Estimating bloodstain age in the short term based on DNA fragment length using nanopore sequencer.

We used a nanopore sequencer to quantify DNA fragments > 10,000 bp in size and then evaluated their relationship with short-term bloodstain age. Moreover, DNA degradation was investigated after bloodstains were wetted once with water. Bloodstain samples on cotton gauze were stored at room temperature and low humidity for up to 6 months. Bloodstains stored for 1 day were wetted with nuclease-free water, allowed to dry, and stored at room temperature and low humidity for up to 1 week. The proportion of fragments > 20,000 bp in dry bloodstains tended to decrease over time, particularly for fragments > 50,000 bp in size. This trend was modeled using a power approximation curve, with the highest R2 value (0.6475) noted for fragments > 50,000 bp in size; lower values were recorded for shorter fragments. The proportion of longer fragments was significantly reduced in bloodstains that were dried after being wetted once, and there was significant difference in fragments > 50,000 bp between dry conditions and once-wetted. This result suggests that even temporary exposure to water causes significant DNA fragmentation, but not extensive degradation. Thus, bloodstains that appear fresh but have a low proportion of long DNA fragments may have been wetted previously. Our results indicate that evaluating the proportion of long DNA fragments yields information on both bloodstain age and the environment in which they were stored.

Simplified detection of the species of origin of antler velvets using single-stranded tag hybridization chromatographic printed-array strip.

In this study, we developed a convenient and easy-to-use origin identification method for antler velvets based on a simple DNA extraction technique and single-stranded tag hybridization chromatographic printed-array strip (STH-PAS). The primer sets used to detect Cervus elaphus, Rangifer tarandus, and 12S rRNA did not engage in non-specific reactions such as primer dimer formation. In both the triplex and singleplex assays, the sensitivity was < 1 ng DNA. Moreover, Cervus elaphus DNA could be detected in OTC crude drug products. Although the detection sensitivity resulting from the simplified extraction was slightly lower than that obtained with extraction by conventional methods, the amount of DNA was sufficient even from a small sample. The choice of a triplex or singleplex assay will depend on the purpose of the test. For example, if it is important to determine whether the antler velvet is derived from Cervus elaphus or Rangifer tarandus, a triplex assay is appropriate. If it is necessary to explore whether antler velvet from Cervus elaphus is included in an OTC crude drug product, a singleplex assay using the Cervus elaphus primer set is informative. If it is necessary to explore whether powdered antler velvet includes counterfeit products (from Rangifer tarandus), a singleplex assay employing the Rangifer tarandus primer is appropriate. The singleplex assay detects minor components even at a 1,000:1 ratio. Our study thus demonstrated the utility of a method combining simple DNA extraction with STH-PAS for efficient identification of the origin of antler velvets.

Fine Particle Adsorption Capacity of Volcanic Soil from Southern Kyushu, Japan.

"Akahoya" is a volcanic soil classified as a special soil deposited in Kyushu, Japan. Many of its properties are not yet clearly understood. We found that Akahoya had the potential to adsorb bacteria in cattle feces, which prompted us to investigate its material properties and perform experiments to comprehensively evaluate its adsorption performance for various fine particles such as acidic and basic dyes, NOx/SOx gas, and phosphoric acid ions, in addition to bacteria. Akahoya had a very high specific surface area owing to the large number of nanometer-sized pores in its structure; it exhibited a high adsorption capacity for both NO2 and SO2. Regarding the zeta potential of Akahoya, the point of zero charge was approximately pH 7.0. The surface potential had a significant effect on the adsorption of acidic and basic dyes. Akahoya had a very high cation exchange capacity when the sample surface was negatively charged and a high anion exchange capacity when the sample surface was positively charged. Akahoya also exhibited a relatively high adsorption capacity for phosphoric acid because of its high level of Al2O3, and the immersion liquid had a very high Al ion concentration. Finally, filtration tests were performed on Escherichia coli suspension using a column filled with Akahoya or another volcanic soil sample. The results confirmed that the Escherichia coli adhered on the Akahoya sample. The results of the Escherichia coli release test, after the filtration test, suggested that this adhesion to Akahoya could be phosphorus-mediated.

A method for determining the origin of crude drugs derived from animals using MinION, a compact next-generation sequencer.

We evaluated whether MinION, an inexpensive portable sequencer, can be used to identify the origin of crude drugs derived from animals. Standard and nonstandard crude drugs with different species of origin were examined. In addition, standards mixed with nonstandard samples were used. As a target gene, cytochrome c oxidase I was amplified and sequenced. The fast mode results had a slightly lower match ratio than high-accuracy mode, but the animals of origin were correctly determined by BLAST for all samples. For antler velvet derived from Rangifer tarandus, even when the sequences were aligned based on Cervus elaphus, the animal of origin was determined correctly. Minor contents could be detected from mixtures of two animals, if the mixtures contained at least 19:1 mtDNA when the coverage allele-fraction threshold was 0.05. By contrast, in fast mode, two sequences could not be separated due to the low accuracy of the base-calling for each read. For fieldwork, the species of origin of crude drugs could be identified with only simple DNA extraction and library preparation. Therefore, MinION appears to be a convenient tool for identifying the origins of crude drugs derived from animals.

Dural Changes Induced by an Ultrasonic Bone Curette in an Excised Porcine Spinal Cord.

In spinal surgery, ultrasonic bone curettes are considered unlikely to cause mechanical injury to the dura; however, there is little evidence to support this claim. We investigated the effect of direct contact with an ultrasonic bone curette on the dura and the protective effect of covering the dura with a cotton pattie using an excised porcine spinal cord. The ultrasonic bone curette was pressed against the porcine spinal cord with constant force and activated for 1 s, with or without covering the dura with a cotton pattie. The dural surface and cross-section were observed using electron and light microscopy. When the ultrasonic bone curette was applied directly against the dura, most specimens showed non-perforating dural injuries. However, none of the specimens showed dural perforation. Histological changes were also observed. The use of a cotton pattie reduced the occurrence of these changes, although it did not prevent them when ultrasonic vibration was applied with a large force. We considered ultrasonic bone curettes to have a low risk of dural perforation and, thus, to be a safe surgical device as long as they did not accidentally make strong contact with the dura.

Dual antibacterial and anti-inflammatory efficacy of a chitosan-chondroitin sulfate-based in-situ forming wound dressing.

None of the currently available wound dressings exhibit combined antibacterial and anti-inflammatory activity. Using polyelectrolyte complexation (PEC) between a cationic polysaccharide chitosan (CH) and an anionic glycosaminoglycan chondroitin sulfate (CS), we have developed a unique in-situ forming scaffold (CH-CS PEC), which develops at the wound site itself to influence the function of the wound bed cells. The current study demonstrated that CH-CS PEC could induce bacterial cell death through membrane pore formation and increased ROS production. Moreover, possibly due to its unique material properties including medium-soft viscoelasticity, porosity, and surface composition, CH-CS PEC could modulate macrophage function, increasing their phagocytic ability with low TNF-α and high IL-10 production. Faster wound closure and decreased CFU count was observed in an in-vivo infected wound model, with reduced NF-κB and increased VE-cadherin expression, indicating reduced inflammation and enhanced angiogenesis. In summary, this study exhibited that CH-CS PEC has substantial antibacterial and immunomodulatory properties.

A sudden infant death associated with atrial septal aneurysm and abnormality of the connecting site between the inferior vena cava and right atrium.

An atrial septal aneurysm (ASA) is a rare cardiac anomaly characterized by varicose bulgingofthe atrial septum (oval fossa) into the left or right atrium. Pathogenesis and clinical significance of ASA are controversial. We report an autopsy case of a huge undiagnosed ASA with abnormality of the connecting site between the inferior vena cava and the right atrial ostium in a 2-month-oldJapanesefemale who died suddenly and unexpectedly. She was born at 36 weeks 4 days (body weight 3,110 g). No abnormality was detected during pregnancy or delivery. The postnatal growth was normal with no cardiac problem detected at the 1-month checkup. The ASA bulged off in a mass to the left atrium (width, 0.8 cm; excursion ratio, 53%), reaching close to the inflow site of the right pulmonary vein, with dilation of the pulmonary vein. The connecting site between the inferior vena cava and the right atrium was atypically located 1.6 cm away from the atrioventricular groove. Although most cases of ASA in an infant resolve physiologically as the infant grows, the infant in the present case is thought to have had an exceptional pathological ASA, possibly causing supraventricular arrhythmia. The abnormality of the connecting site between the inferior vena cava and the right atrium might have affected the development and continuation of the ASA.

A Germanium Catalyst Accelerates the Photoredox α-C(sp3)-H Alkylation of Primary Amines.

Site-selective C(sp3)-H functionalizations using photoredox catalysis (PC) and hydrogen atom transfer (HAT) catalysis have received increasing attention. Here, we report a Ph2GeCl2 cocatalyst that greatly improves the yield of α-C(sp3)-H alkylation of primary amines catalyzed by a PC-HAT hybrid system. The α-position of the amino group selectively reacted even when weaker C-H bonds existed in the substrates. This finding may help the design of a novel site-selective hybrid catalysis.

Estimating individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq.

We tried to estimate individual mtDNA haplotypes in mixed DNA samples by combining MinION and MiSeq. The BAM files produced by MiSeq were viewed using Integrative Genomics Viewer (IGV) to verify mixed bases. By sorting the reads according to base type for each mixed base, partial haplotypes were determined. Then, the BAM files produced by MinKNOW were viewed using IGV. To determine haplotypes with IGV, only mixed bases determined by MiSeq were used as target bases. By sorting the reads according to base type for each target base, each contributor's haplotype was estimated. In mixed samples from two contributors, even a haplotype with a minor contribution of 5% could be distinguished from the haplotype of the major contributor. In mixed samples of three contributors (mixture ratios of 1:1:1 and 4:2:1), each haplotype could also be distinguished. Sequences of C-stretches were determined very inaccurately in the MinION analysis. Although the analysis method was simple, each haplotype was correctly detected in all mixed samples with two or three contributors in various mixture ratios by combining MinION and MiSeq. This should be useful for identifying contributors to mixed samples.

Protein Modification at Tyrosine with Iminoxyl Radicals.

Post-translational modifications (PTMs) of proteins are a biological mechanism for reversibly controlling protein function. Synthetic protein modifications (SPMs) at specific canonical amino acids can mimic PTMs. However, reversible SPMs at hydrophobic amino acid residues in proteins are especially limited. Here, we report a tyrosine (Tyr)-selective SPM utilizing persistent iminoxyl radicals, which are readily generated from sterically hindered oximes via single-electron oxidation. The reactivity of iminoxyl radicals with Tyr was dependent on the steric and electronic demands of oximes; isopropyl methyl piperidinium oxime 1f formed stable adducts, whereas the reaction of tert-butyl methyl piperidinium oxime 1o was reversible. The difference in reversibility between 1f and 1o, differentiated only by one methyl group, is due to the stability of iminoxyl radicals, which is partly dictated by the bond dissociation energy of oxime O-H groups. The Tyr-selective modifications with 1f and 1o proceeded under physiologically relevant, mild conditions. Specifically, the stable Tyr-modification with 1f introduced functional small molecules, including an azobenzene photoswitch, to proteins. Moreover, masking critical Tyr residues by SPM with 1o, and subsequent deconjugation triggered by the treatment with a thiol, enabled on-demand control of protein functions. We applied this reversible Tyr modification with 1o to alter an enzymatic activity and the binding affinity of a monoclonal antibody with an antigen upon modification/deconjugation. The on-demand ON/OFF switch of protein functions through Tyr-selective and reversible covalent-bond formation will provide unique opportunities in biological research and therapeutics.

Transient Antidiuretic Hormone Insufficiency Caused by Severe Hyperosmolar Hyperglycemic Syndrome Based on Nephrogenic Diabetes Insipidus.

BACKGROUND: The hyperosmolar hyperglycemic state (HHS), an acute complication of diabetes mellitus with plasma hyperosmolarity, promotes the secretion of anti-diuretic hormone (ADH) and reduces the storage of ADH. Magnetic resonance T1-weighted imaging reflects ADH storage in the posterior pituitary lobe, which disappears when the storage is depleted. Whether the HHS induces ADH depletion leading to clinical manifestations has been unclear. CASE REPORT: A 55-year-old Japanese woman was admitted to our center because of mental disturbance and hypotension. She had received lithium carbonate for bipolar disorder and presented with polydipsia and polyuria from 15 years of age. On admission, she had mental disturbance (Glasgow Coma Scale, E4V1M1), hypotension (systolic blood pressure, 50 mmHg), and tachycardia (pulse rate, 123/min). Plasma glucose was 697 mg/dL osmolality was 476 mOsm/kg•H2O, and bicarbonate was 23.7 mmol/L. The diagnoses of HHS and hypovolemic shock were made. During treatment with fluid replacement and insulin therapy, the urine volume continued to be approximately 3 to 4 L/day, and an endocrine examination revealed ADH insufficiency and nephrogenic diabetes insipidus. Desmopressin 10 µg/day and trichlormethiazide 2 mg/day were necessary and administered, and the endogenous ADH secretion improved gradually. The signal intensity of the pituitary posterior lobe, initially decreased on magnetic resonance T1 images, was also improved. CONCLUSION: This patient had ADH insufficiency associated with ADH depletion due to hyperosmolarity and nephrogenic diabetes insipidus. Clinicians should be aware of the risk of the development of critical HHS and relative ADH insufficiency in patients being treated with lithium carbonate.

Designer Exosomes: Smart Nano-Communication Tools for Translational Medicine.

Exosomes are the master transporters of genes, RNAs, microRNAs, proteins, and lipids. They have applications in major diseases, including cancer, cardiovascular diseases, neurological disorders, and diabetes mellitus. Delivery of the exosomes to recipient cells is governed by the functional heterogenicity of the tissues. Engineered exosomes are promising tools in tissue regeneration. In addition to their role as intracellular communication cargos, exosomes are increasingly primed as standard biomarkers in the progression of diseases, thereby solving the diagnostic dilemma. Futuristic empowerment of exosomes with OMICS strategy can undoubtedly be a bio-tool in translational medicine. This review discusses the advent transformation of exosomes in regenerative medicine and limitations that are caveats to broader applications in clinical use.

Suicides Among Lesbian, Gay, Bisexual, and Transgender People: Medical Examiner Reports in the Special Wards of Tokyo, Japan, 2009-2018.

Purpose: We aimed to report the occurrence of suicide death and to reveal the characteristics among LGBT people in the special wards of Tokyo. Methods: All suicide deaths between 2009 and 2018 were identified from the Tokyo Medical Examiner's Office database, and the characteristics were compared between LGBT people and non-LGBT people by retrospective review of the medical examiner reports. Results: Among 17,638 people who died by suicide, 84 (0.5%) were identified as LGBT people, of which a large proportion was gay males (51.2%) and transgender people (42.9%). Smaller proportions of lesbian (3.6%) and bisexual (2.4%) females were identified. Compared with suicides among non-LGBT people, there were several differences in the circumstances surrounding suicides among LGBT people, namely method of suicide, source of income, and number of household members (transgender people), and toxic substance in poisoning and medical history (gay males). Conclusions: The results tentatively suggest the risk or supportive factors that should be considered for preventing suicides among LGBT people; however, the present data are likely to be underestimated. To better understand suicides among LGBT people, systematic efforts to collect sexual orientation and gender identity data for suicide are needed.

Unexpected death in a young child associated with anomalous aortic origin of the left main coronary artery without physical exertion: A case of an anomalous coronary artery with highly abundant elastic fibers.

Sudden death due to anomalous aortic origin of a coronary artery is far less common among young children in the absence of exercise stress. This report describes the case of a 2-year-old boy with a lower respiratory tract infection who suffered sudden cardiac arrest in his bed at home. The autopsy revealed that the left coronary artery (LCA) originated from the right sinus of Valsalva with an acute angle takeoff and traveled between the aorta and the pulmonary trunk (an interarterial course). Upon histological examination, the LCA, before reaching its major branches, was located adjacent to the outside of the aortic wall without an intramural passage, and the arterial wall was composed almost exclusively of elastic fibers without media containing smooth muscle cells throughout the entire length of the abnormal running. Screening tests for respiratory virus infection detected enterovirus in the lung tissue. In association with an acute angle takeoff and interarterial course, the wall structure with highly abundant elastic fibers that are more flexible tissues among blood vessel components might suggest their vulnerability to compression during the great vessels' systolic expansion in the sympathetic activation induced by the viral infection, leading to fatal myocardial ischemia without physical exertion.

Fatal intracranial hemorrhage due to infantile acute lymphoblastic leukemia mimicking abusive head trauma.

We report the case of a 2-month-old infant who was found moribund in her crib. Postmortem computed tomography (PMCT) was performed before autopsy. As the baby had a severe subdural hematoma, retinal hemorrhage, and encephalopathy on PMCT, abusive head trauma (AHT) was tentatively diagnosed. At autopsy, no scalp hemorrhages or skull fractures were found; however, the classic triad of AHT was present, mainly on the right side. Additionally, there was dark red discoloration around the heart, and the liver, spleen, and pancreas were enlarged. Peripheral blood was macroscopically cloudy with marked leukocytosis. After careful histological examination, B-cell precursor acute lymphoblastic leukemia (ALL) was diagnosed. All the macroscopic lesions could be attributed to ALL. The manner of death was natural. To the best of our knowledge, this is the first report of infantile ALL mimicking AHT on PMCT images. This case demonstrates the importance of a comprehensive systematic approach to considering differential diagnosis when PMCT shows multiple intracranial hemorrhages suggestive of AHT in an infant.

Dissecting lesions in a culprit artery of a hemorrhagic focus in the basal ganglia: Histopathological analysis by serial sectioning.

In a hypertensive hemorrhagic focus of the basal ganglia, the culprit arteries have been reported to be associated with dissecting lesions, whose topographical relationship to the rupture sites remains to be clarified. Herein we describe multiple dissecting lesions in the culprit artery of hypertensive hemorrhage of the basal ganglia. A 1.0 × 0.8 × 0.8 cm-sized bleeding globe was confirmed at a left lenticulostriate artery and histologically analyzed by serial sectioning. Three independent dissecting lesions were identified in the culprit artery. They were situated near the bifurcations, ranging from 240 to 3200 µm in length. The dissections mainly occurred between the intima and media with disruption of the internal elastic lamina (IEL), forming a fresh thrombus within the false lumen. Two rupture sites causing the cerebral hematoma were confirmed away from the dissecting lesions. One was situated close but not adjacent to the longest dissecting lesion; the other, measuring approximately 150 µm in diameter, was adjacent to the bifurcation of an artery. The histopathological findings suggest that the dissecting lesion resulted from medial detachment following IEL disruption in the process of arterial rupture of the culprit artery. We conclude that this was a secondary manifestation during the rupture rather than a cause of the arterial rupture.

Catalytic and Aerobic Oxidative Biaryl Coupling of Anilines Using a Recyclable Heterogeneous Catalyst for Synthesis of Benzidines and Bicarbazoles.

In this study, a heterogeneous rhodium-catalyzed oxidative homocoupling reaction of anilines utilizing molecular oxygen as the sole oxidant is reported. Employing a commercially available and recyclable Rh/C catalyst enabled the oxidative dimerization of various anilines, including N,N-disubstituted and N-monosubstituted anilines, as well as diarylamines, triarylamines, and carbazoles. Additionally, the catalytic protocol was extended to the ortho-ortho coupling of anilines, affording 2,2'-diaminobiphenyls with high regioselectivity. Notably, the developed approach provides rapid access to diversely functionalized benzidines and diaminobiphenyls in an operationally simple, practical, and environmentally friendly manner.

Probing molecular bond-length using molecular-frame photoelectron angular distributions.

The molecular-frame photoelectron angular distributions (MFPADs) in O 1s photoemission from CO2 molecule were measured. Patterns due to photoelectron diffractions were observed in the MFPADs. The polarization-averaged MFPADs were compared with theoretical calculation and were found to be useful in determining the molecular bond-length, which is a component to determine molecular structures.

Sudden unexpected death due to coronary thrombosis associated with isolated necrotizing vasculitis in the coronary arteries of a young adult.

Coronary arteritis is an uncommon cause of sudden death in non-atherosclerotic coronary diseases, and is mostly associated with systemic vasculitis or systemic autoimmune diseases; therefore, sudden death due to isolated coronary arteritis rarely occurs. The case described in this report is that of a 34-year-old man with no significant personal medical history who died suddenly after presenting with nausea. Postmortem examination revealed a significant infiltration of lymphocytes predominantly on the adventitia and periadventitial tissues of the coronary arteries in the epicardium. The lymphocytic infiltrate partially extended to the thickened intima with fibrosis, destructing the media and internal elastic lamina, and the lumen was occluded by a thrombus in the left main stem and left anterior descending branch. The arterial walls exhibited focal fibrinoid necrosis with regression in the intima and fibrous scars with angiogenesis in the media and adventitia. Focal myocardial infarction was detected in the left ventricle as a fibrotic change of the myocardium. No findings associated with vasculitis were discerned in the aorta, other peripheral arteries, or major organs. Laboratory tests of postmortem blood samples returned negative results for antinuclear antibodies, cryoglobulin, immunoglobulin G4, and cytoplasmic anti-neutrophil cytoplasmic antibodies for myeloperoxidase and proteinase 3. These autopsy findings suggest that the sudden death was caused by isolated necrotizing vasculitis that is assumed to be polyarteritis nodosa localized at the coronary arteries. However, pathological characteristics may not be exactly the same between isolated necrotizing vasculitis in the coronary arteries and polyarteritis nodosa.

C(sp3 )-H Cyanation Promoted by Visible-Light Photoredox/Phosphate Hybrid Catalysis.

Inspired by the reaction mechanism of photo-induced DNA cleavage in nature, a C(sp3 )-H cyanation reaction promoted by visible-light photoredox/phosphate hybrid catalysis was developed. Phosphate radicals, generated by one-electron photooxidation of phosphate salt, functioned as a hydrogen-atom-transfer catalyst to produce nucleophilic carbon radicals from C(sp3 )-H bonds with a high bond-dissociation energy. The resulting carbon radicals were trapped by a cyano radical source (TsCN) to produce the C-H cyanation products. Due to the high functional-group tolerance and versatility of the cyano group, the reaction will be useful for realizing streamlined building block syntheses and late-stage functionalization of drug-like molecules.