Longitudinal Study of Hepatitis A Infection by Saliva Sampling: The Kinetics of HAV Markers in Saliva Revealed the Application of Saliva Tests for Hepatitis A Study

Despite the increasing numbers of studies investigating hepatitis A diagnostic through saliva, the frequency and the pattern of hepatitis A virus (HAV) markers in this fluid still remains unknown. To address this issue, we carried on a longitudinal study to examine the kinetics of HAV markers in saliva, in comparison with serum samples. The present study followed-up ten patients with acute hepatitis A infection during 180 days post diagnosis (dpd). Total anti-HAV was detected in paired serum and saliva samples until the end of the follow-up, showing a peak titer at 90^th. However, total anti-HAV level was higher in serum than in saliva samples. This HAV marker showed a probability of 100% to be detected in both serum and saliva during 180 dpd. The IgM anti-HAV could be detected in saliva up to 150 dpd, showing the highest frequency at 30^th, when it was detected in all individuals. During the first month of HAV infection, this acute HAV marker showed a detection probability of 100% in paired samples. The detection of IgM anti-HAV in saliva was not dependent on its level in serum, HAV-RNA detection and/or viral load, since no association was found between IgM anti-HAV positivity in saliva and any of these parameter (p>0.05). Most of the patients (80%) were found to contain HAV-RNA in saliva, mainly at early acute phase (30^th day). However, it was possible to demonstrate the HAV RNA presence in paired samples for more than 90 days, even after seroconversion. No significant relationship was observed between salivary HAV-RNA positivity and serum viral load, demonstrating that serum viral load is not predictive of HAV-RNA detection in saliva. Similar viral load was seen in paired samples (on average 10⁴ copies/mL). These data demonstrate that the best diagnostic coverage can be achieved by salivary anti-HAV antibodies and HAV-RNA tests during 30–90 dpd. The long detection and high probability of specific-HAV antibodies positivity in saliva samples make the assessment of salivary antibodies a useful tool for diagnosis and epidemiological studies. The high frequency of HAV-RNA in saliva and the probability of detection of about 50%, during the first 30 dpd, demonstrate that saliva is also useful for molecular investigation of hepatitis A cases, mainly during the early course of infection. Therefore, the collection of saliva may provide a simple, cheap and non-invasive means of diagnosis, epidemiological surveys and monitoring of hepatitis A infection purposes.     

Assessing the Consequences of Denoising Marker-Based Metagenomic Data

Early marker-based metagenomic studies were performed without properly accounting for the effects of noise (sequencing errors, PCR single-base errors, and PCR chimeras). Denoising algorithms have been developed, but they were validated using data derived from mock communities, in which the true sequences were known. Since the algorithms were designed to be used in real community studies, it is important to evaluate the results in such cases. With this goal in mind, we processed a real 16S rRNA metagenomic dataset through five denoising pipelines. By reconstituting the sequence reads at each stage of the pipelines, we determined how the reads were being altered. In one denoising pipeline, AmpliconNoise, we found that the algorithm that was designed to remove pyrosequencing errors changed the reads in a manner inconsistent with the known spectrum of these errors, until one of the parameters was increased substantially from its default value. Additionally, because the longest read was picked as the representative for each cluster, sequences were added to the 3′ ends of shorter reads that were often dissimilar from what had been removed by the truncations of the previous filtering step. In QIIME, the denoising algorithm caused a much larger number of changes to the reads unless the parameters were changed from their defaults. The denoising pipeline in mothur avoided some of these negative side-effects because of its strict default filtering criteria, but these criteria also greatly limited the sequence information produced at the end of the pipeline. We recommend that those using these denoising pipelines be cognizant of these issues and examine how their reads are being transformed by the denoising process as a component of their analysis.     

Δ98Δ, a minimalist model of antiparallel β‐sheet proteins based on intestinal fatty acid binding protein

The design of β‐barrels has always been a formidable challenge for de novo protein design. For instance, a persistent problem is posed by the intrinsic tendency to associate given by free edges. From the opposite standpoint provided by the redesign of natural motifs, we believe that the intestinal fatty acid binding protein (IFABP) framework allows room for intervention, giving rise to abridged forms from which lessons on β‐barrel architecture and stability could be learned. In this context, Δ98Δ (encompassing residues 29–126 of IFABP) emerges as a monomeric variant that folds properly, retaining functional activity, despite lacking extensive stretches involved in the closure of the β‐barrel. Spectroscopic probes (fluorescence and circular dichroism) support the existence of a form preserving the essential determinants of the parent structure, albeit endowed with enhanced flexibility. Chemical and physical perturbants reveal cooperative unfolding transitions, with evidence of significant population of intermediate species in equilibrium, structurally akin to those transiently observed in IFABP. The recognition by the natural ligand oleic acid exerts a mild stabilizing effect, being of a greater magnitude than that found for IFABP. In summary, Δ98Δ adopts a monomeric state with a compact core and a loose periphery, thus pointing to the nonintuitive notion that the integrity of the β‐barrel can indeed be compromised with no consequence on the ability to attain a native‐like and functional fold.     

Small Toxic Proteins and the Antisense RNAs That Repress Them

Summary: There has been a great expansion in the number of small regulatory RNAs identified in bacteria. Some of these small RNAs repress the synthesis of potentially toxic proteins. Generally the toxin proteins are hydrophobic and less than 60 amino acids in length, and the corresponding antitoxin small RNA genes are antisense to the toxin genes or share long stretches of complementarity with the target mRNAs. Given their short length, only a limited number of these type I toxin-antitoxin loci have been identified, but it is predicted that many remain to be found. Already their characterization has given insights into regulation by small RNAs, has suggested functions for the small toxic proteins at the cell membrane, and has led to practical applications for some of the type I toxin-antitoxin loci.     

Cellular and subcellular localization of gastrointestinal glutathione peroxidase in normal and malignant human intestinal tissue

The gastrointestinal glutathione peroxidase (GI-GPx) is believed to prevent absorption of hydroperoxides. GI-GPx is expressed in the intestine together with the other three glutathione peroxidase isoenzymes, raising the question of the physiological role of the different GPx types. We therefore studied the cellular and subcellular distribution of GI-GPx in normal and malignant tissue obtained from patients with colorectal cancer or familial polyposis by immunohistochemistry. In healthy ileum epithelium GI-GPx was preferentially enriched in Paneth cells. In unaffected crypts of colon and rectum, it decreased gradually from the ground to the luminal surface. In crypt ground, GI-GPx was uniformly distributed, whereas in cells at the luminal surface it was concentrated in structures capping the nuclei at the apical pole. In colorectal cancer, GI-GPx expression depended on the stage of malignant transformation. In early stages, GI-GPx was increased and pronouncedly associated with the vesicular structures. In progressed stages of malignancy, structures disintegrated and GI-GPx distribution became more diffuse. These observations support the hypothesis that GI-GPx, apart from being a barrier against hydroperoxide absorption, might be involved in cell growth and differentiation.