

Immobilizing metal oxide nanoparticles onto polymer substrate could endow antibacterial performance and enhance mechanical property. In-situ strategy is extensively used to better control loading percentage, uniform distribution and particle size of nanoparticles. However, it still remains challenge in depositing stable bicomponents copper oxide nanoparticles on non-adhesive surface of cellulose hydrogel in high density. In this study, Cu2O@CuO nanospheres were in-situ deposited onto cellulose hydrogels via liquid phase reduction. Particularly, sodium hydroxide in the cellulose hydrogel severed as the precipitant, which not only save the usage of chemicals, but also enhanced binding between nanoparticles and the hydrogel. Furthermore, Cu2O@CuO nanospheres demonstrate biocidal antifouling performance against Escherichia coli by releasing biocide. After hydrolysis of precipitation layer, the exposed cellulose hydrogel exhibits fouling-resistant property for Chlorella Vulgaris due to the hydration layer on its surface. Such composites hold great promise in antifouling coatings and other applications.

Effects of shading on triterpene saponin accumulation and related gene expression of Aralia elata (Miq.) Seem.

Aralia elata (Miq.) Seem is widely used as a medicinal plant and functional food in China. In this study, A. elata plants were exposed to full sunlight (CK), 40% shading (LS), 60% shading (MS), and >80% shading (ES) condition to investigate the effects of shading treatments on growth, stress levels, antioxidant enzymes activity, araloside content and related gene expression. The greatest growth and leaf biomass were achieved in 40% shading, and leaf biomass per plant increased by 16.09% compared to the non-shading treatment. Furthermore, the lowest reactive oxide species (ROS) production and lipid peroxidation resulting from increasing antioxidant enzyme activity were also observed in LS treatment. Overall, shading percentage negatively regulated the expression of key enzymes (squalene synthase, SS; squalene epoxidase, SE and ß-amyrin synthase, bAS) involved in the saponin biosynthesis, resulting in the greatest yields of total and four selected aralosides in A. elata leaves were achieved in sunlight group. However, the greatest yield of total saponin in the leaves was observed in the 40% shading group due to higher leaf biomass. The results suggest that optimizing the field growing conditions would be important for obtaining the greatest yield of bioactive components. Total saponin and selected aralosides also have a significant correlation with ROS production and antioxidant enzyme activity, these indicated the increased yield of these saponins may be part of a defense response. The study concludes that the production of saponin was the interaction of oxidative stress and photosynthesis.

Identification and analysis of CYP450 and UGT supergene family members from the transcriptome of Aralia elata (Miq.) seem reveal candidate genes for triterpenoid saponin biosynthesis.

BACKGROUND: Members of the cytochrome P450 (CYP450) and UDP-glycosyltransferase (UGT) gene superfamily have been shown to play essential roles in regulating secondary metabolite biosynthesis. However, the systematic identification of CYP450s and UGTs has not been reported in Aralia elata (Miq.) Seem, a highly valued medicinal plant. RESULTS: In the present study, we conducted the RNA-sequencing (RNA-seq) analysis of the leaves, stems, and roots of A. elata, yielding 66,713 total unigenes. Following annotation and KEGG pathway analysis, we were able to identify 64 unigenes related to triterpenoid skeleton biosynthesis, 254 CYP450s and 122 UGTs, respectively. A total of 150 CYP450s and 92 UGTs encoding > 300 amino acid proteins were utilized for phylogenetic and tissue-specific expression analyses. This allowed us to cluster 150 CYP450s into 9 clans and 40 families, and then these CYP450 proteins were further grouped into two primary branches: A-type (53%) and non-A-type (47%). A phylogenetic analysis of 92 UGTs and other plant UGTs led to clustering into 16 groups (A-P). We further assessed the expression patterns of these CYP450 and UGT genes across A. elata tissues, with 23 CYP450 and 16 UGT members being selected for qRT-PCR validation, respectively. From these data, we identified CYP716A295 and CYP716A296 as the candidate genes most likely to be associated with oleanolic acid synthesis, while CYP72A763 and CYP72A776 were identified as being the most likely to play roles in hederagenin biosynthesis. We also selected five unigenes as the best candidates for oleanolic acid 3-O-glucosyltransferase. Finally, we assessed the subcellular localization of three CYP450 proteins within Arabidopsis protoplasts, highlighting the fact that they localize to the endoplasmic reticulum. CONCLUSIONS: This study presents a systematic analysis of the CYP450 and UGT gene family in A. elata and provides a foundation for further functional characterization of these two multigene families.

Circulating Memory T Follicular Helper Cells in Patients with Neuromyelitis Optica/Neuromyelitis Optica Spectrum Disorders.

OBJECTIVE: This study aimed to examine the potential role of memory T follicular helper (Tfh) cells in patients with neuromyelitis optica/neuromyelitis optica spectrum disorders (NMO/NMOSD). METHODS: The percentages of different subsets of circulating memory Tfh cells in 25 NMO/NMOSD patients before and after treatment as well as in 17 healthy controls were examined by flow cytometry. The levels of IL-21 and AQP4 Ab in plasma and CSF were measured by ELISA. RESULTS: The percentages and numbers of circulating memory Tfh cells, ICOS(+), CCR7(-), CCR7(-)ICOS(+), CCR7(+), CCR7(+)ICOS(+) memory Tfh cells, and the levels of IL-21 in plasma and CSF were significantly increased in NMO/NMOSD patients. The percentages of CCR7(-) and CCR7(-)ICOS(+) memory Tfh cells were positively correlated with ARR, plasma IL-21, and AQP4 Ab levels. The percentages of CCR7(+) and CCR7(+)ICOS(+) memory Tfh cells were positively correlated with CSF white blood cell counts, proteins, and IL-21 levels. Treatment with corticosteroids significantly reduced the numbers of CCR7(-)ICOS(+) and CCR7(+)ICOS(+) memory Tfh cells as well as plasma IL-21 levels in patients with partial remission. CONCLUSIONS: Our findings indicate that circulating memory Tfh cells may participate in the relapse and development of NMO/NMOSD and may serve as a new therapeutic target.

Let-7a gene knockdown protects against cerebral ischemia/reperfusion injury.

The microRNA (miRNA) let-7 was one of the first miRNAs to be discovered, and is highly conserved and widely expressed among species. let-7 expression increases in brain tissue after cerebral ischemia/reperfusion injury; however, no studies have reported let-7 effects on nerve injury after cerebral ischemia/reperfusion injury. To investigate the effects of let-7 gene knockdown on cerebral ischemia/reperfusion injury, we established a rat model of cerebral ischemia/reperfusion injury. Quantitative reverse transcription-polymerase chain reaction demonstrated that 12 hours after cerebral ischemia/reperfusion injury, let-7 expression was up-regulated, peaked at 24 hours, and was still higher than that in control rats after 72 hours. Let-7 gene knockdown in rats suppressed microglial activation and inflammatory factor release, reduced neuronal apoptosis and infarct volume in brain tissue after cerebral ischemia/reperfusion injury. Western blot assays and luciferase assays revealed that mitogen-activated protein kinase phosphatase-1 (MKP1) is a direct target of let-7. Let-7 enhanced phosphorylated p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) expression by down-regulating MKP1. These findings suggest that knockdown of let-7 inhibited the activation of p38 MAPK and JNK signaling pathways by up-regulating MKP1 expression, reduced apoptosis and the inflammatory reaction, and exerted a neuroprotective effect following cerebral ischemia/reperfusion injury.

Double Roles of Macrophages in Human Neuroimmune Diseases and Their Animal Models.

Macrophages are important immune cells of the innate immune system that are involved in organ-specific homeostasis and contribute to both pathology and resolution of diseases including infections, cancer, obesity, atherosclerosis, and autoimmune disorders. Multiple lines of evidence point to macrophages as a remarkably heterogeneous cell type. Different phenotypes of macrophages exert either proinflammatory or anti-inflammatory roles depending on the cytokines and other mediators that they are exposed to in the local microenvironment. Proinflammatory macrophages secrete detrimental molecules to induce disease development, while anti-inflammatory macrophages produce beneficial mediators to promote disease recovery. The conversion of the phenotypes of macrophages can regulate the initiation, development, and recovery of autoimmune diseases. Human neuroimmune diseases majorly include multiple sclerosis (MS), neuromyelitis optica (NMO), myasthenia gravis (MG), and Guillain-Barré syndrome (GBS) and macrophages contribute to the pathogenesis of these neuroimmune diseases. In this review, we summarize the double roles of macrophage in neuroimmune diseases and their animal models to further explore the mechanisms of macrophages involved in the pathogenesis of these disorders, which may provide a potential therapeutic approach for these disorders in the future.

Cerebrolysin improves sciatic nerve dysfunction in a mouse model of diabetic peripheral neuropathy.

To examine the effects of Cerebrolysin on the treatment of diabetic peripheral neuropathy, we first established a mouse model of type 2 diabetes mellitus by administering a high-glucose, high-fat diet and a single intraperitoneal injection of streptozotocin. Mice defined as diabetic in this model were then treated with 1.80, 5.39 or 8.98 mL/kg of Cerebrolysin via intraperitoneal injections for 10 consecutive days. Our results demonstrated that the number, diameter and area of myelinated nerve fibers increased in the sciatic nerves of these mice after administration of Cerebrolysin. The results of several behavioral tests showed that Cerebrolysin dose-dependently increased the slope angle in the inclined plane test (indicating an improved ability to maintain body position), prolonged tail-flick latency and foot-licking time (indicating enhanced sensitivity to thermal and chemical pain, respectively, and reduced pain thresholds), and increased an index of sciatic nerve function in diabetic mice compared with those behavioral results in untreated diabetic mice. Taken together, the anatomical and functional results suggest that Cerebrolysin ameliorated peripheral neuropathy in a mouse model of type 2 diabetes mellitus.

Follicular Helper CD4+ T Cells in Human Neuroautoimmune Diseases and Their Animal Models.

Follicular helper CD4(+) T (TFH) cells play a fundamental role in humoral immunity deriving from their ability to provide help for germinal center (GC) formation, B cell differentiation into plasma cells and memory cells, and antibody production in secondary lymphoid tissues. TFH cells can be identified by a combination of markers, including the chemokine receptor CXCR5, costimulatory molecules ICOS and PD-1, transcription repressor Bcl-6, and cytokine IL-21. It is difficult and impossible to get access to secondary lymphoid tissues in humans, so studies are usually performed with human peripheral blood samples as circulating counterparts of tissue TFH cells. A balance of TFH cell generation and function is critical for protective antibody response, whereas overactivation of TFH cells or overexpression of TFH-associated molecules may result in autoimmune diseases. Emerging data have shown that TFH cells and TFH-associated molecules may be involved in the pathogenesis of neuroautoimmune diseases including multiple sclerosis (MS), neuromyelitis optica (NMO)/neuromyelitis optica spectrum disorders (NMOSD), and myasthenia gravis (MG). This review summarizes the features of TFH cells, including their development, function, and roles as well as TFH-associated molecules in neuroautoimmune diseases and their animal models.

Circulating CCR7+ICOS+ Memory T Follicular Helper Cells in Patients with Multiple Sclerosis.

OBJECTIVE: This study is aimed at examining the potential roles of circulating memory T follicular helper (Tfh) cells in patients with multiple sclerosis (MS). METHODS: The numbers of different subsets of circulating memory Tfh cells in 25 patients with relapsed MS before and after treatment as well as 14 healthy controls (HC) were examined by flow cytometry. The levels of plasma IL-21 in all patients and cerebrospinal fluid (CSF) IL-21 in some MS patients and controls with non-inflammatory neuronal diseases (NND) were measured by ELISA. RESULTS: In comparison with that in the HC, the numbers of circulating CD3+CD4+CXCR5+CD45RA-, ICOS+, CCR7+ and CCR7+ICOS+ memory Tfh cells and the levels of plasma IL-21 significantly increased in MS patients, but significantly decreased in the patients with complete remission (CR). The levels of CSF IL-21 were significantly higher in the MS patients than that in the NND patients. The numbers of CCR7+ICOS+ memory Tfh cells were positively correlated with the EDSS scores, the levels of plasma and CSF IL-21, IgG, MBP-Ab or MOG-Ab. CONCLUSIONS: Our findings indicated that circulating memory Tfh cells, especially CCR7+ICOS+ memory Tfh cells, may be associated with the relapse of MS and may serve as a new therapeutic target.

Myoclonic epilepsy with ragged-red fibers: A case report.

Myoclonic epilepsy with ragged-red fibers is a maternally inherited disease that is characterized by myoclonic epilepsy, cerebellar ataxia and progressive muscular weakness. The present study reports the case of a 25-year-old male who presented with paroxysmal left upper limb tics and weakness for two years. Neurological examination revealed intact cranial nerves, decreased deep tendon reflexes and decreased sensation of touch, pain and vibration. The gait of the patient was broad and he was unable to walk in a straight line. Local cortical atrophy was also observed in the left temporal-occipital cortex on a magnetic resonance imaging scan. The muscle biopsy revealed ragged-red fibers. Therefore, the present study hypothesized that imaging observations and follow-up examinations are important in patients with myoclonic epilepsy.

Deficiency of Trim27 protects dopaminergic neurons from apoptosis in the neurotoxin model of Parkinson's disease.

Parkinson's disease (PD) is an age-related neurodegenerative movement disorder, characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The MPTP/MPP+ model is often used to investigate the signaling mechanisms of dopamine (DA) degeneration, both in vivo and in vitro. The identification of specific genetic and environmental factors responsible for PD has bolstered evidence for a shared pathway of neuronal death -apoptosis. Trim27 is reported to promote apoptosis. However, little evidence exists to indicate a linkage between Trim27 and PD. In this study, we found that compared to healthy individuals, Trim27 was significantly upregulated in patients with PD. We further showed that Trim27 expression was dramatically induced in PC12 cells and in the SNpc of the PD mouse model. RNAi-mediated knockdown of Trim27 in PC12 cells showed obvious suppression of apoptosis. There are reduced dopaminergic neuron loss and lower apoptotic protein expression levels in MPTP-treated Trim27-/- mice, compared with MPTP-treated WT mice. These data demonstrated that Trim27 deficiency decreases apoptosis and protects dopaminergic neurons in the neurotoxin model of PD, suggesting that Trim27 may be an effective potential target during the treatment of PD.

A rare case of critical illness polyneuropathy and literature review.

A 40- year-old Male was admitted to the first hospital of Jilin University with the complaint of 4 days of fever and headache and aggravation of weakness in his lower extremities accompanied with dysuria and disturbance of consciousness for one day. He had tachycardia, tachypnea and elevated white blood cell counts. General status of the patient got better day by day, while weakness and pain in his lower extremities had developed and gradually quadriplegia arose. When intensive care unit history, weaning difficulty from mechanical ventilator, clinical manifestations in intensive care unit associated with SIRS, symmetrical paresis pronounced in distal lower extremities, absence of deep tendon reflexes, evidence of distal sensory impairment, presence of electrophysiologic results indicating axonal sensorimotor polyneuropathy and muscle and nerve biopsy results were taken into consideration, he was diagnosed as critical illness polyneuropathy.

Intraepidermal nerve fiber density of healthy human.

OBJECTIVES: To analyze intraepidermal nerve fiber density (IEFND) by skin biopsy, evaluate the effect of age, anatomical sites, and ethnic origin on IEFND and develop a reference range of IENFD at the distal leg of healthy human. METHODS: Seventy skin biopsy specimens from surgical procedures involving 70 patients were analyzed. Specimens were fixed routinely in formalin and thereafter embedded in paraffin. Nerve fibers of 10-µm-thick sections were observed using immunoperoxidase staining with panaxonal antibody protein gene product 9·5 (PGP 9·5). The morphology of intraepidermal nerve fibers (IENFs) and the IENFD was determined using light microscope. The statistical analysis was performed with SPSS 16·0 software. RESULTS: No significant correlation was observed between IENFD and age (Pwrist  =  0·830, Pdistal leg  =  0·478). The significant correlation was observed between IENFD and anatomic site (P  =  0·001), the IENFD of upper arm and proximal thigh were significantly higher than that of wrist and distal leg. The reference range for IENFD of distal leg in normal Chinese humans was 40·55 fibers/mm(2). The IEFND of Chinese healthy human was significantly lower than that of Finnish (62·87 ± 15·25 vs 114·617 ± 32·322 fibers/mm(2), P < 0·05). DISCUSSION: Skin biopsy may be a useful tool in sensory neuropathies. IENFD is independent of age, but varies in different parts of the body. The proximal sites have a higher IENFD, but no significant difference is found between the wrist and distal leg.

Human mesenchymal stem cells increases expression of α-tubulin and angiopoietin 1 and 2 in focal cerebral ischemia and reperfusion.

Angiogenesis is associated with improved neurologic recovery after cerebral ischemia. Human bone marrow mesenchymal stem cells (hMSCs) have been successfully used to treat ischemic stroke and were shown to induce the expression of a number of neurotrophic factors including VEGF, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) in a rat middle cerebral artery occlusion (MCAO) ischemia model. In this study, we aimed to understand the mechanism underlying the improvement of neurological function following hMSCs transplantation into MCAO rats. We established a rat MCAO model and used immunofluorescence to evaluate α-tubulin expression in the hippocampus. We used RT-PCR to determine the expression of Ang-1 and Ang-2 mRNAs after transplantation of hMSCs into MCAO rats. We showed a significant decrease in α-tubulin expression in rats with cerebral ischemia, suggesting that α-tubulin is a protective protein in cerebral ischemia Transplantation of hMSCs significantly upregulated α-tubulin levels in the hippocampus. Transplantation of hMSCs also resulted in a significant upregulation of Ang-1 and Ang-2 mRNAs in MCAO rats. Ang-2 expression was upregulated earlier than Ang-1, suggesting that (1) transplantation of hMSCs promotes angiogenesis and that (2) Ang-2 may be an initiator of angiogenesis. Our results provide a theoretical basis for the therapeutic use of hMSCs in cerebral ischemia.

The impact of V30A mutation on transthyretin protein structural stability and cytotoxicity against neuroblastoma cells.

Single point mutations in the transthyretin (TTR) gene may cause a hereditary neurodegenerative disease termed familial amyloidotic polyneuropathy (FAP) due to accelerated deposition of amyloid fibrils, resulting in peripheral and autonomic nervous system dysfunction. Recently, we found a Chinese FAP family involving a TTR V30A mutation. To understand the pathogenic mechanisms of this V30A TTR, we investigated the effects of this mutation on TTR quaternary and tertiary structural stabilities and cytotoxicities against neuroblastoma cells along with the most common variant V30M TTR and the wild-type (WT) TTR. Our results showed that the V30A mutation impaired the thermodynamic and kinetic stabilities of the TTR protein by increasing the extent and rate of tetramer dissociation and unfolded monomer and amyloid fibril formation, even to a greater extent than the V30M mutation under several experimental conditions. Further, an obviously cytotoxic effect of the V30A TTR on the human neuroblastoma cell line, IMR-32, was observed. The V30A TTR induced apoptosis and autophagy concomitant with the accumulation of reactive oxygen species (ROS) and DNA double-strand breaks, reflected in the induction of phosphor-H2A.X. These results suggest that the V30A mutation in the TTR gene promotes the formation of unfolded monomers and amyloid fibrils, which potentially contribute to the increased neurotoxicity and the pathology associated with this FAP family.