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In the past decade, RNA oxidation has caught the attention of many researchers, working to uncover its role in the pathogenesis of neurodegenerative diseases. It has been well documented that RNA oxidation is involved in a wide variety of neurological diseases and is an early event in the process of neurodegeneration. The analysis of oxidized RNA species revealed that at least messenger RNA (mRNA) and ribosomal RNA (rRNA) are damaged in several neurodegenerative diseases, including Alzheimer's disease and amyotrophic lateral sclerosis (ALS). The magnitude of the RNA oxidation, at least in mRNA, is significantly high at the early stage of the disease. Oxidative damage to mRNA is not random but selective and many oxidized mRNAs are related to the pathogenesis of the disease. Several studies have suggested that oxidative modification of RNA affects the translational process and consequently produces less protein and/or defective protein. Furthermore, several proteins have been identified to be involved in handling of damaged RNA. Although a growing body of studies suggests that oxidative damage to RNA may be associated with neuron deterioration, further investigation and solid evidence are needed. In addition, further uncovering of the consequences and cellular handling of the oxidatively damaged RNA should be important focuses in this area and may provide significant insights into the pathogenesis of neurodegenerative diseases.

Transgenic mice expressing mutant Cu2+/Zn2+ superoxide dismutase SOD1(G93A) develop similar clinical and pathological phenotypes to amyotrophic lateral sclerosis (ALS) patients. Here, we utilize representational difference analysis to identify the transcripts that are up-regulated in the presymptomatic stage of SOD1(G93A) mice. Unexpectedly, three predominant clones were 18S or 28S ribosomal RNA (rRNA) segments. One of these clones corresponded to a capped and polyadenylated transcript containing a large portion of 18S rRNA, named MSUR1 (mutant SOD1-up-regulated RNA 1). In vitro expression experiments show that MSUR1 is able to rescue SOD1(G93A)-mediated cell death. Expression of MSUR1 significantly reduces SOD1(G93A)-induced free radical levels and oxidative damage. Further, MSUR1 can reduce hydrogen peroxide-mediated cytotoxicity. MSUR1 does not encode a protein, suggesting its role as a functional noncoding RNA. It is widely expressed in various tissues. Searching the database of GenBank revealed that a large number of expressed sequence tag (EST) clones contain large portions of rRNA sequence, potentially indicating a heretofore overlooked class of mRNAs with functional significance.

We previously reported that up to 50% of messenger RNAs (mRNA) are oxidatively damaged in the affected area of Alzheimer's disease (AD) brains. The role of RNA oxidation in the cell death process is unknown. In the present study, we used cortical primary dissociated cultures to investigate the relationship between RNA oxidation and neuron degeneration induced by various insults, including hydrogen peroxide, glutamate, and amyloid beta peptide. These insults mediate the production of reactive oxygen species and thus induce oxidative stress. The results showed that RNA oxidation was an early event far preceding cell death, not merely a consequence of dying cells. RNA oxidation occurred primarily in a distinct group of neurons that died later. Identification of oxidized RNA species revealed that significant amounts of mRNAs were oxidized and that some mRNA species were more susceptible to oxidative damage, consistent with findings in the AD brain. The level of protein corresponding to the oxidized mRNA species was significantly decreased. Polyribosome analysis indicated that oxidized bases in mRNAs caused ribosome stalling on the transcripts, which led to a decrease of protein expression. These results suggest that RNA oxidation may be directly associated with neuronal deterioration, rather than harmless epiphenomena, during the process of neurodegeneration.

Glutamate is the major excitatory neurotransmitter in the central nervous system. Its activity is carefully modulated in the synaptic cleft by glutamate transporters. The glial glutamate transporter EAAT2 is the main mediator of glutamate clearance. Reduced EAAT2 function could lead to accumulation of extracellular glutamate, resulting in a form of cell death known as excitotoxicity. In amyotrophic lateral sclerosis and Alzheimer disease, EAAT2 protein levels are significantly decreased in affected areas. EAAT2 mRNA levels, however, remain constant, indicating that alterations in EAAT2 expression are due to disturbances at the post-transcriptional level. In the present study, we found that some EAAT2 transcripts contained 5'-untranslated regions (5'-UTRs) greater than 300 nucleotides. The mRNAs that bear long 5'-UTRs are often regulated at the translational level. We tested this possibility initially in a primary astrocyte line that constantly expressed an EAAT2 transcript containing the 565-nt 5'-UTR and found that translation of this transcript was regulated by many extracellular factors, including corticosterone and retinol. Moreover, many disease-associated insults affected the efficiency of translation of this transcript. Importantly, this translational regulation of EAAT2 occurred in vivo (i.e. both in primary cortical neurons-astrocytes mixed cultures and in mice). These results indicate that expression of EAAT2 protein is highly regulated at the translational level and also suggest that translational regulation may play an important role in the differential EAAT2 protein expression under normal and disease conditions.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder that is characterized by progressive degeneration of motor neurons in the spinal cord, motor cortex, and brainstem. There is currently no effective treatment for ALS. Transgenic mice expressing familiar ALS-linked mutant superoxide dismutase (SOD1) have been commonly used for preclinical trials; however, successful trails in mice mostly fail in subsequent human trials. Most preclinical trials start treatment before the onset of symptoms, while ALS patients in clinical trials have surpassed the onset of disease. In addition, ALS is not only a multi-factorial disease but also a multi-systemic disease that affects several cell types [1]. Therefore, therapies should be aimed at the interception of multiple mechanisms. In the present study, we investigated (1) whether the therapeutic agents, which were previously tested starting before disease onset, still showed protective effects when the treatments were initiated after disease onset and (2) whether combination therapies offered better effects after disease onset. Minocycline, which inhibits microglia activation and apoptotic cascade, was previously found to be effective at ameliorating motor impairment and increasing the lifespan of SOD1G93A mice when administered at 5 weeks of age [2]. However, minocycline fails in human trials [3]. We assessed whether similar beneficial effects could be seen when treatment began at 90 days of age. This time point was chosen because SOD1G93A mice [4] exhibited 10~20% decline in motor function at this age, which may be equivalent to the stage that ALS patients first visit a doctor. The results showed no delay in impaired motor function (Fig. 1A&B, Mino) and no extension in survival (Fig. 1C&D, Mino). This result is consistent with a recent report [5]. Ceftriaxone, which reduces excitotoxicity via activation of glial glutamate transporter EAAT2, was found to slow disease progression in SOD1G93A mice when administered at 12 weeks of age [6]. Ceftriaxone is currently in clinical trials for ALS. We found that when ceftriaxone was administered starting from 90 days of age, treatment no longer slowed disease progression (Fig. 1A&B, Cfx) and survival (Fig. 1C&D, Cfx). We then assessed whether the combined treatments of ceftriaxone and minocycline, starting at 90 days of age, would have protective effects. The results showed no delay in impaired motor function (Fig. 1A&B, Double) but a significant increase in survival by ~12 days (Fig. 1C&D, Double). Figure 1 Effects of single and combination treatments on disease onset and survival of SOD1G93A mice. SOD1G93A mice (B6SJL-Tg(SOD1-G93A)1Gur/J with high copy number of the mutant human SOD1 gene, Jackson Laboratory) were used in this study (the cited previous ... Vitamin E, which reduces oxidative damage, can delay disease onset when administered at 30 days of age, but has no effect in extending the lifespan of SOD1G93A mice [7], only slightly slowing disease progression in human ALS trials [8,9]. We found that when mice were treated with vitamin E starting at 30 days of age and then with ceftriaxone and minocycline starting at 90 days of age, a significant delay in motor dysfunction by ~18 days (Fig. 1A&B, Triple D30) and an extension in survival by ~16 days (Fig. 1C&D, Triple D30) were observed. Furthermore, we assessed the combined three treatments starting at 90 days of age. This triple treatment resulted in greater improvement of motor performance than that seen in the double treatment (ceftriaxone and minocycline) (Fig. 1A&B, Triple D90). The prolonged survival for the triple treatment was similar to that seen in the double treatment (Fig. 1C&D, Triple D90). To confirm that the protective effects of combined treatments were due to the inhibition of targeted mechanisms, we performed immunofluorescent staining on lumbar spinal cord sections prepared from treated SOD1G93A mice and wild-type littermates at 115 days of age. As shown in Fig. 2A, EAAT2 protein levels were restored in treated mice (in the double and triple groups). Oxidative damage as measured RNA oxidation (15A3 staining) was also reduced [7]. Astrogliosis (GFAP staining) and microglial activation (CD11b staining) in SOD1G93A mice were increased compared to that seen in wild-type littermates. There was even greater enhanced glial activation by the cocktail treatments - the double and triple groups, which is consistent with a recent report [5]. Furthermore, to confirm that the delayed disease onset and prolonged survival were associated with reduced neuronal loss, we performed cresyl violet staining on lumbar spinal cord adjacent sections. A significant reduction of motor neuron loss was observed in the triple groups (Fig. 2B). Figure 2 Effects of combination treatments on oxidative damage, EAAT2 expression, glial activation, and motor neuron loss in SOD1G93A mice. Immunofluorescent (A) and Cresyl violet (B) stainings were performed on lumbar spinal cord sections prepared from treated ... Transition from preclinical mouse studies to human clinical trials is difficult for most diseases. As the first step, proper design of preclinical trials is very important. The results of this study suggest that inappropriate pre-symptomatic treatment in mice may partially provide explanations for the failure of some clinical trials in ALS patients. In addition, combination treatments may serve as a therapeutic strategy for human trials. The agents that have been unsuccessful in human trials may prove to be more beneficial when used in combination with one another.

Introduction There is strong evidence that B-cell receptor (BCR) signaling has a critical role in the pathogenesis of B-cell malignancies such as non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). The BCR signalosome, B-cell receptor signaling protein complex, is therefore a rational therapeutic target. This has already been proven by success in clinical trials of B-cell signaling inhibitors such as Ibrutinib and Idelalisib. It is becoming more important to know what combinations of new and traditional agents is best for each patient. Although genetic profiles may help to predict sensitivity to treatment to some degree, it is ideal to profile the BCR signaling state of each case to select most effective B-cell signaling inhibitors. Our goal is to develop a BCR signalosome-oriented molecular marker and investigate its clinical value. We focus on protein-protein interactions in the B-cell signalosome that are regulated by tyrosine kinases, their substrates, and SH2 domains. We hypothesized that the global tyrosine phosphorylation state determined by SH2 profiling, an SH2 domain-based molecular diagnostic approach, may meaningfully represent the B-cell signaling state of B-cell malignancies. Here we conducted SH2 profiling of 1) BCR signalosome peptides to determine specificity of BCR SH2 domain probes and 2) CLL patient samples to determine the presence of patient specific BCR SH2 profiles. Methods For microarrays, phosphorylation site databases were extensively searched and 368 tyrosine phosphorylation sites from the core 38 proteins which make up the BCR-signalosome were selected for peptide synthesis. Replicated peptide microarrays containing pairs of phosphorylated and unphosphorylated tyrosine motifs were separately probed with a set of BCR SH2 domains including BTK, BLNK, LYN, PI3K, SYK, and PLCg2. For clinical sample experiments, PBMC samples were collected from 35 CLL patients who visited UConn Health (UCH) and Saint Francis Hospital (SFH) between 2008 and 2014. The median age at study enrollment was 67 (46-98 years). Male patients constituted 63%. Binet stage A disease was present in 76% of the patients. Reverse-phase SH2 domain binding assay was performed as previously described using the BCR SH2 domains. Results According to the microarray results, 94% of BCR signalosome phosphotyrosines were bound by at least one SH2 domain (median 5 domains). A group of proteins including CD22, CD79A, CD45, and PLCg2 protein harbour tyrosine sites that can bind to more than 10 SH2 domains suggesting competition between these SH2 domains may exist in the cell. Specificity of BCR SH2 domains could be grouped into three levels: very specific (BTK, BLNK), medium (Lyn, PLCg2, SHP-1, etc) and broad (SHIP). We found a number of previously undocumented SH2-ligand interactions that may be involved in specific downstream signaling pathways. A clustering analysis of CLL samples revealed the presence of different patient groups such as BLNK-dominant and PLC-dominant. Of those clusters, we observed that a cluster with low BLNK signal and high LYN signal was enriched with clinically progressive type CLL cases. To test the prognostic impact of the BLNK/LYN profile, the CLL cases were divided into four groups by high (+) and low (-) BLNK and LYN SH2 binding and compared with PFS. There was a significant difference between the groups in a log-rank test, indicating that patients with the BLNK-low & LYN-high profile progressed more rapidly. Of note, in the microarray experiments we identified that a group of BCR signaling proteins/peptides such as CD19, CD79A, and Dok1 show a similar BLNK-low & LYN-high profile. Their involvements in the SH2 profile of CLL samples remain to be determined. Conclusion Aiming to develop a new molecular marker based on the BCR signaling state of B-cell malignancies, we applied SH2 profiling to BCR peptide microarrays and CLL/NHL patient samples. We confirmed that BCR signalosome-oriented SH2 probes have sufficient specificity to distinguish various signalosome tyrosine sites. SH2 profiling of CLL indicated that the BCR SH2 probes are able to distinguish CLL subgroups, one of which was correlated with a poor PFS. Further efforts are underway to determine the clinical marker value of the BCR signalosome profile, such as its utility in risk stratification, early detection of disease progression, and prediction or assessment of response to B-cell signaling inhibitor therapy. Disclosures Wasser: Amgen, Inc: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Accumulating evidence has shown that various lengths of ribosomal RNA (rRNA) sequences are widely present in polyadenylated RNA. This review article will discuss these polyadenylated rRNA containing transcripts (PART). PART are highly abundant and widely expressed in various tissues. It appears that there may be two types of PART. One type, type I, contains the rRNA segments (from approximately 10 nucleotides up to several hundred nucleotides) located within the transcripts. It has been demonstrated that short rRNA sequences within type I PART may function as cis-regulatory elements that regulate translational efficiency. The other type, type II, contains large portions or almost entire sequences of rRNA with a cap at the 5' end and poly(A) at 3' end. Recent work has shown that some type II PART have functional significance for some neurodegenerative disease processes and may play an important role in the pathogenesis of diseases. Further investigation in this area is critical to understanding the basic biology of PART and the potential role of PART in diseases.

Desertification is one of the most serious environment and socioeconomic problems in the world and China today. Land of sandy desertification almost covers everywhere in Ejin Banner (County), Inner Mongolia. Using remote sensing method can expediently acquire some information such as desertification’s status, and effect of harnessing desertification. In this paper, ecological change situation from 1986 to 2000 in Ejina Oasis is researched. The result is followed: (1) Area of water body of Lakes in Northeastern Ejin Banner are distinctly reduced. (2) Area of the oasis is rapidly shrunk. From 1986 to 2000, the area is reduced 61.48%, average 4.39% annually. (3) Condition of oasis growing is declined. The oasis’s ecological environment trends to bad. Its main reason is what human activities exceeded the load capacity of natural resources such as water and land. By means of self-restorative ability of natural vegetation and human’s scientific conservation, that the oasis’s ecosystem maintains dynamical balance and the environment recovers is possible.

Based on radar remote sensing and sediment facies analyzing, this paper studies the features of environmental evolution of North-eastern Ejin Banner since 60 Ka BP(Before Present). The conclusions are listed as follows: (1) The evolution of Gaxunnur Lake, Sugunur Lake, Tiane Lake is dominated by faults and regional climate; (2) By analyzing deposit of old Juyanze Lake, it is a large outlet lake about 5OKa BP. Just about 5OKa BP, there was a rapid decline of temperature in the Northwestern of China. The events caused those lakes shrinkage. (3) By fault activity arising uplift in the north of old Juyan Lake and depression in the south, the lake's water flowed out from north to south at around 35Ka BP, which is reversed to the former flow direction. So, old Juyanze fluvial fan was formed. At the same time, Juyan Lake separated from Sugunur lake and Wentugunr old channel was abandoned. (4) Near 2000 years , Ruoshui River is a wandering river. Due to intense influence of human activities, the oasis ecosystem is rapidly degenerated in recently 15 years.

Background: Accumulating evidence indicates that RNA oxidation is involved in a wide variety of neurological diseases and may be associated with neuronal deterioration during the process of neurodegeneration. However, previous studies were done in postmortem tissues or cultured neurons. Here, we used transgenic mice to demonstrate the role of RNA oxidation in the process of neurodegeneration. Methodology/Principal Findings: We demonstrated that messenger RNA (mRNA) oxidation is a common feature in amyotrophic lateral sclerosis (ALS) patients as well as in many different transgenic mice expressing familial ALS-linked mutant copper-zinc superoxide dismutase (SOD1). In mutant SOD1 mice, increased mRNA oxidation primarily occurs in the motor neurons and oligodendrocytes of the spinal cord at an early, pre-symptomatic stage. Identification of oxidized mRNA species revealed that some species are more vulnerable to oxidative damage, and importantly, many oxidized mRNA species have been implicated in the pathogenesis of ALS. Oxidative modification of mRNA causes reduced protein expression. Reduced mRNA oxidation by vitamin E restores protein expression and partially protects motor neurons. Conclusion/Significance: These findings suggest that mRNA oxidation is an early event associated with motor neuron deterioration in ALS, and may be also a common early event preceding neuron degeneration in other neurological diseases.