Fragile X premutation rCGG repeats cause the nuclear accumulation of Hsp70 mRNA.

<p>A. Quantitative analysis of <i>Hsp70</i> mRNA levels by real-time PCR from the adult heads of genotypes: <i>+/+</i> (wild-type (WT)); <i>elav; rCGG₆₀</i> (rCGG-expressing homozygous flies); <i>elav/+; rCGG₆₀/+</i> (rCGG-heterozygous flies); <i>Rm62^LOF/+</i> (Rm62 mutation heterozygous flies); and <i>rCGG₆₀/+; Rm62^LOF/+</i> (interaction). Housekeeping ribosomal protein 32 (<i>Rpl32</i>) mRNA was used as an internal control. *: p<0.05 B. Western blot with anti-histone 3 antibody, and α tubulin. C. Quantitative analysis of <i>Hsp70</i> mRNA levels by real-time PCR on cytoplasmic and nuclear RNA fractions obtained from adult heads of wild-type (WT) and rCGG-expressing flies. <i>Rpl32</i> mRNA was used as control. D. Quantitative analysis of <i>Hsp70</i> mRNA levels in total RNA fractions upon heat shock. Both wild-type (WT) and rCGG-expressing flies were heat shocked for 30 min. No heat shock (NHS) represents non-heat shocked controls. Flies were decapitated at the indicated time after heat shock. Heads were collected and total RNA isolated from them. Both WT and rCGG-expressing flies displayed robust expression of <i>Hsp70</i> in response to heat shock. After the removal of heat shock, <i>Hsp70</i> transcripts declined radically in the WT, whereas in rCGG-expressing flies, <i>Hsp70</i> transcripts show prolonged accumulation. Control samples do not display any overt differences in the timing or expression levels of <i>Hsp70</i> during the initial response to a short heat shock. Real time against <i>Fmr1</i> serves as a control on fractionated samples. The data represent mean ± SEM, n = 3.</p

Pur α Interactome.

<p>Pur α Interactome.</p

Mutant Alleles Corresponding to Pur α-Interacting Proteins Used for Genetic Screen.

<p><b><i>—</i></b>: Very Mild or No effect.</p

Identification via microarray analyses of selective mRNAs that accumulate in the nucleus as a result of fragile X premutation rCGG repeats.

<p>A. The three sets of microarray experiments were carried out in triplicate (three biological replicates) with equal amounts of total RNA obtained from the <i>Drosophila</i> heads of control total, nuclear, and cytoplasmic fractions and the corresponding experimental premutation rCGG repeat samples of total, nuclear, and cytoplasmic fractions. Microarray analyses were carried out by identifying significantly changed genes at the 0.001 level of the univariate test. B. Scatter plot of mean log intensities of each sample demonstrating differentially expressed significant genes with fold-change of 2 or more between the classes within a particular set. C. The numbers of unique differentially expressed genes generated by the different cellular compartments of rCGG sample (comparison of total, cytoplasmic, and nuclear fraction) are displayed in Venn diagrams. D. Fold enrichment depicted by ratios between the intensities of normalized log-transformed gene expressions for 45 genes unique to CGG nuclear fractions in various classes is displayed using the Cluster and TreeView programs for the wild-type and premutation rCGG datasets. The fold of the change is indicated on both sides of the scale bar E. Validation of nuclear enrichment by real-time PCR analysis of the selective genes. Real time against <i>Fmr1</i> serves as a control. The data represent mean ± SEM, n = 3.</p

Identification of Pur α-interacting proteins.

<p>A. Silver-staining gel with distinct bands for recombinant proteins and affinity-purified proteins. The captured proteins were further analyzed by mass spectrometry, and distinct classes of proteins were identified. B. <i>Rm62</i> and <i>Hts</i> mutants enhance rCGG-mediated neurodegeneration in fly. Column 1: wild-type fly; Column 2: fly expressing (CGG)₉₀-EGFP only; Column 3: fly expressing (CGG)₉₀-EGFP in the heterozygous background of <i>Hts</i>⁰¹¹⁰³ Loss-of-Function (LOF) mutation; Column 4: fly expressing CGG₉₀-EGFP in the heterozygous background of <i>Rm62</i>⁰¹⁰⁸⁴ Loss-of-Function (LOF) mutation; Column 5: fly expressing CGG₉₀-EGFP in the heterozygous background of <i>Rm62</i>^(3)3607 overexpression (Gain-of-Function (GOF). SEM eye images are shown.</p

Rm62 and Hts directly interact with Pur α and Fragile X premutation rCGG repeats decrease the expression of Rm62 posttranscriptionally.

<p>A. Pull-down assay between GST-tagged dPur α and <i>in vitro</i>-translated Rm62 or <i>in vitro</i>-translated Hts or luciferase (negative control). Indicated are samples treated or untreated with RNase prior to binding reaction. In the Input lanes, we loaded 25% of the translation products used in a reaction. Both Rm62 and Hts, but not luciferase, interacts with dPur α. B. Western blot shows the interaction between endogenous Rm62 or endogenous Hts and affinity-purified GST-Pur α. Indicated are samples treated or untreated with RNase prior to binding reaction. C. Western blot shows the interaction between affinity-purified GST-Rm62 and endogenous Pur α in RNase untreated and treated samples. D. Western blot shows the interaction between endogenous mammalian p68 and affinity-purified fly GST-Pur α. E. Protein levels of Rm62 and Hts in wild-type and rCGG-expressing flies. Quantitative analysis of Rm62 and Hts protein levels by Western blot on adult head extracts of the following genotypes: wild-type (WT); <i>elav</i>; rCGG₆₀-expressing flies. Proteins are indicated to the right, corresponding molecular weights to the left. α actin represents a loading control. F. Quantitative analysis of <i>Rm62</i> mRNA levels by real-time PCR on total RNA obtained from adult heads of wild-type (WT) and rCGG-expressing flies. Quantification is relative to the housekeeping ribosomal protein 32 (<i>Rpl32</i>) mRNA. (E; mean ± SEM n = 3). G. Statistical evaluation of the percent viability displayed by various genotypes: <i>elav/+; +/+; Rm62^LOF/+</i> (Rm62 heterozygous); <i>elav/+; rCGG₆₀-EGFP/+; TM3Sb/+</i> (Premutation heterozygous), <i>elav/+; +/+; TM3Sb/+</i> (Internal control); <i>elav/+; rCGG₆₀-EGFP/+; Rm62^LOF/+</i> (interaction). Mean of three data sets was used.</p

Fragile X premutation rCGG repeats display genetic interaction with the nuclear export factor, small bristles, and the expression of molecular chaperone <i>Hsp70</i> suppresses rCGG repeat-mediated neurodegeneration <i>in vivo</i>.

<p>A. Statistical evaluation of the percent viability displayed by Sbr homozygous and heterozygous flies or premutation heterozygous flies, and their respective interaction experiments <i>Sbr12^LOF/elav; rCGG₆₀-EGFP/+.</i> Genotypes: <i>+/+</i> (wild-type); <i>Sbr12^LOF/Sbr12^LOF</i>; <i>Sbr12^LOF/+</i>; <i>elav/+; rCGG₆₀-EGFP/+</i> and <i>Sbr12^LOF/elav; rCGG₆₀-EGFP/+</i> (interaction). Mean of three data sets was used. Error bars indicate SEM. **: p<0.001. B. Shown are SEM pictures of the eyes of adult flies expressing gmr: (CGG)₉₀-EGFP/+ only (left), in comparison to <i>Gmr:</i> (CGG)₉₀-EGFP/+; <i>Hsp70^GOF</i>/+. C. Model representing various interactions involving fragile X premutation rCGG repeats and its potential impact on mRNA nuclear export.</p

Image_1_Adherence to the Dietary Approaches to Stop Hypertension diet reduces the risk of breast cancer: A systematic review and meta-analysis.TIF

BackgroundDespite increasing evidence for the association of adherence to the Dietary approaches to stop hypertension (DASH) diet with breast cancer risk, the results remain inconclusive. The purpose of the current systematic review was to summarize the evidence from previous observational studies and explore the potential association between DASH diet and breast cancer risk using meta-analysis.MethodsA comprehensive literature search was conducted using the databases of PubMed, Web of Science, CNKI and Wanfang Data to identify the relevant publications from inception up to July 2022. The pooled relative risks (RRs) and 95% confidence intervals (CIs) were calculated for the highest versus the lowest categories of DASH score in relation to breast cancer risk, using a random-effects model. The Cochran’s Q test and I-squared (I2) statistic were used to detect the sources of heterogeneity among the included studies.ResultsOverall, eleven studies, involving 23,254 breast cancer cases and 449,273 participants, were included in this systematic review and meta-analysis. Combining 16 effect sizes from 11 studies, a significant inverse association between adherence to the DASH diet and risk of breast cancer was observed (RR = 0.79; 95% CI: 0.70, 0.90, P ConclusionThe results of this systematic review and meta-analysis indicate a significant inverse association between adherence to the DASH diet and risk of breast cancer. Further large prospective studies and randomized controlled trials are required to confirm our findings.</p

Galectin-3 Is a Candidate Biomarker for Amyotrophic Lateral Sclerosis: Discovery by a Proteomics Approach

The discovery of biomarkers for neurodegenerative diseases will have a major impact on the efficiency of therapeutic clinical trials and may be important for understanding basic pathogenic mechanisms. We have approached the discovery of protein biomarkers for amyotrophic lateral sclerosis (ALS) by profiling affected tissues in a relevant animal model and then validating the findings in human tissues. Ventral roots from SOD1G93A “ALS” mice were analyzed by label-free quantitative mass spectrometry, and the resulting data were compared with data for matched samples from nontransgenic littermates and transgenic mice carrying wild-type human SOD1 (SOD1WT). Of 1299 proteins, statistical inference of the data in the three groups identified 14 proteins that were dramatically altered in the ALS mice compared with the two control groups. The protein galectin-3 emerged as a lead biomarker candidate on the basis of its differential expression as assessed by immunoblot and immunocytochemistry in SOD1G93A mice as compared to controls and because it is a secreted protein that could potentially be measured in human biofluids. Spinal cord tissue from ALS patients also exhibited increased levels of galectin-3 when compared to controls. Further measurement of galectin-3 in cerebrospinal fluid samples showed that ALS patients had approximately twice as much galectin-3 as normal and disease controls. These results provide the proof of principle that biomarker identification in relevant and well-controlled animal models can be translated to human disease. The challenge is to validate our biomarker candidate proteins as true biomarkers for ALS that will be useful for diagnosis and/or monitoring disease activity in future clinical trials

Table_1_Adherence to the Dietary Approaches to Stop Hypertension diet reduces the risk of breast cancer: A systematic review and meta-analysis.doc

BackgroundDespite increasing evidence for the association of adherence to the Dietary approaches to stop hypertension (DASH) diet with breast cancer risk, the results remain inconclusive. The purpose of the current systematic review was to summarize the evidence from previous observational studies and explore the potential association between DASH diet and breast cancer risk using meta-analysis.MethodsA comprehensive literature search was conducted using the databases of PubMed, Web of Science, CNKI and Wanfang Data to identify the relevant publications from inception up to July 2022. The pooled relative risks (RRs) and 95% confidence intervals (CIs) were calculated for the highest versus the lowest categories of DASH score in relation to breast cancer risk, using a random-effects model. The Cochran’s Q test and I-squared (I2) statistic were used to detect the sources of heterogeneity among the included studies.ResultsOverall, eleven studies, involving 23,254 breast cancer cases and 449,273 participants, were included in this systematic review and meta-analysis. Combining 16 effect sizes from 11 studies, a significant inverse association between adherence to the DASH diet and risk of breast cancer was observed (RR = 0.79; 95% CI: 0.70, 0.90, P ConclusionThe results of this systematic review and meta-analysis indicate a significant inverse association between adherence to the DASH diet and risk of breast cancer. Further large prospective studies and randomized controlled trials are required to confirm our findings.</p