Sudden death of a 2-year-old child due to alpha-ketoadipic aciduria.

Alpha-ketoadipic acid is one of the metabolic intermediates of lysine and tryptophan, and it is known as the biochemical hallmark of alpha-ketoadipic aciduria (α-KA). α-KA is a rare autosomal recessive disorder. Its pathophysiology is reduced alpha-ketoadipic acid dehydrogenase activity, and that makes it difficult to metabolize lysine and tryptophan. The symptoms of this disease are multiple, e.g., psychomotor retardation, epilepsy, and ataxia, and it can even be asymptomatic. We present a case of sudden death in a 2-year-old boy with alpha-ketoadipic aciduria. Postmortem computed tomography (CT) and autopsy were performed to elucidate the cause of death. No obvious lesions could be identified except for a marked fatty liver. Urinalysis showed elevated excretion of α-ketoadipic acid.

Proposal of a liver histology-based scoring system for bile salt export pump deficiency.

AIM: Bile salt export pump (BSEP) deficiency manifests a form of progressive intrahepatic cholestasis. This study aimed to establish a scoring system of liver histology for the uncommon genetic condition. METHODS: After a roundtable discussion and histology review, a scoring system for BSEP deficiency was established. Eleven tissue samples were independently evaluated by three pathologists based on the proposed standard for an interobserver agreement analysis. In four cases with serial tissue samples available, correlation between changes in histology scores and clinical outcome was examined. RESULTS: Of 14 initially listed histopathological findings, 12 were selected for scoring and grouped into the following four categories: cholestasis, parenchymal changes, portal tract changes and fibrosis. Each category consisted of two to four microscopic findings that were further divided into three to six scores; therefore, each category had a maximum score of 8-11. Interobserver agreement was highest for pericellular fibrosis (κ = 0.849) and lowest for hepatocellular cholestasis (κ = 0.241) with the mean and median κ values of the 12 parameters being 0.561 and 0.602, respectively. For two patients whose clinical features worsened, score changes between two time points were interpreted as deteriorated. In two patients, who showed a good clinical response to preprandial treatment with sodium 4-phenylbutyrate, histological changes were evaluated as improved or unchanged. CONCLUSIONS: The proposed histology-based scoring system for BSEP deficiency with moderate interobserver agreement may be useful not only for monitoring microscopic changes in clinical practice but also for a surrogate endpoint in clinical trials.

CYP7A1 expression in hepatocytes is retained with upregulated fibroblast growth factor 19 in pediatric biliary atresia.

AIM: Bile acid biosynthesis is strictly regulated under physiological conditions. The expression of fibroblast growth factor (FGF) 19 is induced when bile acids bind to the farnesoid X receptor in the intestinal epithelium. Fibroblast growth factor 19 is then transported by the portal flow, causing transcriptional inhibition of cytochrome P450, family 7, subfamily A, polypeptide 1 (CYP7A1), a key enzyme in bile acid biosynthesis, through the extracellular signal-regulated kinase (ERK) pathway. However, the regulatory mechanisms of these signaling pathways in hepatocytes under chronic cholestasis remain unclear. We investigated the regulation of these signaling pathways in patients with biliary atresia (BA). METHODS: We analyzed the regulation of molecules in these signaling pathways using liver and serum samples from eight BA children and four non-cholestatic disease controls. RESULTS: CYP7A1 mRNA expression was not inhibited in BA microdissected hepatocyte-enriched tissue (HET) despite high serum bile acid concentrations. The FGF19 protein was synthesized in BA HET, and its serum concentration was elevated. Fibroblast growth factor receptor 4 was phosphorylated in BA livers. However, ERK phosphorylation was significantly reduced. We examined SPRY2 expression to determine how the ERK pathway was inactivated downstream of the FGF receptor; the expression was significantly increased in BA HET. CONCLUSIONS: This is the first study to measure the CYP7A1 mRNA levels in human BA HET. Fibroblast growth factor 19 was increased in BA hepatocytes. By focusing on its regulation in hepatocytes, we showed that the FGF19 pathway did not suppress bile acid synthesis, probably due to an altered mechanism involving upregulated SPRY2 in BA patients.

Circulating tricarboxylic acid cycle metabolite levels in citrin-deficient children with metabolic adaptation, with and without sodium pyruvate treatment.

Citrin deficiency causes adult-onset type II citrullinemia (CTLN-2), which later manifests as severe liver steatosis and life-threatening encephalopathy. Long-standing energy deficit of the liver and brain may predispose ones to CTLN-2. Here, we compared the energy-driving tricarboxylic acid (TCA) cycle and fatty acid ß-oxidation cycle between 22 citrin-deficient children (age, 3-13years) with normal liver functions and 37 healthy controls (age, 5-13years). TCA cycle analysis showed that basal plasma citrate and α-ketoglutarate levels were significantly higher in the affected than the control group (p<0.01). Conversely, basal plasma fumarate and malate levels were significantly lower than those for the control (p<0.001). The plasma level of 3-OH-butyrate derived from fatty acid ß-oxidation was significantly higher in the affected group (p<0.01). Ten patients underwent sodium pyruvate therapy. However, this therapy did not correct or attenuate such deviations in both cycles. Sodium pyruvate therapy significantly increased fasting insulin secretion (p<0.01); the fasting sugar level remained unchanged. Our results suggest that citrin-deficient children show considerable deviations of TCA cycle metabolite profiles that are resistant to sodium pyruvate treatment. Thus, long-standing and considerable TCA cycle dysfunction might be a pivotal metabolic background of CTLN-2 development.

Estimation of cadaveric age in crime scenes using Raman spectroscopy.

The number of unidentified corpses has been increasing in recent years. There is a need for an objective and readily applicable method to estimate age, which is important information for identification. In previous reports, we reported that the protein folding ratio (RPF) of skin, as measured by Raman spectroscopy using cross sections of skin samples, is highly correlated with age. In this study, we investigated the possibility of estimating age by measuring Raman spectra from the skin surface of cadavers using a portable device. The resultant intercept, slope, and root mean square error were 97.9, - 63.7 (p < 0.0001), and 11.68, respectively. We evaluated this regression formula by using 10-fold cross-validation, resulting in a coefficient of determination of 0.51. The portable Raman spectrometer may be of assistance in estimating age at death of corpses at the scene of discovery.

Clinical symptoms, biochemistry, and liver histology during the native liver period of progressive familial intrahepatic cholestasis type 2.

BACKGROUND: Progressive familial intrahepatic cholestasis type 2 (PFIC2) is an ultra-rare disease caused by mutations in the ABCB11 gene. This study aimed to understand the course of PFIC2 during the native liver period. METHODS: From November 2014 to October 2015, a survey to identify PFIC2 patients was conducted in 207 hospitals registered with the Japanese Society of Pediatric Gastroenterology, Hepatology, and Nutrition. Investigators retrospectively collected clinical data at each facility in November 2018 using pre-specified forms. RESULTS: Based on the biallelic pathogenic variants in ABCB11 and/or no hepatic immunohistochemical detection of BSEP, 14 Japanese PFIC2 patients were enrolled at seven facilities. The median follow-up was 63.2 [47.7-123.3] months. The median age of disease onset was 2.5 [1-4] months. Twelve patients underwent living donor liver transplantation (LDLT), with a median age at LDLT of 9 [4-57] months. Two other patients received sodium 4-phenylbutyrate (NaPB) therapy and survived over 60 months with the native liver. No patients received biliary diversion. The cases that resulted in LDLT had gradually deteriorated growth retardation, biochemical tests, and liver histology since the initial visit. In the other two patients, jaundice, growth retardation, and most of the biochemical tests improved after NaPB therapy was started, but pruritus and liver fibrosis did not. CONCLUSIONS: Japanese PFIC2 patients had gradually worsening clinical findings since the initial visit, resulting in LDLT during infancy. NaPB therapy improved jaundice and growth retardation but was insufficient to treat pruritus and liver fibrosis.

Vinculin functions as regulator of chondrogenesis.

To identify the genes involved in chondrocytic differentiation, we applied gene trap mutagenesis to a murine mesenchymal chondrogenic cell line ATDC5 and isolated a clone in which the gene encoding vinculin was trapped. The trapped allele was assumed to express a fusion protein containing a truncated vinculin lacking the tail domain and the geo product derived from the trap vector. The truncated vinculin was suggested to exert a dominant negative effect. Impaired functioning of vinculin caused by gene trapping in ATDC5 cells or knockdown in primary chondrocytes resulted in the reduced expression of chondrocyte-specific genes, including Col2a1, aggrecan, and Col10a1. The expression of Runx2 also was suppressed by the dysfunctional vinculin. On the other hand, the expression of Sox9, encoding a key transcription factor for chondrogenesis, was retained. Knockdown of vinculin in metatarsal organ cultures impaired the growth of the explants and reduced the expression of Col2a1 and aggrecan. Gene trapping or knockdown of vinculin decreased the phosphorylation of ERK1/2 but increased that of Src homology 2 domain-containing tyrosine phosphatase 2 (SHP2) and Akt during chondrocytic differentiation, suggesting a disturbance of signaling by insulin-like growth factor I (IGF-I). Knockdown of vinculin in the metatarsal organ culture abrogated the IGF-I-induced growth and inhibited the up-regulation of Col2a1 and aggrecan expression by IGF-I. Loss of vinculin function in differentiating chondrocytes impaired the activation of the p38 MAPK pathway also, suggesting its involvement in the regulation of chondrogenesis by vinculin. Our results indicate a tissue-specific function of vinculin in cartilage whereby it controls chondrocytic differentiation.

Sodium-coupled neutral amino acid transporter 4 functions as a regulator of protein synthesis during liver development.

AIM: The molecular mechanisms by which hepatocyte nuclear factor (HNF)4α regulates fetal liver development have not been fully elucidated. We screened the downstream molecules of HNF4α during liver development and identified sodium-coupled neutral amino acid transporter (SNAT)4. The aim of this study is to investigate the regulation of SNAT4 by HNF4α and to clarify its roles in differentiating hepatocytes. METHODS: HNF4α was overexpressed in cultured liver buds using adenovirus, and suppression subtractive hybridization screening was performed. Temporal and spatial expression of SNAT4 during liver development was investigated. Regulation of SNAT4 by HNF4α was examined by promoter analyses and electrophoretic mobility shift assays (EMSA). Metabolic labeling and western blotting were carried out using primary hepatoblasts with SNAT4 overexpression. RESULTS: The expression of Slc38a4 encoding SNAT4 showed a marked perinatal increase, and was predominant among system A amino acid transporters. It was first detected in embryonic day 18.5 liver, and found in most hepatocytes after birth. Three alternative first exons were found in the SNAT4 gene. Promoter analyses using approximately 3-kb fragments corresponding to each first exon (AP1, AP2, AP3) revealed that AP1 and AP2 exhibited strong promoter activity in mouse hepatoblasts with endogenous HNF4α. Transactivation of AP2 was upregulated by HNF4α in HeLa cells without endogenous HNF4α. EMSA has demonstrated that HNF4α directly binds to cis-elements in AP2. Overexpression of SNAT4 facilitated amino acid uptake and de novo protein synthesis in primary hepatoblasts. CONCLUSION: SNAT4 functions downstream of HNF4α and plays significant roles in liver development through mechanisms of amino acid uptake and protein synthesis.

A suicide case by hanging without fixing the end of the rope.

Hanging is a method of death in which one end of a cord is tied around the neck and the other end is tied to an unmovable object and hung down, compressing the neck with weight. We have identified a rare case in which death was accomplished without tying one end of the rope. The individual was successfully hanged by the frictional force between the upward rope and the downward rope. The police concluded the case as a suicide.

Artificial intelligence-based forensic sex determination of East Asian cadavers from skull morphology.

Identification of unknown cadavers is an important task for forensic scientists. Forensic scientists attempt to identify skeletal remains based on factors including age, sex, and dental treatment remains. Forensic scientists commonly consider skull or pelvic shape to evaluate the sex; however, these evaluations require sufficient experience and knowledge and lack objectivity and reproducibility. To ensure objectivity and reproducibility for sex evaluation, we applied a gated attention-based multiple-instance learning model to three-dimensional (3D) skull images reconstructed from postmortem head computed tomography scans. We preprocessed the images, trained with 864 training data, validated the model with 124 validation data, and evaluated the performance of our model in terms of accuracy with 246 test data. Furthermore, three forensic scientists evaluated the 3D skull images, and their performances were compared with those of the model. Our model showed an accuracy of 0.93, which was higher than that of the forensic scientists. Our model primarily focused on the entire skull owing to visualization but focused less on the areas often investigated by forensic scientists. In summary, our model may serve as a supportive tool to identify cadaver sex based on skull shape. Further studies are required to improve the model's performance.

Deep Neural Networks-Based Age Estimation of Cadavers Using CT Imaging of Vertebrae.

Although age estimation upon death is important in the identification of unknown cadavers for forensic scientists, to the best of our knowledge, no study has examined the utility of deep neural network (DNN) models for age estimation among cadavers. We performed a postmortem computed tomography (CT) examination of 1000 and 500 male and female cadavers, respectively. These CT slices were converted into 3-dimensional images, and only the thoracolumbar region was extracted. Eighty percent of them were categorized as training datasets and the others as test datasets for both sexes. We fine-tuned the ResNet152 models using the training datasets. We conducted 4-fold cross-validation, and the mean absolute error (MAE) of the test datasets was calculated using the ensemble learning of four ResNet152 models. Consequently, the MAE of the male and female models was 7.25 and 7.16, respectively. Our study shows that DNN models can be useful tools in the field of forensic medicine.

Utility of a rapid assay for prostaglandin E-major urinary metabolite as a biomarker in pediatric ulcerative colitis.

Prostaglandin E-major urinary metabolite (PGE-MUM) is a urinary biomarker reflecting ulcerative colitis (UC) activity. This prospective observational study aimed to evaluate the usefulness of PGE-MUM via rapid chemiluminescent enzyme immunoassay in detecting endoscopic remission (ER) and histologic remission (HR) in pediatric UC (6-16 years) in comparison with fecal calprotectin (FCP). ER and HR were defined as Mayo endoscopic score (MES) of 0 and Matts' histological grades (Matts) of 1 or 2, respectively. A total of 104 UC and 39 functional gastrointestinal disorder (FGID) were analyzed. PGE-MUM levels were significantly higher in the UC group than in the FGID group (P < 0.001). FCP levels were significantly elevated in the group without ER and HR than in the group with ER and HR (P < 0.001 and P = 0.001), whereas PGE-MUM levels were significantly higher in the group without ER compared to the group with ER (P < 0.001). No significant differences were noted in the AUCs for PGE-MUM and FCP in detecting ER and HR. Although PGE-MUM was inferior to FCP for the detection of HR, it might have the potential for application as a biomarker of endoscopic activity in pediatric UC owing to its noninvasive and rapid method.

Humoral immunity is involved in the development of pericentral fibrosis after pediatric live donor liver transplantation.

Although LT can be successful for treating end-stage liver disease in children, some patients develop fibrosis around the central vein area (PCF). This raises the possibility that PCF could lead to later cirrhosis and graft failure. Here, we report a retrospective immunohistochemical study of 28 patients who received a live donor liver transplant. We assessed the incidence and etiology of PCF using CD3, CD20, HLA-DR, and C4d-specific antibodies. Histological evidence of PCF was found in 13 cases (46.4%), of which 11 (84.6%) had experienced ACR and/or CP events post-transplant. Immunohistochemical evaluation revealed significantly stronger staining with these antibodies in the central vein area in PCF, especially for CD20 and C4d. This implies humoral immunopathology and suggests involvement of humoral immunity in the development of PCF. These results further imply that suppression of cellular immunity alone is insufficient to prevent PCF. We therefore suggest that suppression of both humoral and cellular immunity in combination would be required for prevention of PCF.

Estimating the age at death for forensic cases using quantitative computed tomography.

Estimation of the age at death is an important task for forensic scientists. Although the correlation between age and bone mineral density is already known, including for cadavers, to our knowledge, there are no published studies on age estimation with quantitative computed tomography. Quantitative computed tomography can be used to measure bone mineral density based on the mean computed tomography value of the cancellous bone. As this value cannot be calculated in putrefied cases, we modified quantitative computed tomography to calculate the bone mineral density from regions of the bone with mean computed tomography values of 50-350 Hounsfield units. We aimed to examine whether this method could be used for age estimation. We examined 171 male and 106 female cadavers, some of which were putrefied. We performed univariate linear regression analysis for age at death and bone mineral density. The resultant intercept, slope, and root mean square error were 91.3, - 0.20 (p < 0.0001), and 11.4, respectively, for male cadavers, and 96.1, - 0.23 (p < 0.0001), and 11.0, respectively, for female cadavers. We evaluated this regression formula by using 10-fold cross-validation, resulting in a coefficient of determination of 0.33 for male cadavers and 0.42 for female cadavers. The modified quantitative computed tomography method may be of assistance in estimating age at death, even in putrefied cases.

Rapid infusion of excessive phenytoin: A newborn autopsy case.

The patient was a two-day-old female infant. The patient's mother was a primigravid in her 20 s who developed premature abruption of the normal placenta on the first day of the 33rd week of gestation. The infant was born by emergency cesarean section with severe neonatal asphyxia with a birth weight of 1928 g. Spontaneous circulation was returned 11 min after birth. The infant was treated under mechanical ventilation in the neonatal intensive care unit, and phenobarbital was administered for repeated seizures. On day 2, spontaneous respiration was observed; however, the patient developed seizures repeatedly. The dose of phenobarbital reached the maximum and was switched to midazolam. In the early morning of day 3, while midazolam was administered up to the maximum dose, the infant developed status epilepticus, and the anticonvulsant drug was changed to phenytoin. Due to a calculation error, the intravenous administration of phenytoin was started at 400 mg/30 min, which is 10-fold of the normal dose. Six minutes later, after 80 mg was administered, the administration was stopped due to a drop in blood pressure; however, the infant died of cardiac arrest. An autopsy, which was performed approximately 25 h after death, revealed the blood phenytoin concentration in the heart was 63.85 µg/mL. The cause of death was determined to be acute phenytoin toxicity. This is the first fatal case reported of the blood concentration of phenytoin caused by rapid intravenous administration.

The coronary artery calcification severity on postmortem CT could be a clue for the diagnosis of sudden cardiac death.

About two-thirds of sudden deaths are sudden cardiac deaths (SCD), and ischemic heart disease (IHD) accounts for 60% of these. Although an autopsy needs to be performed to prove SCD, the forensic autopsy rate is very low in Japan. To diagnose the cause of death, postmortem computed tomography (PMCT) is often performed. Because coronary artery calcification (CAC) is a risk factor in cardiac diseases such as IHD and its severity can be evaluated with CT, we examined its ability to diagnose SCD. We collected 104 autopsy cases with CT scans. On the basis of the autopsy report, we separated the cases into two groups: SCD suspected as the cause of death and SCD not suspected. We calculated each CAC severity with the Agatston score from the CT images. Cases with Agatston scores of more than 400 were labeled as severe. The relationship between SCD and CAC severity was confirmed with Fisher's exact test (p < 0.05). The sensitivity and specificity of CAC severity for SCD were 20.3% and 97.5%, respectively, and the positive likelihood ratio was 8.1. Severe CAC can increase the probability of SCD. In cases in which only PMCT can be performed, this finding can be helpful for diagnosing SCD.