A fluorescence-microscopic and cytofluorometric system for monitoring the turnover of the autophagic substrate p62/SQSTM1

Autophagic flux can be measured by determining the declining abundance of autophagic substrates such as sequestosome 1 (SQSTM1, better known as p62), which is sequestered in autophagosomes upon its direct interaction with LC3. However, the total amount of p62 results from two opposed processes, namely its synthesis (which can be modulated by some cellular stressors including autophagy inducers) and its degradation. To avoid this problem, we generated a stable cell line expressing a chimeric protein composed by p62 and the HaloTag (?) protein, which serves as a receptor for fluorescent HaloTag (?) ligands. Upon labeling with HaloTag (?) ligands (which form covalent, near-to-undissociable bonds with the Halotag (?) receptor) and washing, the resulting fluorescent labeling is not influenced by de novo protein synthesis, therefore allowing for the specific monitoring of the fusion protein decline without any interference by protein synthesis. We demonstrate that a HaloTag (?) -p62 fusion protein stably expressed in suitable cell lines can be used to monitor autophagy by flow cytometry and automated fluorescence microscopy. We surmise that this system could be adapted to high-throughput applications.     

Methodology: a modified method for high-quality DNA extraction for molecular analysis in cereal plants

Cereal crops that have rigid non-cellulose components in the cell wall tissues of leaves and high starch and protein content in grains face limitations in DNA extraction. Advanced molecular genetic techniques such as mapping and marker-assisted selection programs require pure and quick DNA extraction. In this study, we developed methods for isolating high-quality genomic DNA from leaves and seeds of major cereal crops with minor modifications. DNA yields ranged from 300 to 1800 ng for 0.01 g seed or leaf tissue.     

Introgression of cotton leaf curl virus-resistant genes from Asiatic cotton (Gossypium arboreum) into upland cotton (G. hirsutum)

Cotton is under the constant threat of leaf curl virus, which is a major constraint for successful production of cotton in the Pakistan. A total of 3338 cotton genotypes belonging to different research stations were screened, but none were found to be resistant against the Burewala strain of cotton leaf curl virus (CLCuV). We explored the possibility of transferring virus-resistant genes from Gossypium arboreum (2n = 26) into G. hirsutum (2n = 52) through conventional breeding techniques. Hybridization was done manually between an artificial autotetraploid of G. arboreum and an allotetraploid G. hirsutum, under field conditions. Boll shedding was controlled by application of exogenous hormones, 50 mg/L gibberellic acid and 100 mg/L naphthalene acetic acid. Percentage pollen viability in F(1) hybrids was 1.90% in 2(G. arboreum) x G. hirsutum and 2.38% in G. hirsutum x G. arboreum. Cytological studies of young buds taken from the F(1) hybrids confirmed that they all were sterile. Resistance against CLCuV in the F(1) hybrids was assessed through grafting, using the hybrid plant as the scion; the stock was a virus susceptible cotton plant, tested under field and greenhouse conditions. All F(1) cotton hybrids showed resistance against CLCuV, indicating that it is possible to transfer resistant genes from the autotetraploid of the diploid donor specie G. arboreum into allotetraploid G. hirsutum through conventional breeding, and durable resistance against CLCuV can then be deployed in the field.     

Validation of statistical models for estimating hospitalization associated with influenza and other respiratory viruses

Background

Reliable estimates of disease burden associated with respiratory viruses are keys to deployment of preventive strategies such as vaccination and resource allocation. Such estimates are particularly needed in tropical and subtropical regions where some methods commonly used in temperate regions are not applicable. While a number of alternative approaches to assess the influenza associated disease burden have been recently reported, none of these models have been validated with virologically confirmed data. Even fewer methods have been developed for other common respiratory viruses such as respiratory syncytial virus (RSV), parainfluenza and adenovirus.

Methods and Findings

We had recently conducted a prospective population-based study of virologically confirmed hospitalization for acute respiratory illnesses in persons <18 years residing in Hong Kong Island. Here we used this dataset to validate two commonly used models for estimation of influenza disease burden, namely the rate difference model and Poisson regression model, and also explored the applicability of these models to estimate the disease burden of other respiratory viruses. The Poisson regression models with different link functions all yielded estimates well correlated with the virologically confirmed influenza associated hospitalization, especially in children older than two years. The disease burden estimates for RSV, parainfluenza and adenovirus were less reliable with wide confidence intervals. The rate difference model was not applicable to RSV, parainfluenza and adenovirus and grossly underestimated the true burden of influenza associated hospitalization.

Conclusion

The Poisson regression model generally produced satisfactory estimates in calculating the disease burden of respiratory viruses in a subtropical region such as Hong Kong.     

Denoising two-photon calcium imaging data

Two-photon calcium imaging is now an important tool for in vivo imaging of biological systems. By enabling neuronal population imaging with subcellular resolution, this modality offers an approach for gaining a fundamental understanding of brain anatomy and physiology. Proper analysis of calcium imaging data requires denoising, that is separating the signal from complex physiological noise. To analyze two-photon brain imaging data, we present a signal plus colored noise model in which the signal is represented as harmonic regression and the correlated noise is represented as an order autoregressive process. We provide an efficient cyclic descent algorithm to compute approximate maximum likelihood parameter estimates by combing a weighted least-squares procedure with the Burg algorithm. We use Akaike information criterion to guide selection of the harmonic regression and the autoregressive model orders. Our flexible yet parsimonious modeling approach reliably separates stimulus-evoked fluorescence response from background activity and noise, assesses goodness of fit, and estimates confidence intervals and signal-to-noise ratio. This refined separation leads to appreciably enhanced image contrast for individual cells including clear delineation of subcellular details and network activity. The application of our approach to in vivo imaging data recorded in the ferret primary visual cortex demonstrates that our method yields substantially denoised signal estimates. We also provide a general Volterra series framework for deriving this and other signal plus correlated noise models for imaging. This approach to analyzing two-photon calcium imaging data may be readily adapted to other computational biology problems which apply correlated noise models.     

A novel mutation in the HSD17B10 gene of a 10-year-old boy with refractory epilepsy, choreoathetosis and learning disability

Hydroxysteroid (17beta) dehydrogenase 10 (HSD10) is a mitochondrial multifunctional enzyme encoded by the HSD17B10 gene. Missense mutations in this gene result in HSD10 deficiency, whereas a silent mutation results in mental retardation, X-linked, syndromic 10 (MRXS10). Here we report a novel missense mutation found in the HSD17B10 gene, namely c.194T>C transition (rs104886492), brought about by the loss of two forked methyl groups of valine 65 in the HSD10 active site. The affected boy, who possesses mutant HSD10 (p.V65A), has a neurological syndrome with metabolic derangements, choreoathetosis, refractory epilepsy and learning disability. He has no history of acute decompensation or metabolic acidosis whereas his urine organic acid profile, showing elevated levels of 2-methyl-3-hydroxybutyrate and tiglylglycine, is characteristic of HSD10 deficiency. His HSD10 activity was much lower than the normal control level, with normal β-ketothiolase activity. The c.194T>C mutation in HSD17B10 can be identified by the restriction fragment polymorphism analysis, thereby facilitating the screening of this novel mutation in individuals with intellectual disability of unknown etiology and their family members much easier. The patient's mother is an asymptomatic carrier, and has a mixed ancestry (Hawaiian, Japanese and Chinese). This demonstrates that HSD10 deficiency patients are not confined to a particular ethnicity although previously reported cases were either Spanish or German descendants.     

Role of NQO1 609C>T and NQO2-3423G>A polymorphisms in susceptibility to gastric cancer in Kashmir valley

NADPH: quinone oxidoreductase 1 (NQO1) and dihydronicotinamide riboside: quinone oxidoreductase 2 (NQO2) are cytosolic enzymes that catalyze reductive activation of carcinogens from cigarette smoke, such as nitrosamines and heterocyclic amines. These enzymes also protect cells against oxidative damage from reactive oxygen species. The present study investigated the associations of genetic variants of NQO1 609C>T and NQO2 -3423G>A polymorphisms with susceptibility to gastric cancer (GC) as well as their interactions with known risk factors in Kashmir valley. A case control study was performed in 303 subjects (108 GC and 195 healthy controls). All subjects were genotyped using polymerase chain reaction-restriction fragment length polymorphism method. Data were statistically analyzed by chi-square test and logistic regression model. The NQO1 609C>T TT genotype and T allele were significantly associated with increased risk for GC, whereas NQO2 -3423G>A polymorphism did not show any association with GC. Also, NQO1 609C>T TT genotype showed significant association with gastric adenocarcinoma. The interaction of NQO1/NQO2 genotypes with high consumption of salted tea, a known risk factor, did not further modulate the risk of GC. In conclusion, NQO1 609C>T polymorphism shows association with GC risk in Kashmir valley.     

Sleep habits of Iranian pre-school children in an urban area: Late sleeping and sleep debt in children

Using BEARS (Bedtime problems, Excessive sleepiness, Awakenings during the night, Regularity of sleep, Snoring), and CSHQ (Children’s Sleep Habits Questionnaires) screening tools with 224 participants revealed that Iranian children have shorter night sleep duration than expected (9.54 vs 12 h) for their age group. Earlier sleepers had longer night sleep duration (10:36 ± 1:12; 9:12 ± 1:06 h, P > 0.001), and total daily sleep time (11:36 ± 1:42; 10:36 ± 1:30 h, P = 0.005) than late sleepers. A majority (85%) of naptakers had sleep bedtime of 22:00 or later. The poor sleep quality of Iranian preschool children is probably due to cultural characteristics, climate differences, or harmful sleep habits.