Development and comparison of forensic interval age prediction models by statistical and machine learning methods based on the methylation rates of ELOVL2 in blood DNA.

Age estimation can be useful information for narrowing down candidates of unidentified donors in criminal investigations. Various age estimation models based on DNA methylation biomarkers have been developed for forensic usage in the past decade. However, many of these models using ordinary least squares regression cannot generate an appropriate estimation due to the deterioration in prediction accuracy caused by an increased prediction error in older age groups. In the present study, to address this problem, we developed age estimation models that set an appropriate prediction interval for all age groups by two approaches: a statistical method using quantile regression (QR) and a machine learning method using an artificial neural network (ANN). Methylation datasets (n = 1280, age 0-91 years) of the promoter for the gene encoding ELOVL fatty acid elongase 2 were used to develop the QR and ANN models. By validation using several test datasets, both models were shown to enlarge prediction intervals in accordance with aging and have a high level of correct prediction (>90 %) for older age groups. The QR and ANN models also generated a point age prediction with high accuracy. The ANN model enabled a prediction with a mean absolute error (MAE) of 5.3 years and root mean square error (RMSE) of 7.3 years for the test dataset (n = 549), which were comparable to those of the QR model (MAE = 5.6 years, RMSE = 7.8 years). Their applicability to casework was also confirmed using bloodstain samples stored for various periods of time (1-14 years), indicating the stability of the models for aged bloodstain samples. From these results, it was considered that the proposed models can provide more useful and effective age estimation in forensic settings.

TIPE2 (Tumor Necrosis Factor α-induced Protein 8-like 2) Is a Novel Negative Regulator of TAK1 Signal.

TIPE2 (TNF-α-induced protein 8-like 2) is a novel death effector domain protein and is a negative regulator of the innate and adaptive immune response. Although it has been demonstrated that caspase-8 contributes to the negative regulation of TIPE2, the negative regulatory mechanism is not entirely understood. Here, we demonstrate that TIPE2 interacts with TGF-ß-activated kinase 1 (TAK1), a crucial regulatory molecule of inflammatory and immune signals, and consequently acts as a powerful negative regulator of TAK1. The interaction between endogenous TIPE2 and TAK1 was observed in RAW264.7 macrophage-like cells and mouse primary cells derived from spleen and thymus. The TIPE2 amino acid 101-140 region interacted with TAK1 by binding to the amino acid 200-291 region of the internal kinase domain of TAK1. TIPE2 interfered with the formation of the TAK1-TAB1-TAB2 complex and subsequently inhibited activation of TAK1 and its downstream molecules. Importantly, silencing TIPE2 through RNA interference attenuated the inhibitory action of TIPE2 on LPS- and TNF-α-stimulated TAK1 activity. Exogenous TIPE2 101-140, the region that interacts with TAK1, also inhibited LPS- and TNF-α-stimulated NF-κB reporter activity. Interestingly, cell-permeable TIPE2 protein maintained its binding ability with TAK1 and exhibited the same inhibitory action of native TIPE2 on TLR4 signaling in vitro Thus, cell-permeable TIPE2 protein is a potential candidate for intracellular protein therapy for TAK1-related diseases. The present study demonstrates that TIPE2 acts as a novel negative regulator of inflammatory and immune responses through TAK1 signaling.

Synthetic peptides of actin-tropomyosin binding region of troponin I and heat shock protein 20 modulate the relaxation process of skinned preparations of taenia caeci from guinea pig.

To explore the possible role of the thin filament-linked regulation of cross-bridge cycling in living smooth muscle contraction, we studied the effects of TnIp and HSP20p, a synthetic peptide originating from an actin tropomyosin binding region of rabbit cardiac troponin I (residues 136-147; GKFKRPTLRRVR), and that of human heat shock protein 20 (residues 110-121; GFVAREFHRRYR) on the relaxation of skinned (cell membrane ilized) preparations from guinea pig taenia caeci. An active stress of the skinned preparations, resulting from actin-myosin interaction, rapidly decayed following Ca(2+) removal (relaxation). TnIp accelerated the initial rapid phase and slowed the following slow phase of the relaxation. On the other hand, HSP20p only slowed the whole process of the relaxation. The relaxation time courses were well fitted in a double exponential manner, and the double exponential decay of the stress could be explained as a portion of fast-detaching cross bridges not to dissociate rapidly by Ca(2+) removal, but to transfer to latch bridges dissociating very slowly. Our present results suggested that (i) TnIp and HSP20p accelerated transferring from fast-detaching cross bridges to slow-detaching (latch) bridges, and (ii) TnIp accelerated dissociation of the fast-detaching cross bridges and the latch bridges, while HSP20p slowed dissociation the fast-detaching cross bridges. Since TnIp and HSP20p are thought to bind to actin and tropomyosin, but not to myosin, we concluded that through thin-filament-dependent mechanisms these peptides regulated the formation and/or deformation of latch bridges in smooth muscle. The thin-filament-dependent regulation might physiologically control the stress maintenance and relaxation in smooth muscle cells.

The squamous cell carcinoma antigen 2 inhibits the cysteine proteinase activity of a major mite allergen, Der p 1.

The squamous cell carcinoma antigens 1 (SCCA1) and SCCA2 belong to the ovalbumin-serpin family. Although SCCA1 and SCCA2 are closely homologous, these two molecules have distinct properties; SCCA1 inhibits cysteine proteinases such as cathepsin K, L, and S, whereas SCCA2 inhibits serine proteinases such as cathepsin G and human mast cell chymase. Although several intrinsic target proteinases for SCCA1 and SCCA2 have been found, the biological roles of SCCA1 and SCCA2 remain unknown. A mite allergen, Der p 1, is one of the most immunodominant allergens and also acts as a cysteine proteinase probably involved in the pathogenesis of allergic diseases. We have recently shown that both SCCA1 and SCCA2 are induced by two related Th2-type cytokines, IL-4 and IL-13, in bronchial epithelial cells and that SCCA expression is augmented in bronchial asthma patients. In this study, we explored the possibility that SCCA proteins target Der p 1, and it turned out that SCCA2, but not SCCA1, inhibited the catalytic activities of Der p 1. We furthermore analyzed the inhibitory mechanism of SCCA2 on Der p 1. SCCA2 contributed the suicide substrate-like mechanism without formation of a covalent complex, causing irreversible impairment of the catalytic activity of Der p 1, as SCCA1 does on papain. In addition, resistance to cleavage by Der p 1 also contributed to the inhibitory mechanism of SCCA2. These results suggest that SCCA2 acts as a cross-class serpin targeting an extrinsic cysteine proteinase derived from house dust mites and that it may have a protective role against biological reactions caused by mites.

Effects of hydrogen peroxide on contraction of skinned aorta from guinea pigs.

We examined the effects of hydrogen peroxide (H2O2) on the contractile force of cell membrane permeabilized descending thoracic aorta from guinea pigs. H2O2 enhanced generated force in the Ca2+-induced contraction at any given concentration of Ca2+, and maintained force level in the 30 mM Mg2+-induced Ca2+-independent force maintenance. H2O2 seems to directly enhance the cross-bridge interaction of aortic smooth muscles.