A high‐throughput and quantitative hierarchical oligonucleotide primer extension (HOPE)‐based approach to identify sources of faecal contamination in water bodies

As faecal contamination of recreational and drinking water impairs the water quality and threatens public health, water bodies are routinely monitored for faecal coliforms to detect contamination. However, faecal coliforms are facultative anaerobes that survive and reproduce in ambient waters, and their presence does not depict the origin of contamination. Therefore, the use of Bacteroides‐Prevotella 16S rRNA gene to perform faecal source tracking has been proposed and applied. Here, we demonstrate the use of a new molecular method termed hierarchical oligonucleotide primer extension (HOPE) to simultaneously detect human‐associated Bacteroides spp. and three clusters of cow‐, pig‐ and dog‐specific uncultivated Bacteroidales. The method correctly identifies the origin of faecal contamination when tested against human, cow, pig and dog faeces (n = 17, 17, 16 and 13 respectively), and in waters contaminated with faeces of known origins. Subsequent tests with a total of 21 blind samples show that HOPE is able to accurately indicate single or multiple sources of faecal contamination originating from pigs, cows and humans in 81% of the blind samples. HOPE can further correctly detect and identify faecal contamination in five sampling sites located along a canal in southern Taiwan, and the results are validated against conventional faecal coliform tests and quantitative PCR. Overall, this study demonstrates HOPE as a quantitative and high‐throughput method that can identify sources of faecal contamination.     

A Novel Preprocessing Method Using Hilbert Huang Transform for MALDI-TOF and SELDI-TOF Mass Spectrometry Data

Motivation

Mass spectrometry is a high throughput, fast, and accurate method of protein analysis. Using the peaks detected in spectra, we can compare a normal group with a disease group. However, the spectrum is complicated by scale shifting and is also full of noise. Such shifting makes the spectra non-stationary and need to align before comparison. Consequently, the preprocessing of the mass data plays an important role during the analysis process. Noises in mass spectrometry data come in lots of different aspects and frequencies. A powerful data preprocessing method is needed for removing large amount of noises in mass spectrometry data.

Results

Hilbert-Huang Transformation is a non-stationary transformation used in signal processing. We provide a novel algorithm for preprocessing that can deal with MALDI and SELDI spectra. We use the Hilbert-Huang Transformation to decompose the spectrum and filter-out the very high frequencies and very low frequencies signal. We think the noise in mass spectrometry comes from many sources and some of the noises can be removed by analysis of signal frequence domain. Since the protein in the spectrum is expected to be a unique peak, its frequence domain should be in the middle part of frequence domain and will not be removed. The results show that HHT, when used for preprocessing, is generally better than other preprocessing methods. The approach not only is able to detect peaks successfully, but HHT has the advantage of denoising spectra efficiently, especially when the data is complex. The drawback of HHT is that this approach takes much longer for the processing than the wavlet and traditional methods. However, the processing time is still manageable and is worth the wait to obtain high quality data.     

Reticulon 3 interacts with NS4B of the hepatitis C virus and negatively regulates viral replication by disrupting NS4B self‐interaction

The non‐structural protein 4B (NS4B) of the hepatitis C virus (HCV) is an endoplasmic reticulum (ER) membrane protein comprising two consecutive amphipathic α‐helical domains (AH1 and AH2). Its self‐oligomerization via the AH2 domain is required for the formation of the membranous web that is necessary for viral replication. Previously, we reported that the host‐encoded ER‐associated reticulon 3 (RTN3) protein is involved in the formation of the replication‐associated membranes of (+)RNA enteroviruses during viral replication. In this study, we demonstrated that the second transmembrane region of RTN3 competed for, and bound to, the AH2 domain of NS4B, thus abolishing NS4B self‐interaction and leading to the downregulation of viral replication. This interaction was mediated by two crucial residues, lysine 52 and tyrosine 63, of AH2, and was regulated by the AH1 domain. The silencing of RTN3 in Huh7 and AVA5 cells harbouring an HCV replicon enhanced the replication of HCV, which was counteracted by the overexpression of recombinant RTN3. The synthesis of viral RNA was also increased in siRNA‐transfected human primary hepatocytes infected with HCV derived from cell culture. Our results demonstrated that RTN3 acted as a restriction factor to limit the replication of HCV.     

Spatial Repolarization Heterogeneity Detected by Magnetocardiography Correlates with Cardiac Iron Overload and Adverse Cardiac Events in Beta-Thalassemia Major

Background

Patients with transfusion-dependent beta-thalassemia major (TM) are at risk for myocardial iron overload and cardiac complications. Spatial repolarization heterogeneity is known to be elevated in patients with certain cardiac diseases, but little is known in TM patients. The purpose of this study was to evaluate spatial repolarization heterogeneity in patients with TM, and to investigate the relationships between spatial repolarization heterogeneity, cardiac iron load, and adverse cardiac events.

Methods and Results

Fifty patients with TM and 55 control subjects received 64-channel magnetocardiography (MCG) to determine spatial repolarization heterogeneity, which was evaluated by a smoothness index of QT_c (SI-QT_c), a standard deviation of QT_c (SD-QT_c), and a QT_c dispersion. Left ventricular function and myocardial T2* values were assessed by cardiac magnetic resonance. Patients with TM had significantly greater SI-QT_c, SD-QT_c, and QT_c dispersion compared to the control subjects (all p values<0.001). Spatial repolarization heterogeneity was even more pronounced in patients with significant iron overload (T2*<20 ms, n = 20) compared to those with normal T2* (all p values<0.001). Log_e cardiac T2* correlated with SI-QT_c (r = −0.609, p<0.001), SD-QT_c (r = −0.572, p<0.001), and QT_c dispersion (r = −0.622, p<0.001), while all these indices had no relationship with measurements of the left ventricular geometry or function. At the time of study, 10 patients had either heart failure or arrhythmia. All 3 indices of repolarization heterogeneity were related to the presence of adverse cardiac events, with areas under the receiver operating characteristic curves (ranged between 0.79 and 0.86), similar to that of cardiac T2*.

Conclusions

Multichannel MCG demonstrated that patients with TM had increased spatial repolarization heterogeneity, which is related to myocardial iron load and adverse cardiac events.     

N-Acetylcysteine ameliorates lipopolysaccharide-induced organ damage in conscious rats

Lipopolysaccharide is strongly associated with septic shock, leading to multiple organ failure. It can activate monocytes and macrophages to release proinflammatory mediators such as tumor necrosis factor- (TNF-), interleukin-1 (IL-1), and nitric oxide (NO). The present experiments were designed to induce endotoxin shock by an intravenous injection ofKlebsiella pneumoniae lipopolysaccharide (LPS, 10 mg/kg) in conscious rats. Arterial pressure and heart rate (HR) were continuously monitored for 48 h after LPS administration. N-Acetyl-cysteine was used to study its effects on organ damage. Biochemical substances were measured to reflect organ functions. Biochemical factors included blood urea nitrogen (BUN), creatinine (Cre), lactic dehydrogenase (LDH), creatine phosphokinase (CPK), aspartate transferase (GOT), alanine transferase (GPT), TNF-, IL-1, methyl guanidine (MG), and nitrites/nitrates. LPS caused significant increases in blood BUN, Cre, LDH, CPK, GOT, GPT, TNF-, IL-1, MG levels, and HR, as well as a decrease in mean arterial pressure and an elevation of nitrites/nitrates. N-Acetylcysteine suppressed the release of TNF-, IL-1, and MG, but enhanced NO production. These actions ameliorate LPS-induced organ damage in conscious rats. The beneficial effects may suggest a potential chemopreventive effect of this compound in sepsis prevention and treatment.     

Effects of low dose rate (0.003-0.025 Gy/h) chronic X-irradiation on radioresistant and radiosensitive L5178Y mouse lymphoma cells

Cultures of radioresistant (LY-R) and radiosensitive (LY-S) strains of L5178Y mouse lymphoma cells were exposed continuously to X-rays delivered at dose rates ranging from 0.003 to 0.025 Gy/h for up to 35 days. Populations of both strains proliferated actively during the exposure, but the growth rates were reduced in a dose rate-dependent manner. The reduction of growth rate occurred for strain LY-S earlier during the exposure and at lower dose rates than for strain LY-R. The survival (as measured by colony forming ability) of strain LY-R was affected only slightly at all dose rates applied. For strain LY-S, a decrease in the surviving fraction was observed in the initial part of the exposure. This decrease was followed by a plateau and eventually by an increase, in some cases to values close to the control level. The increase in the surviving fraction indicated that the radioresistance of the exposed LY-S cells had increased. This pattern was particularly clear for dose rates greater than 0.014 Gy/h. The pre-irradiated cells exhibited radioresistance when exposed to acute X-radiation after termination of the chronic exposure. The increase in radiation resistance was stable for at least 70 days after termination of the protracted exposure. These results show that mutagenic and/or selective phenomena leading to an increase in radiation resistance of mammalian cells can be caused by protracted exposures to X-rays at dose rates permitting active proliferation.     

Impact of lek mating on the sterile insect technique: A modeling study

Modelling studies are presented which describe the effect of lek mating on the control of a wild population by sterile male release. The mixed leks are assumed to follow a Poisson-binomial distribution and the system includes three parts: territory defense, matings inside a lek and matings outside a lek. The effects of parameters on the hatchability are discussed. Among the parameters, sterile type effect (W_s), female choice (f_s) and mating competitiveness (C_m) are the most important. The application to determining the effects of sterile male release and on the proportion of sterile males required for eradication are also discussed.     

Tetrapod phylogeny inferred from 18S and 28S ribosomal RNA sequences and a review of the evidence for amniote relationships [published erratum appears in Mol Biol Evol 1991 May;8(3):398]

The 18S ribosomal RNAs of 21 tetrapods were sequenced and aligned with five published tetrapod sequences. When the coelacanth was used as an outgroup, Lissamphibia (living amphibians) and Amniota (amniotes) were found to be statistically significant monophyletic groups. Although little resolution was obtained among the lissamphibian taxa, the amniote sequences support a sister-group relationship between birds and mammals. Portions of the 28S ribosomal RNA (rRNA) molecule in 11 tetrapods also were sequenced, although the phylogenetic results were inconclusive. In contrast to previous studies, deletion or down- weighting of base-paired sites were found to have little effect on phylogenetic relationships. Molecular evidence for amniote relationships is reviewed, showing that three genes (beta-hemoglobin, myoglobin, and 18S rRNA) unambiguously support a bird-mammal relationship, compared with one gene (histone H2B) that favors a bird- crocodilian clade. Separate analyses of four other genes (alpha- crystallin A, alpha-hemoglobin, insulin, and 28S rRNA) and a combined analysis of all sequence data are inconclusive, in that different groups are defined in different analyses and none are strongly supported. It is suggested that until sequences become available from a broader array of taxa, the molecular evidence is best evaluated at the level of individual genes, with emphasis placed on those studies with the greatest number of taxa and sites. When this is done, a bird-mammal relationship is most strongly supported. When regarded in combination with the morphological evidence for this association, it must be considered at least as plausible as a bird-crocodilian relationship.