Transcriptional modulator H2A histone family, member Y (H2AFY) marks Huntington disease activity in man and mouse.

Huntington disease (HD) is a progressive neurodegenerative disease that affects 30,000 individuals in North America. Treatments that slow its relentless course are not yet available, and biomarkers that can reliably measure disease activity and therapeutic response are urgently needed to facilitate their development. Here, we interrogated 119 human blood samples for transcripts associated with HD. We found that the dynamic regulator of chromatin plasticity H2A histone family, member Y (H2AFY) is specifically overexpressed in the blood and frontal cortex of patients with HD compared with controls. This association precedes the onset of clinical symptoms, was confirmed in two mouse models, and was independently replicated in cross-sectional and longitudinal clinical studies comprising 142 participants. A histone deacetylase inhibitor that suppresses neurodegeneration in animal models reduces H2AFY levels in a randomized phase II clinical trial. This study identifies the chromatin regulator H2AFY as a potential biomarker associated with disease activity and pharmacodynamic response that may become useful for enabling disease-modifying therapeutics for HD.

Development of a gold nanoparticle-based lateral flow immunoassay for the fast detection of diafenthiuron in cabbage and apples.

Due to its unique insecticidal and acaricidal mechanism of action, and ability to mix with most insecticides and fungicides, diafenthiuron (DIAF) is widely used in the cultivation of fruits and vegetables. However, this insecticide can cause unacceptable harm to organisms, making the detection of DIAF residues in fruits and vegetables crucial. In this study, a novel hapten based on the structure of DIAF was utilized to prepare a monoclonal antibody (mAb) with high specificity and sensitivity. The half maximum inhibitory concentration (IC50) of the anti-DIAF mAb was 20.96 µg kg-1 as determined by ic-ELISA and little cross-reactivity with other analogues. Next, a GNP-based lateral flow immunoassay (LFIA) was developed to detect DIAF in cabbages and apples. The optimized LFIA, for cabbage samples, showed a visual limit of detection (vLOD), cut-off value and calculated limit of detection (cLOD) of 0.1 mg kg-1, 10 mg kg-1 and 1.5 µg kg-1, respectively, and for apples 0.1 mg kg-1, 5 mg kg-1 and 3.4 µg kg-1, respectively. Recovery rates in cabbage and apples were 89.4-105.0% and 105.3-112.0%, with a coefficient of variation of 2.73-5.71% and 2.15-7.56%, respectively. These results indicated that the established LFIA based on our anti-DIAF mAb was a reliable method for in situ rapid detection of DIAF in cabbage and apple samples.

A gold nanoparticle based colorimetric sensor for the rapid detection of Yersinia enterocolitica serotype O:8 in food samples.

Foodborne diseases caused by Yersinia enterocolitica serotype O:8 represent global public health problems. We need rapid Y. enterocolitica O:8 detection methods to ensure food safety. In this study, we developed a colloidal gold-based immunochromatographic strip test (ICST) for the rapid detection of Y. enterocolitica O:8. A sandwich detection format was applied to Y. enterocolitica O:8 detection, where a monoclonal antibody (mAb) labeled with colloidal gold and mAb immobilized on the test line were used as the capture antibody and detection antibody, respectively. The limit of detection of the colloidal gold ICST was 1.3 × 103, 3.0 × 102 and 8.0 × 102 CFU mL-1 for Y. enterocolitica O:8 CICC 21669, CICC 21681 and CICC 21567, respectively. The method developed by us had no cross-reactivity with other foodborne pathogens. Using ICST, we detected Y. enterocolitica O:8 strains at a low level (5 CFU mL-1) in milk and pork samples after 4 h and 6 h of separate incubation. The results were obtained within 10 min without sophisticated instruments. Therefore, the colloidal gold ICST is an accurate and sensitive method for the detection of Y. enterocolitica O:8 in food samples.

Association of SNCA with Parkinson: replication in the Harvard NeuroDiscovery Center Biomarker Study.

BACKGROUND: Mutations in the α-synuclein gene (SNCA) cause autosomal dominant forms of Parkinson's disease, but the substantial risk conferred by this locus to the common sporadic disease has only recently emerged from genome-wide association studies. METHODS: We genotyped a prioritized noncoding variant in SNCA intron 4 in 344 patients with Parkinson's disease and 275 controls from the longitudinal Harvard NeuroDiscovery Center Biomarker Study. RESULTS: The common minor allele of rs2736990 was associated with elevated disease susceptibility (odds ratio, 1.40; P = .0032). CONCLUSIONS: This result increases confidence in the notion that in many clinically well-characterized patients, genetic variation in SNCA contributes to "sporadic" disease.

Sensitive detection of RNA based on concatenated self-fuelled strand displacement amplification and hairpin-AgNCs.

Herein, a self-fuelled amplification strategy (SFAS) is proposed, in which two strand displacement amplification (SDA) processes were concatenated for the proliferation of ssDNA. The ssDNA then initiated a polymerase action and caused the destruction of hairpin-templated silver nanoclusters (AgNCs), resulting in decreased fluorescence for sensing miRNA-21. This SFAS-based sensor is less complicated in design and facile in operation, because of the easy concatenation of SDA and mutual enzymes used in the signal output process. The sensitivity of this SFAS-based miRNA sensor was 1.78 × 10-11 M with a linear relationship in the range 0.02-1.0 × 10-9 M, and the recoveries of this method ranged from 82.07% to 106.58% with an average RSD of 10.96%.

Gold nanoparticle-based immunochromatographic assay for detection Pseudomonas aeruginosa in water and food samples.

Pseudomonas aeruginosa (P. aeruginosa) is the common infection-causing bacterial pathogen. Conventional methods for the detection of P. aeruginosa are time-consuming, and therefore, a more rapid analytical method is required. Here, monoclonal antibodies (Mabs) against P. aeruginosa (CICC 10419) were prepared and based on paired Mabs, an immunochromatographic assay (ICA) was developed. The ICA strip showed a limit of detection of 2.41 × 104 CFU/mL and the linear range of detection was 3.13 × 104-1.0 × 106 CFU/mL. No cross-reactivity was observed when other common Gram-negative and Gram-positive bacteria were used. The analytical performance of the ICA strip indicated that the developed ICA had good specificity and stability. Moreover, the feasibility of the ICA strip was verified by detecting P. aeruginosa (CICC 10419) in spiked water and food samples. The ICA strip could detect samples contaminated with a low-level of P. aeruginosa (CICC 10419) after 8 h enrichment.

An immunochromatographic sensor for ultrasensitive and direct detection of histamine in fish.

To ensure food quality and prevent histamine (HA) toxicity, a rapid and direct method of detecting HA is required. In this work, we prepared a monoclonal antibody (mAb) against HA using a hapten produced by the introduction of a phenyl-containing linker. The novel mAb exhibited high sensitivity against HA as determined by ELISA, with a half-maximal inhibitory concentration of 21.51 ng/mL. A gold nanoparticle-based immunosensor was fabricated for rapid detection of HA in fish samples. After optimizing the immunosensor, a visual limit of detection (LOD) and a calculated LOD were 0.25 mg/kg and 10.48 µg/kg for HA, respectively. Recovery rates from the spiked fish samples ranged from 87.33% to 104.67% with the coefficient of variation below 10.82%. Concurrently, the whole process in testing real sample was completed within 15 min, and all results were well confirmed and comparable by liquid chromatography-mass spectrometry and the commercial test strip. These data revealed that the proposed immunosensor could be used as a monitoring tool for the rapid and direct detection of HA in fish samples.

Changes of procedural learning in Chinese patients with non-demented Parkinson disease.

To study procedural learning changes in patients with non-demented Parkinson disease (PD) but without depression. The Nissen serial reaction time task (SRTT) software version II (as a task of procedural learning), the Wechsler Memory Scale-Chinese version (WMS-CR), and two tasks of implicit memory were applied to 20 PD patients with a Hoehn-Yahr score at I-II degrees and 20 matched healthy controls were enrolled for the Nissen Version test. In the explicit WMS-CR and the implicit (word stem completion and degraded picture naming) tasks, the patients' scores fell within normal limits. In the SRTT, healthy controls displayed significantly reduced response times and error rates across the blocks of repeated sequence trials. In contrast, PD patients only showed a reduction in error rates but no change in response times. Impairment of nigrostriatal pathways selectively affects the performance in visuo-motor learning tasks such as the SRTT, but not in both the explicit tasks of WMS-CR and the implicit tasks.

Designing siRNA that distinguish between genes that differ by a single nucleotide.

Small interfering RNAs (siRNAs), the guides that direct RNA interference (RNAi), provide a powerful tool to reduce the expression of a single gene in human cells. Ideally, dominant, gain-of-function human diseases could be treated using siRNAs that specifically silence the mutant disease allele, while leaving expression of the wild-type allele unperturbed. Previous reports suggest that siRNAs can be designed with single nucleotide specificity, but no rational basis for the design of siRNAs with single nucleotide discrimination has been proposed. We systematically identified siRNAs that discriminate between the wild-type and mutant alleles of two disease genes: the human Cu, Zn superoxide dismutase (SOD1) gene, which contributes to the progression of hereditary amyotrophic lateral sclerosis through the gain of a toxic property, and the huntingtin (HTT) gene, which causes Huntington disease when its CAG-repeat region expands beyond approximately 35 repeats. Using cell-free RNAi reactions in Drosophila embryo lysate and reporter assays and microarray analysis of off-target effects in cultured human cells, we identified positions within an siRNA that are most sensitive to mismatches. We also show that purine:purine mismatches imbue an siRNA with greater discriminatory power than other types of base mismatches. siRNAs in which either a G:U wobble or a mismatch is located in the "seed" sequence, the specialized siRNA guide region responsible for target binding, displayed lower levels of selectivity than those in which the mismatch was located 3' to the seed; this region of an siRNA is critical for target cleavage but not siRNA binding. Our data suggest that siRNAs can be designed to discriminate between the wild-type and mutant alleles of many genes that differ by just a single nucleotide.

Synergistical accumulation for electrochemical sensing of 1-hydroxypyrene on electroreduced graphene oxide electrode.

A graphene modified glassy carbon electrode was fabricated for sensitive and convenient electrochemical detection of 1-hydroxypyrene (1-OHP). The graphene modified electrode was prepared by eletrochemical reduction of graphene oxide on glass carbon electrode. l-OHP was synergistically accumulated on the graphene modified electrode via π-π conjugate adsorption and electrochemical oxidation. Electrochemical behavior of l-OHP on the graphene modified glass carbon electrode was studied in detail. The developed method gave a linear calibration curve for the determination of 1-OHP in the range of 5-300 nM (R2 = 0.997), a limit of detection of 0.84 nM (S/N = 3). The proposed method was also successfully applied to the analysis of urine samples for l-OHP with the recoveries of 97.3-101.1%.

Is coronary artery disease a multifactorial inherited disorder with a sex-influenced trait?

The genetic linkage of coronary artery disease is well-established. However, the transmission of this disease is not clearly defined. Although the Mendelian autosomal dominant pattern has been seen in familial hypercholesterolemia and mutant MEF2A induced familiar myocardial infarction, and a multifactorial genetic model has been proposed for non-familial CAD, the gender difference in this disease is not well explained. We hypothesized that CAD is a multifactorial inherited disorder with a sex-influenced trait, which shows an autosomal dominant pattern in men and autosomal recessive transmission in women. This hypothesis is supported by the facts including an age-dependent higher prevalence in men, the autosomal locations of CAD associated genes, the gender difference seen even in familiar CAD, and the potential gene-gene interactions between CAD associated genes on autosomal chromosomes and those found on the X chromosome. Further investigation of genetic components will provide not only the critical information about the etiology of CAD, but also help to clarify the confusion in the use of exogenous female hormones in the prevention and/or the treatment of the disease.

Bone health in postmenopausal women with early breast cancer: how protective is tamoxifen?

Breast cancer is a leading threat to women's health. Tamoxifen, the most successful selective estrogen receptor modulator, has been used in hormonal therapy for three decades. Along with its therapeutic effect on breast cancer, tamoxifen also demonstrates potential benefits for bone health. However, the extent and quality of such benefits have not been systematically evaluated. We conducted a comprehensive literature search and identified 27 peer-reviewed articles investigating the relationship between tamoxifen and bone health in postmenopausal women with early stage breast cancer. The majority of studies reported that tamoxifen therapy alone protected against the loss of spinal bone mineral density. The bones in the hip also benefited from tamoxifen treatment while there was no evidence demonstrating tamoxifen's protection against bone loss in arms. When tamoxifen was combined with chemotherapy, it was found to partially prevent or reverse the bone loss resulting from chemotherapy. Patients with a history of hormone replacement therapy experienced bone loss while patients without the history had increased bone mineral density during tamoxifen therapy. Despite an apparent impact of tamoxifen on bone mineral density, the few available studies of tamoxifen and bone fractures appear to suggest no protective effect but an increase in fracture incidence. More investigation is necessary to clarify the discrepancy between bone mineral density and fracture in postmenopausal breast cancer patients treated with tamoxifen.

Radiolabeling small RNA with technetium-99m for visualizing cellular delivery and mouse biodistribution.

To develop a noninvasive direct method for the in vivo tracking of small interfering RNA (siRNA) used in RNA interference, two 18-nucleotide oligoribonucleotides were radiolabeled with technetium-99m ((99m)Tc-RNA). The ability of (99m)Tc-RNA to track delivery was tested in cultured cells and living mice. The cellular delivery of (99m)Tc-RNAs could be quantified by gamma counting and could be visualized by microautoradiography. Radiolabeled RNAs can be efficiently delivered into cells by reaching up to 3x10(5) molecules of small RNAs per cell. Moreover, RNAs were internalized with homogeneous distribution throughout the cytoplasm and nucleus. In tumor-bearing mice, whole-body images and biodistribution studies showed that (99m)Tc-RNAs were delivered to almost all tissues after intravenous injection. The imaging of living animals allowed noninvasive and longitudinal monitoring of the in vivo delivery of these small RNAs. In conclusion, using (99m)Tc radiolabeling, the delivery of small RNAs could be measured quantitatively in cultured cells and could be noninvasively visualized in living animals using a gamma camera. The results of this study could open up a new approach for measuring the in vivo delivery of small RNAs that might further facilitate the development of siRNAs as targeted therapies.

Initial mechanistic studies of antisense targeting in cells.

UNLABELLED: The continued development of antisense targeting will require a better understanding of the mechanism. METHODS: We performed initial studies of the mechanism of intracellular antisense targeting through measurements of in situ transcription, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), 32P-labeled uridine-5'-triphosphate (alpha-32P-UTP) incorporation, nuclear accumulations of 99mTc-labeled DNAs, and messenger RNA (mRNA) transcription rate. As reported earlier, an antisense DNA against the mdr1 mRNA coding for P-glycoprotein (Pgp) and its sense DNA control were used in KB-G2 (Pgp++) cells. RESULTS: Definitive evidence for antisense targeting was obtained by in situ transcription showing complementary DNA elongation in cells exposed to antisense DNA, acting therefore as an intracellular PCR primer of mdr1 mRNA, but not in cells exposed to sense DNA. Immunofluorescence staining showed higher accumulations of antisense versus sense DNAs in KB-G2 cells. Transnuclear migration was confirmed by higher accumulations in the nucleus compared with the cytoplasm in cells incubated with 99mTc-labeled antisense DNA. However, the observed specific accumulations of antisense DNAs of about 10(6) per cell over 10 h could not be explained by a feedback mechanism upregulating transcription in cells exposed to antisense DNA as no increase in mRNA levels was detected by both RT-PCR and 32P-UTP in these cells. To explore an alternative hypothesis, a novel approach using 99mTc-labeled antisense DNA as a probe of total mRNA from cells previously saturated with unlabeled antisense DNA was used to estimate the transcription rate. Compared with controls, mdr1 mRNA levels were found to be initially low after saturation and to recover at about 2,000 copies per minute per cell. If persistent, this transcription rate would provide 10(6) mRNAs in 10 h. CONCLUSION: The results of all studies are consistent with antisense as the mechanism of targeting. Though a feedback mechanism leading to upregulation of mRNA transcription is an unlikely explanation for the high specific accumulations, our results may be explained if antisense DNAs are targeting mdr1 mRNAs produced at high transcription rates. If the target is primarily pre-mRNA in the nucleus rather than mature mRNA in the cytoplasm, this would provide as well an explanation for the observed migration of 99mTc-labeled antisense DNA into the nucleus.

An enhanced U6 promoter for synthesis of short hairpin RNA.

Short hairpin RNAs (shRNAs) transcribed by RNA polymerase III (Pol III) promoters can trigger sequence-selective gene silencing in culture and in vivo and, therefore, may be developed to treat diseases caused by dominant, gain-of-function type of gene mutations. These diseases develop in people bearing one mutant and one wild-type gene allele. While the mutant is toxic, the wild-type performs important functions. Thus, the ideal therapy must selectively silence the mutant but maintain the wild-type expression. To achieve this goal, we designed an shRNA that selectively silenced a mutant Cu,Zn superoxide dismutase (SOD1(G93A)) allele that causes amyotrophic lateral sclerosis. However, the efficacy of this shRNA was relatively modest. Since the allele-specific shRNA has to target the mutation site, we could not scan other regions of SOD1 mRNA to find the best silencer. To overcome this problem, we sought to increase the dose of this shRNA by enhancing the Pol III promoter. Here we demonstrate that the enhancer from the cytomegalovirus immediate-early promoter can enhance the U6 promoter activity, the synthesis of shRNA and the efficacy of RNA interference (RNAi). Thus, this enhanced U6 promoter is useful where limited choices of shRNA sequences preclude the selection of a highly efficient RNAi target region.

Assessing the Public Health Impact and Effectiveness of Interventions To Prevent Salmonella Contamination of Sprouts.

Sprouts have been a recurring public health challenge due to microbiological contamination, and Salmonella has been the major cause of sprout-associated outbreaks. Although seed treatment and microbiological testing have been applied as risk reduction measures during sprout production, the extent to which their effectiveness in reducing the public health risks associated with sprouts has not been well investigated. We conducted a quantitative risk assessment to measure the risk posed by Salmonella contamination in sprouts and to determine whether and how mitigation strategies can achieve a satisfactory risk reduction based on the assumption that the risk reduction achieved by a microbiological sampling and testing program at a given sensitivity is equivalent to that achieved by direct inactivation of pathogens. Our results indicated that if the sprouts were produced without any risk interventions, the health impact caused by sprouts contaminated with Salmonella would be very high, with a median annual estimated loss of disability-adjusted life years (DALYs) of 691,412. Seed treatment (with 20,000 ppm of calcium hypochlorite) or microbiological sampling and testing of spent irrigation water (SIW) alone could reduce the median annual impact to 734 or 4,856 DALYs, respectively. Combining seed treatment with testing of the SIW would further decrease the risk to 58 DALYs. This number could be dramatically lowered to 3.99 DALYs if sprouts were produced under conditions that included treating seeds with 20,000 ppm of calcium hypochlorite plus microbiological testing of seeds, SIW, and finished products. Our analysis shows that the public health impact due to Salmonella contamination in sprouts could be controlled if seeds are treated to reduce pathogens and microbiological sampling and testing is implemented. Future advances in intervention strategies would be important to improve sprout safety further.

Mutant Cu, Zn superoxide dismutase that causes motoneuron degeneration is present in mitochondria in the CNS.

Mutations in Cu, Zn superoxide dismutase (SOD1) cause a fraction of amyotrophic lateral sclerosis (ALS), which involves motoneuron degeneration, paralysis, and death. An acquired activity by mutant SOD1 is responsible for the cellular toxicity, but how mutant SOD1 kills motoneurons is unclear. In transgenic mouse models of ALS, mitochondrial degeneration occurs early, before disease onset, raising the question of how mutant SOD1 damages mitochondria. Here we investigate the intracellular localization of SOD1 in the CNS to determine whether SOD1 is present in mitochondria, where it could directly damage this organelle. We show that endogenous mouse SOD1, wild-type human, and mutant human SOD1 (G93A), when expressed as transgenes, are colocalized with mitochondria in spinal cord by immunofluorescence confocal microscopy. By immunoelectron microscopy, we show that SOD1 is present within mitochondria at similar concentrations as in the cytoplasm. Thus SOD1, in addition to being a cytosolic enzyme, is present inside mitochondria in the CNS.

Selective silencing by RNAi of a dominant allele that causes amyotrophic lateral sclerosis.

RNA interference (RNAi) can achieve sequence-selective inactivation of gene expression in a wide variety of eukaryotes by introducing double-stranded RNA corresponding to the target gene. Here we explore the potential of RNAi as a therapy for amyotrophic lateral sclerosis (ALS) caused by mutations in the Cu, Zn superoxide dismutase (SOD1) gene. Although the mutant SOD1 is toxic, the wild-type SOD1 performs important functions. Therefore, the ideal therapeutic strategy should be to selectively inhibit the mutant, but not the wild-type SOD1 expression. Because most SOD1 mutations are single nucleotide changes, to selectively silence the mutant requires single-nucleotide specificity. By coupling rational design of small interfering RNAs (siRNAs) with their validation in RNAi reactions in vitro and in vivo, we have identified siRNA sequences with this specificity. A similarly designed sequence, when expressed as small hairpin RNA (shRNA) under the control of an RNA polymerase III (pol III) promoter, retains the single-nucleotide specificity. Thus, RNAi is a promising therapy for ALS and other disorders caused by dominant, gain-of-function gene mutations.

Behavior of Shiga toxigenic Escherichia coli relevant to lettuce washing processes and consideration of factors for evaluating washing process surrogates.

Postharvest processes for fresh produce commonly include washing in water containing antimicrobial chemicals, such as chlorine; however, if the antimicrobials are not present in sufficient levels, washing can promote the spread of contamination that might be present. To understand cross-contamination risk during washing, we tested a collection of Shiga toxigenic Escherichia coli (STEC), including O157:H7 and other non-O157 strains, for certain traits during washing of fresh-cut lettuce, i.e., sensitivity to sublethal chlorine levels and ability to cross-contaminate (detach from and attach to) lettuce in the presence of sublethal chlorine levels. Nonpathogenic E. coli Nissle 1917 (EcN) and Pediococcus pentosaceus lactic acid bacterial species (LAB) were included as potential washing process validation surrogates. As measured by extension of the lag phase of growth in media containing 0.15 ppm of chlorine, chlorine sensitivity varied among the STECs. Cross-contamination was assessed by evaluating transfer of bacteria from inoculated to uninoculated leaves during washing. Without chlorine, similar transfer to wash water and uninoculated leaves was shown. In 1 ppm of chlorine, cross-contamination was not detected with most strains, except for the substantial transfer by a STEC O111 strain and EcN in some replicates. Strain O111 and EcN showed less inactivation in 0.25 ppm of chlorine water compared with O157 (P < 0.05). LAB showed similar transfer and similar chlorine inactivation to O157. Considering together the sublethal chlorine sensitivity and detachment/attachment traits, neither EcN nor LAB displayed optimal characteristics as washing process surrogates for the STEC strains, although further evaluation is needed. This work demonstrated a range of behaviors of STEC strains during lettuce washing and may be helpful in hazard characterization, identifying factors to consider for evaluating washing process efficacy, and identifying phenotypic traits to select surrogates to validate washing processes.

Microbial contamination in sprouts: how effective is seed disinfection treatment?

Microbial contamination of sprouts by Salmonella and Escherichia coli O157 : H7 has been a common cause of foodborne diseases and a continuing challenge to the sprout industry. Seed disinfection treatment has been recommended as a major intervention step in a multihurdle approach to reduce the risk of illness associated with contaminated sprouts. U.S. Food and Drug Administration cited 20000 ppm calcium hypochlorite as an example treatment in its recommendation for seed treatment and this treatment has been considered the reference standard for seed disinfection treatment for over a decade. However, promising new disinfection treatments have emerged in recent years. In this study, we summarized published data and compared the efficacies of different disinfection methods in the reduction of microbial contamination on seeds. Our findings suggest that while biological interventions such as competitive exclusion and certain chemical treatments appear to be similar to 20000 ppm calcium hypochlorite for seed disinfection, physical methods especially high pressure may be more effective than the reference standard regardless of the type of bacteria or seed. The combination of 2 or more treatments, sequentially or simultaneously, may further improve disinfection results. Since treatments with high levels of chemical disinfectants, especially 20000 ppm calcium hypochlorite, can pose environmental and worker safety risks, alternative intervention approaches should be considered. Additional studies to confirm the greater efficacy of certain physical and combined seed disinfection treatments and to identify other effective management strategies are needed to further improve sprout safety.