Evaluation of a high-speed but low-throughput RT-qPCR system for detection of SARS-CoV-2.

With the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), a high-speed and convenient detection technology should be at the forefront of medical care worldwide. This study evaluated the usefulness of GeneSoC, a compact, high-speed reciprocal flow quantitative reverse transcription polymerase chain reaction system, for the detection of SARS-CoV-2. The results support the use of this system for the rapid identification of SARS-CoV-2. This approach can contribute to the strategic selection of initial management strategies for patients with COVID-19.

Electric and magnetic phenomena observed before the volcano-seismic activity in 2000 in the Izu Island Region, Japan.

Significant anomalous changes in the ultra low frequency range (approximately 0.01 Hz) were observed in both geoelectric and geomagnetic fields before the major volcano-seismic activity in the Izu Island region, Japan. The spectral intensity of the geoelectric potential difference between some electrodes on Niijima Island and the third principal component of geomagnetic field variations at an array network in Izu Peninsula started to increase from a few months before the onset of the volcano-seismic activity, culminating immediately before nearby magnitude 6 class earthquakes. Appearance of similar changes in two different measurements conducted at two far apart sites seems to provide information supporting the reality of preseismic electromagnetic signals.

Induction of nitric oxide synthase by traumatic brain injury.

We investigated the dynamic induction/expression of inducible nitric oxide synthase (iNOS) using human brains made available through death by traumatic brain injury (TBI). Astrocytes, microglia, and neutrophils were identified in tissue using immunohistochemical staining with antibodies against glial fibrillary acidic protein (GFAP), MHC class II antigen, and neutrophil elastase, respectively. The localization of iNOS protein in each of these cell types was evaluated using immunohistochemistry. Within 2 days of injury, iNOS immunoreactivity was not detected. However, after 2 days, immunoreactivity was detected in the traumatized brain. The iNOS immunoreactivity was localized on neutrophils and microglia/macrophages in the areas around the tissue necrosis in the traumatized cortical hemisphere, in the deep part of the cortex and the dentate gyri of the hippocampi adjacent to the hemorrhage, and within the cytoplasm of vascular smooth muscle cell of a small artery or arteriole surrounding the injured region. This reactivity was absent after 8 days post-injury.These observations confirmed the prolonged induction of iNOS within various cells in the injured brain. These responses suggest that iNOS plays a crucial role in cerebrovascular damage and/or secondary brain damage subsequent to traumatic brain injury. Furthermore, the dense nitric oxide (NO) generated by iNOS may play a role in neuronal cell death after injury.

The expression of excitatory amino acid transporter 2 (EAAT2) in forensic autopsy cases.

Glutamate is the major excitatory neurotransmitter and the greater part of this amino acid is removed from the synaptic cleft by excitatory amino acid transporter 2 (EAAT2) located on perisynaptic astrocytes. Recently, it was reported that the EAAT2 protein content changed in rats following forebrain ischemia and administration of methamphetamine. We planned to demonstrate the immunohistochemical distribution of EAAT2 in the human brain and discuss the significance of its pathophysiological roles. Thirty-two cases were used from forensic autopsies. The tissues were sampled from the cerebral cortex, striatum and hippocampus. The distribution of EAAT2 was difficult to identify in cases of electrical fatalities. However, continuous and extensive staining of EAAT2 was observed in cases of death from hypothermia. In almost all asphyxia death, we were able to observe a weak stain of EAAT2. In case of solvent abuse, EAAT2 staining was continuous and extensive as in the cases of hypothermia, and patchy negative zones were mixed. This study clearly showed the differences in EAAT2 localization according to the cause of death. These findings suggested that the differences in EAAT2 staining depended on the cause and course (pathophysiological conditions) of death.

An autopsy case of neuroleptic malignant syndrome (NMS) and its immunohistochemical findings of muscle-associated proteins and mitochondria.

Neuroleptic malignant syndrome (NMS) is a rare but potentially fatal disorder. In forensic cases, post-mortem diagnosis of NMS is sometimes difficult if ante-mortem information, such as neuroleptic ingestion or signs and symptoms, cannot be obtained. A 39-year-old Japanese male on a neuroleptic treatment regimen suddenly became agitated and died. Autopsy revealed muscle rigidity and hyperthermia. Post-mortem examination of blood revealed elevation of creatine phosphokinase-MM (CK-MM) and lactate dehydrogenase-4 and dehydrogenase-5 (LDH-4 and LDH-5). In renal glomeruli and tubules, myoglobin was stained immunohistochemically. From these findings, the cause of death was considered to be NMS. To support the diagnosis of NMS, both skeletal and cardiac muscles were stained with actin, myoglobin, desmin and mitochondria antibodies immunohistochemically. Actin, myoglobin, desmin, and mitochondria had been lost from skeletal, but not from the cardiac muscle, which suggested that only the skeletal muscle was damaged. Moreover, because mitochondria had disappeared only from the skeletal muscle, it was considered that skeletal muscle degeneration was caused by mitochondrial damage. Therefore, it is suggested that immunostaining of skeletal muscle by antibodies for muscle-associated proteins and mitochondria is useful to corroborate a diagnosis of NMS.

Optimization and application of hybrid-level binary zone plates.

The binary zone plate (BZP) is a diffractive optical element whose wide-ranging development is expected to have a strong effect on fields such as optical communications and information processing. With the increasing demand for more-compact systems and devices the BZP needs to be efficient as well as small. It is well known that fabrication errors strongly influence the characteristics of BZPs. To mitigate the influence of fabrication errors and obtain an efficient BZP with a high numerical aperture, we propose a design, called the hybrid-level BZP (HBZP), that combines zones with different numbers of phase levels. A method to correct the phase mismatch generated by such a combination is described. We furthermore discuss the optimum design of HBZPs in the presence of fabrication errors and report on its experimental evaluation.

Spontaneous luxation of encapsulated intraocular lens onto the retina after a triple procedure of vitrectomy, phacoemulsification, and intraocular lens implantation.

PURPOSE: To report the clinical and histological findings of a luxated intraocular lens (IOL) in the capsular bag. METHODS: Review of a case. RESULTS: Twenty-three months after a triple procedure of vitrectomy, phacoemulsification, and IOL implantation for diabetic vitreous hemorrhage and cataract, the encapsulated IOL spontaneously luxated. Scanning electron microscopy showed sparsely distributed anterior and equatorial zonules, with only a few posterior zonules on the surface of the removed capusular bag. CONCLUSION: The absence of the anterior hyaloid membrane and posterior zonules and contraction of the lens capsule may cause dialysis of the zonules. Therefore, the anterior hyaloid membrane should be left in place in patients at low risk for the development of postoperative proliferation to maintain the long-term stability of the IOL.

Cloning of an auxin-responsive 1-aminocyclopropane-1-carboxylate synthase gene (CMe-ACS2) from melon and the expression of ACS genes in etiolated melon seedlings and melon fruits.

Two cDNA fragments (pCMe-ACS2 and 3) encoding auxin-responsive 1-aminocyclopropane-1-carboxylate synthase (ACS; EC.4.4.1.14) have been isolated from melon, and the expression patterns of the genes in etiolated melon seedlings and melon fruit have been determined by RT-PCR analysis. The deduced amino acid sequences of pCMe-ACS2 and 3 were homologous to those of AT-ACS6 and 4, which were auxin-responsive ACS genes of Arabidopsis. Both CMe-ACS2 and 3 were auxin-responsive ACS genes and their expressions in roots and hypocotyls were induced by treatment with indole acetic acid (IAA, 100 µM). The mRNA level of CMe-ACS2 in the fruit increased after pollination. Those of both CMe-ACS2 and 3 temporarily increased in the mesocarp tissues at the preclimacteric stage (from day 3 to day 5 after harvest) during ripening, while that of CMe-ACS3 was lower than that of CMe-ACS2. The increase in the mRNA level of CMe-ACS1 (wound- and ripening-induced gene, T. Miki, M. Yamamoto, N. Nakagawa, O. Ogura, H. Mori, H. Imaseki, T. Sato, Nucleotide sequence of a cDNA for 1-aminocyclopropane-1-carboxylate synthase from melon fruits, Plant Physiol. 107 (1995) 297-298.) in the mesocarp tissue was not observed until 5 days after harvest. A genomic DNA encoding CMe-ACS2 was isolated and its nucleotide sequence was determined. Nucleotide sequences resembling the auxin-responsive elements (AuxRE) D1 and D4 (the TGTCTC element) in the GH3 gene from soybean, and the auxin-responsive domain (AuxRD) B in PS-IAA4/5 from pea were found in the 5'-flanking region of the CMe-ACS2 gene.

Geoelectric potential changes: possible precursors to earthquakes in Japan.

Whether electromagnetic precursors to earthquakes (EQs) exist is an important question not only for EQ prediction but also for understanding the physical processes of EQ generation. Slow transient geoelectric potential changes have been observed before several recent EQs in Japan. In most cases, they appeared 1-19 days before the EQs, and their duration and intensity were several minutes to 1 h and 1-2 mV/100 m. The changes appeared before five of all six EQs with magnitude >/= 5 that occurred within 20 km of our stations during the observation period. Changes were also detected at greater epicentral distances (up to 75 km) before two other EQs, including one EQ of magnitude 4.7, which was preceded by a signal simultaneously recorded at three widely separated stations. These geoelectric potential changes have been distinguished through the following criteria from a multitude of other changes, which were noise of various origins. (i) The selected changes were proportional in amplitude to the length of the recording station's short ( approximately 100 m) dipoles and were simultaneously detected also on long (1-10 km) dipoles when the latter were in operation. (ii) No such changes occurred during the observation period that were not followed by EQs. Although the EQ precursory nature of these geoelectric potential changes is admittedly unproven, it seems that the present results warrant continued serious research into the occurrence, generation, and transmission of these signals and their possible causal relationship to EQs.

HSP70 and c-Fos expression of brain stem hypoglossal nucleus in drowning.

The brain stem hypoglossal nucleus (HN) is the center of nerves innervating the upper respiratory tract and is related to control of mastication, deglutition, speech and respiration. To elucidate the relationship between asphyxia and the HN, we investigated the change of hypoglossal neurons in cases of hanging, strangulation, smothering, choking, drowning and respiratory failure. Using immunohistochemical techniques, we observed the brain stem HN with antibodies against microtubule-associated protein 2 (MAP2), muscarinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). MAP2, a cytoskeletal protein of the neuron, is a marker of neuronal damage. Muscarinic AChR was used as a marker of neuronal membrane and ACh signaling. We employed both HSP70 and c-Fos as markers of stress- or damage-related events. We measured the percentage of immunopositive neurons in total neurons of HN. Drowning produced higher expression of HSP70 and c-Fos than other causes of asphyxia, suggesting that drowning induces more severe damage in HN neurons. Furthermore, it was suspected that neuronal changes in drowning might relate to functions of the HN. These observations indicate that immunohistochemical examination of the brain stem HN could provide useful information for determining the cause of asphyxia.

[Forensic pathological significance of iNOS with regard to brain and myocardial damage].

Inducible nitric oxide synthase (iNOS) does not express within cells under normal conditions. But, it is induced by inflammatory cytokines and the activity of iNOS is independent of elevated Ca2+. It is implied that nitric oxide (NO) produced by iNOS has a close relation with pathological conditions as a mediator of cell damages. We aimed at the crucial property of iNOS, therefore we have searched the expression of iNOS for the purpose to establish forensic pathological significance in the cases with damages of neuronal cells or cardiomyocytes. At first, we examined induction and expression of iNOS in the human brains whose causes of death was traumatic brain injury (TBI). These findings demonstrated prolonged induction of iNOS in neutrophils, microglia/macrophages, and vascular smooth muscle cells in traumatized brain. Such a response may indicate that iNOS could play a crucial role on cerebrovascular disturbances and secondary brain injury subsequent to TBI. Secondly, we evaluated the induction and expression of iNOS in the human hearts with myocardial cell damage. The expressions of iNOS were localized to the following areas, the cardiomyocytes in the zone adjacent to the infarcted area, the preserved cytoplasm of infarcted cardiomyocytes, the endothelial cells in arteriolar walls and macrophages in viable myocardium. These findings have led to the speculation that the NO might play a significant role during myocardial ischemia and reperfusion. Furthermore, representative expression of iNOS was identified in coronary artery, that is the portion of shoulder and broken part of coronary atherosclerotic plaque. These findings may indicate that the iNOS might have a close relation to the coronary atherosclerotic lesion. The physiopathological mechanism of iNOS might involve in the close relation with inflammatory cytokines. The precise elucidation of this participation may make it possible to explicate the presumed time of injury in an earlier stage, and to clarify the forensic pathological significance of these inflammatory factors.

Japanese population study of a Y-linked dinucleotide repeat DNA polymorphism.

A polymorphic CA repeats (YCA II) was previously reported on the human Y chromosome. We have used a simple technique based on polymerase chain reaction amplification followed by native polyacrylamide gel electrophoresis to study the inheritance, the genetic stability, and the allele frequency distribution of this polymorphism in the Japanese. We found seven haplotypes which were tentatively designated as: A[(CA)19/(CA)21], B[(CA)19/(CA)22], C[(CA)19/(CA)23], D[(CA)19/(CA)19], E[(CA)21/(CA)21], F[(CA)22/(CA)22], and G[(CA)23/(CA)23]. The frequencies of these haplotypes were: A, 0.21; B, 0.29; C, 0.37; D, 0.02; E, 0.02; F, 0.07; G, 0.01. There was complete concordance with each father-son pairs. The results indicate the dinucleotide system YCA II is very useful for investigation of forensic samples, especially mixed stains in sexual offence cases.

Electrolyte concentration differences between left and right vitreous humor samples.

Between-eye differences in electrolyte concentrations were studied in 200 medico-legal autopsies using an ion-specific electrode system. Taking the highest of the paired vitreous potassium concentrations, cases < 15 mM/L were classified as biochemically nonputrefied (Cat.1, n = 124), cases 15 to 20 mM/L as early putrefaction (Cat.2, n = 51), and cases > 20 mM/L as biochemically putrefied (Cat.3, n = 25). Mean paired vitreous sodium for all cases (n = 200) was 112 to 173 mM/L (mean 148, standard deviation (SD) = 8.9); between-eye differences were 0 to 8 mM/L (0% to 5.1% of mean), averaging 1.5 mM/L (1%) and with only one case (in Cat.3) outside instrument accuracy (+/- 3 mM/L). Mean paired vitreous chloride for all cases was 73 to 124 mM/L (mean 109, SD = 7.8); between-eye differences were 0 to 9 mM/L (0% to 8.8% of mean), averaging 1.7 mM/L (1.5%) and with 5 of 200 cases outside instrument accuracy (+/- 3 mM/L). Thus between-eye concentration differences of sodium and chloride are tolerable using this methodology. Previous reports of greater variability likely reflect errors introduced by sample manipulation prior to analysis. By contrast, between-eye differences in potassium in Cat.1 cases were 0 to 2.34 mM/L (0% to 21.8% of mean) averaging 0.37 mM/L (3.3%). Significant and erratic between-eye differences in potassium undermine the usefulness of vitreous potassium in estimation of time of death.

Immunohistochemical diagnosis and significance of forensic neuropathological changes.

Immunohistochemistry is very useful when investigating the cause of death. Ischemic cell changes in the hippocampal neurons were not obvious in the brains damaged by hypoxic injury. However, it is suggested that even a moderate hypoxia, which may affect the neuronal proteins and metabolism, induced astrocytosis in the CA3 and CA4 regions, and that in patients with a history of hypoxic attacks neuronal damage may be severe even several hours after ischemic injury. Furthermore, hsp70 expression was found in the CA2, CA3 and CA4 regions of long-term survivors after severe hypoxic/ischemic injury. In forensic practice, detailed information about the duration and extent of a hypoxic/ischemic injury is often unavailable, so that immunohistochemical detection of hsp70 and glial cell staining can be of great value in diagnosing not only the hypoxic/ischemic injury during the process of death but also the victim's past history of hypoxic attacks. In diffuse axonal injury, degeneration of axon and myelin, such as swelling and waving, were observed in survivors of more than 8 hours. Retraction balls appeared in survivors of more than 1 days. In longer term survivors, such as 3 or 5 months, breakdown of myelin and fat-granule cells were observed. In addition, retraction balls were also found. Immunohistochemical staining of 200 kD neurofilament was a very useful method to examine axonal changes, because antisera is specific for degenerative neurofilaments. In our study, all cases which had pathological findings of diffuse axonal injury (DAI) were associated with focal head injuries. From the immunohistochemical staining of neurons in the hippocampus, it was suggested that neurons in the hippocampus were injured by diffuse brain damage. Furthermore, repairing and protective mechanisms occurred especially from CA2 to CA4. It was considered that neuronal damage in diffuse brain injury was elucidated not only morphologically but also functionally. Therefore, in cases of suspected diffuse brain damage, it is recommended to examine the neuronal changes in addition to observing the findings of diffuse axonal injury. Immunohistochemical staining of the carotid body is potentially very useful for necropsy diagnosis, since it provides a method to detect evidence of mechanical asphyxia in suspected cases of manual and/or ligature strangulation.

[Immunohistochemical studies on neuronal changes in brain stem nucleus of forensic autopsied cases. I. Various cases of asphyxia and respiratory disorder].

Several nuclei in brain stem are well known to play an important role in supporting human life. However, the connection between neural changes of brain stem and the cause of death is not yet fully understood. To investigate the correlation of brain stem damage with various cause of respiratory disorders, neural changes of the arcuate nucleus (ARC), the hypoglossal nucleus (HN) and the inferior olivary nucleus (IO) were examined using immunohistochemical technique. Based on the cause of death, the forensic autopsy cases were divided into 5 groups as follows. Group I: hanging, ligature strangulation and manual strangulation, Group II: smothering and choking, Group III: drowning, Group IV: respiratory failure, control group: heat stroke and sun stroke. Brain was fixed with phosphate-buffer formalin, and the brain stem was horizontally dissected at the level of apex, then embedded in paraffin. The sections were stained with the antibodies against microtubule-associated protein 2 (MAP2), muscalinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). Three nuclei showed no obvious morphological changes in all examined groups. However, in case of asphyxia (Group I to III), neurons in HN were positively stained with both HSP70 and c-Fos antibodies. This may indicate that the occlusion of upper airway results in the neuronal damage of HN without their morphological changes. Positive staining of HSP70 and c-Fos in IO was more frequently observed in Group III than other 4 groups. Since IO is involved in maintaining body balance which is often disturbed by drowning, it seems possible that neuronal damage in IO observed in drowning may be related to the disturbance of body balance. These observations indicate that immunohistochemical study on the damage to neurons in brain stem nuclei can provide useful information for determining the cause of death.

[Immunohistochemical studies on neuronal changes in brain stem nucleus of forensic autopsied cases. II. Sudden infant death syndrome].

Several nuclei in brain stem are well known to play an important role in supporting human life. However, the connection between neural changes of brain stem and the cause of death is not yet fully understood. Previously, in sudden infant death syndrome (SIDS) it has been suggested that impaired cardioventilatory control might contribute to cause of death. So, to investigate the brain stem damage in SIDS, neural changes of the arcuate nucleus (ARC), the hypoglossal nucleus (HN) and the inferior olivary nucleus (IO) was examined using immunohistochemical technique. Brain was fixed with phosphate-buffer formalin, and the brain stem was horizontally dissected at the level of apex, then embedded in paraffin. The sections were stained with the antibodies against microtubule-associated protein 2 (MAP2), muscalinic acetylcholine receptor (mAChR), c-fos gene product (c-Fos) and 72 kD heat-shock protein (HSP70). Morphological changes of neurons in three nuclei were not evident. Moreover, because MAP2 degeneration and expression of HSP70 and c-Fos were not observed, neuronal damage in those nuclei was not suspected. However, although there was no abnormality of mAChR immunostaining in HN and IO, the rate of mAChR-immunopositive neurons in ARC was less than that in control. These observations indicate that immunohistochemical study on the neuronal changes in ARC can provide useful information for diagnosing SIDS.

Detection of white restorative dental materials using an alternative light source.

We assessed the value of an alternative light source for detecting white composite dental materials in burned and unburned teeth. Teeth filled with 18 different restorative materials (composite, glass ionomer or hybrid composite), were viewed with a Polilight. Between 415 nm and 555 nm, the glass ionomers showed distinctly different optical properties from the other materials: they either fluoresced or appeared darker. Wavelengths 415 nm to 530 nm gave a general enhancement in composite detection (17 of 18 materials). Light above 590 nm was of little value, enhancing detection in only 2 of 18 materials. After simulated burning of the teeth, there was enhanced visibility of 8 of 18 materials at wavelengths under 350 nm. Burning destroyed the previously distinct optical properties of the glass ionomers. Overall, this alternative light source aids the identification of white composite dental materials and could be used in routine forensic odontology practice.

Isolated adrenocorticotropic hormone deficiency: an autopsy case of adrenal crisis. A case report.

We present a case of fatal adrenal crisis due to isolated adrenocorticotropic hormone (ACTH) deficiency. Autopsy revealed each adrenal gland weighed 0.9 g and the adrenal cortexes were very thin and atrophic. Additionally, cortisol could not be observed in the adrenal cortex by immunohistochemical staining. Furthermore, urine cortisol and 17-OHCS concentration had decreased to a very low level, 20 mg/L and 0.8 mg/L respectively. The anterior pituitary gland was atrophic, and showed fibrosis and lymphocytosis was suspected. Immunohistochemically growth hormone (GH)-stained pituitary gland cells were observed, but there were no cells stained with anti-ACTH antibody. From the history and pathological findings, no other deficiencies of pituitary hormones were evident. Therefore, isolated ACTH deficiency was suspected. Furthermore, as the thyroid gland showed lymphocytic thyroiditis, is was considered that isolated ACTH deficiency was associated with an autoimmune cause. Generally, as patients of chronic adrenocortical insufficiency are exposed to stress and, therefore, have an increased requirement for glucocorticoids, the blood pressure falls, leading to hypovolemic shock called " an adrenal crisis." Without treatment, patients die in crisis within several hours. In our case, the deceased had drunk alcohol without sleep for 2 days. We believe that the stress of drinking and sleeplessness induced adrenal crisis and caused his death.

The fates of the callosal neurons in neocortex after bisection of the corpus callosum, using the technique of retrograde neuronal labeling with two fluorescent dyes.

The fate of callosal neurons after callosotomy is yet unclear although this has become a common surgical procedure for intractable generalized epilepsies. Using retrograde neuronal labeling with two fluorescent dyes, we demonstrated that callosal neurons in the parietal cortex of the adult rat survive up to 20 weeks after callosotomy. Our data suggest that these neurons possess numerous ipsilateral axon collaterals with indispensable functions in the ipsilateral hemisphere.