A rapid nuclear staining test using cationic dyes contributes to efficient STR analysis of telogen hair roots.

Telogen hairs presented in the crime scene are commonly encountered as trace evidence. However, short tandem repeat (STR) profiling of the hairs currently have low and limited use due to poor success rate. To increase the success rate of STR profiling of telogen hairs, we developed a rapid and cost-effective method to estimate the number of nuclei in the hair roots. Five cationic dyes, Methyl green (MG), Harris hematoxylin (HH), Methylene blue (MB), Toluidine blue (TB), and Safranin O (SO) were evaluated in this study. We conducted a screening test based on microscopy and the percentage of loss with nuclear DNA, in order to select the best dye. MG was selected based on its specific nuclei staining and low adverse effect on the hair-associated nuclear DNA. We examined 330 scalp and 100 pubic telogen hairs with MG. Stained hairs were classified into five groups and analyzed by STR. The fast staining method revealed 70% (head hair) and 33.4% (pubic hair) of full (30 alleles) and high partial (18-29 alleles) STR profiling proportion from the lowest nuclei count group (one to ten nuclei). The results of this study demonstrated a rapid, specific, nondestructive, and high yield DNA profiling method applicable for screening telogen hairs.

Enhanced expression and purification of inositol 1,4,5-trisphosphate 3-kinase A through use of the pCold1-GST vector and a C-terminal hexahistidine tag in Escherichia coli.

Inositol 1,4,5-trisphosphate 3-kinase A (IP3K-A, alternative name: ITPKA) is a neuron-specific enzyme that converts 1,4,5-trisphosphate (IP3) into inositol 1,3,4,5-tetrakisphosphate (IP4) through its kinase domain. In addition, transient overexpression of IP3K-A induces morphological changes in dendritic spines of excitatory synapses in a kinase-independent manner, apparently by modulating the organization of the neuronal cytoskeleton. Although the procurement of a purified recombinant IP3K-A protein would be indispensable for the biochemical elucidation of its physiological roles, production of recombinant IP3K-A has proven technically challenging in conventional Escherichia coli expression systems. These difficulties stem from low enzyme solubility, as well as poor protein quality caused by the tendency of IP3K-A to split into partial fragments. In present study, we newly introduced cold-shock expression vector (pCold1) together with a C-terminal hexahistidine tag (C-HIS) to enhance the expression levels of recombinant IP3K-A in E. coli. Importantly, when compared with other commonly-employed bacterial expression systems, the pCold1 system improved the yield and the purity of full-length IP3K-A due to the exclusion of truncated enzyme forms, and also enhanced the solubility of the enzyme. Furthermore, the functional integrity of purified IP3K-A was confirmed in both kinase activity assay and microtubule binding assay. Recombinant IP3K-A acquired via this modified protocol will be expected to facilitate the exploration of the enzyme's biochemical profile, both structurally and functionally.

Current development of therapeutic vaccines for the treatment of chronic infectious diseases.

Chronic infectious diseases refer to diseases that require a long period of time from onset to cure or death, the use of therapeutic vaccines has recently emerged to eradicate diseases. Currently, clinical research is underway to develop therapeutic vaccines for chronic infectious diseases based on various vaccine formulations, and the recent success of the messenger RNA vaccine platform and efforts to apply it to therapeutic vaccines are having a positive impact on conquering chronic infectious diseases. However, since research on the development of therapeutic vaccines is still relatively lacking compared to prophylactic vaccines, there is a need to focus more on the development of therapeutic vaccines to overcome threats to human health caused by chronic infectious diseases. In order to accelerate the development of therapeutic vaccines for chronic infectious diseases in the future, it is necessary to establish a clear concept of therapeutic vaccines suitable for the characteristics of each chronic infectious disease, as well as standardize vaccine effectiveness evaluation methods, secure standards/reference materials, and simplify the vaccine approval procedure.

Microbiota Composition and Pulmonary Surfactant Protein Expression as Markers of Death by Drowning.

Pathological diagnosis of drowning remains a challenge for forensic science, because of a lack of pathognomonic findings. We analyzed microbiota and surfactant protein in the lungs for a novel diagnosis of drowning. All rats were divided into drowning, postmortem submersion, and control groups. The water, lungs, closed organs (kidney and liver), and cardiac blood in rats were assayed by targeting 16S ribosomal RNA of Miseq sequencing. Lung samples were analyzed by immunohistochemical staining for surfactant protein A. The closed organs and cardiac blood of drowned group have a lot of aquatic microbes, which have not been detected in postmortem submersion group. Furthermore, intra-alveolar granular staining of surfactant protein A (SP-A) was severely observed in the drowned group than the postmortem submersion and control groups. The findings suggested that the presence of aquatic microbiota in the closed organs and increased expression of SP-A could be markers for a diagnosis of drowning.