In vitro functional analysis of four variants of human asparagine synthetase.

The loss-of-function variants of the human asparagine synthetase (ASNS) gene cause asparagine synthetase deficiency (ASNSD). Diagnosis of ASNSD requires genetic tests because a specific biochemical diagnostic for ASNSD is not available. There are a few reports describing the functional evaluation of ASNS variants. Therefore, in vitro methods are needed to evaluate the detected variants in patients. In this report, five types of human ASNS proteins (wild-type and our reported four variants: p.Leu145Ser, p.Leu247Trp, p.Val489Asp, and p.Trp541Cysfs*5) were expressed in silkworm using a baculoviral expression system. An enzymatic activity assay of ASNS was performed, and the concentration of asparagine by ninhydrin and High Performance Liquid Chromatography methods using the purified recombinant proteins was measured. We established ASNS deficient HEK293 cells using the CRISPR/Cas9 method and evaluated the growth of cells without asparagine after transduction of ASNS variants with a lentiviral expression system. The four ASNS variants displayed significantly low enzymatic activity. The ASNS deficient HEK293 cells transduced with wild-type ASNS grew without asparagine, whereas cells transduced with the variants did not grow or showed significantly slower growth than cells transduced with wild-type ASNS. Herein, we established a method for evaluating the enzymatic activity of the recombinant human ASNS variants. The results of the cell-based assay corroborated the results of the enzymatic activity. These methods should enable the evaluation of the pathogenicity of ASNS variants.

Deficiency of 3-hydroxybutyrate dehydrogenase (BDH1) in mice causes low ketone body levels and fatty liver during fasting.

d-3-Hydroxy-n-butyrate dehydrogenase (BDH1; EC 1.1.1.30), encoded by BDH1, catalyzes the reversible reduction of acetoacetate (AcAc) to 3-hydroxybutyrate (3HB). BDH1 is the last enzyme of hepatic ketogenesis and the first enzyme of ketolysis. The hereditary deficiency of BDH1 has not yet been described in humans. To define the features of BDH1 deficiency in a mammalian model, we generated Bdh1-deficient mice (Bdh1 KO mice). Under normal housing conditions, with unrestricted access to food, Bdh1 KO mice showed normal growth, appearance, behavior, and fertility. In contrast, fasting produced marked differences from controls. Although Bdh1 KO mice survive fasting for at least 48 hours, blood 3HB levels remained very low in Bdh1 KO mice, and despite AcAc levels moderately higher than in controls, total ketone body levels in Bdh1 KO mice were significantly lower than in wild-type (WT) mice after 16, 24, and 48 hours fasting. Hepatic fat content at 24 hours of fasting was greater in Bdh1 KO than in WT mice. Systemic BDH1 deficiency was well tolerated under normal fed conditions but manifested during fasting with a marked increase in AcAc/3HB ratio and hepatic steatosis, indicating the importance of ketogenesis for lipid energy balance in the liver.

Recent advances in understanding beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) deficiency.

Beta-ketothiolase (mitochondrial acetoacetyl-CoA thiolase, T2) deficiency (OMIM #203750, *607809) is an inborn error of metabolism that affects isoleucine catabolism and ketone body metabolism. This disorder is clinically characterized by intermittent ketoacidotic crises under ketogenic stresses. In addition to a previous 26-case series, four series of T2-deficient patients were recently reported from different regions. In these series, most T2-deficient patients developed their first ketoacidotic crises between the ages of 6 months and 3 years. Most patients experienced less than three metabolic crises. Newborn screening (NBS) for T2 deficiency is performed in some countries but some T2-deficient patients have been missed by NBS. Therefore, T2 deficiency should be considered in patients with severe metabolic acidosis, even in regions where NBS for T2 deficiency is performed. Neurological manifestations, especially extrapyramidal manifestations, can occur as sequelae to severe metabolic acidosis; however, this can also occur in patients without any apparent metabolic crisis or before the onset of metabolic crisis.

Differential Metabolic Responses to Adipose Atrophy Associated with Cancer Cachexia and Caloric Restriction in Rats and the Effect of Rikkunshito in Cancer Cachexia.

Despite the similar phenotypes, including weight loss, reduction of food intake, and lower adiposity, associated with caloric restriction (CR) and cancer cachexia (CC), CC is a progressive wasting syndrome, while mild CR improves whole body metabolism. In the present study, we compared adipose metabolic changes in a novel rat model of CC, mild CR (70% of the food intake of control rats, which is similar to the food consumption of CC rats), and severe CR (30% of the food intake of controls). We show that CC and severe CR are associated with much smaller adipocytes with significantly lower mitochondrial DNA content; but, that mild CR is not. CC and both mild and severe CR similarly upregulated proteins involved in lipolysis. CC also downregulated proteins involved in fatty acid biosynthesis, but mild CR upregulated these. These findings suggest that CC might impair de novo fatty acid biosynthesis and reduce mitochondrial biogenesis, similar to severe CR. We also found that rikkunshito, a traditional Japanese herbal medicine, does not ameliorate the enhanced lipolysis and mitochondrial impairment, but rather, rescues de novo fatty acid biosynthesis, suggesting that rikkunshito administration might have partially similar effects to mild CR.

Heterozygous carriers of succinyl-CoA:3-oxoacid CoA transferase deficiency can develop severe ketoacidosis.

Succinyl-CoA:3-oxoacid CoA transferase (SCOT, gene symbol OXCT1) deficiency is an autosomal recessive disorder in ketone body utilization that results in severe recurrent ketoacidotic episodes in infancy, including neonatal periods. More than 30 patients with this disorder have been reported and to our knowledge, their heterozygous parents and siblings have had no apparent ketoacidotic episodes. Over 5 years (2008-2012), we investigated several patients that presented with severe ketoacidosis and identified a heterozygous OXCT1 mutation in four of these cases (Case1 p.R281C, Case2 p.T435N, Case3 p.W213*, Case4 c.493delG). To confirm their heterozygous state, we performed a multiplex ligation-dependent probe amplification analysis on the OXCT1 gene which excluded the presence of large deletions or insertions in another allele. A sequencing analysis of subcloned full-length SCOT cDNA showed that wild-type cDNA clones were present at reasonable rates to mutant cDNA clones. Over the following 2 years (2013-2014), we analyzed OXCT1 mutations in six more patients presenting with severe ketoacidosis (blood pH ≦7.25 and total ketone body ≧10 mmol/L) with non-specific urinary organic acid profiles. Of these, a heterozygous OXCT1 mutation was found in two cases (Case5 p.G391D, Case6 p.R281C). Moreover, transient expression analysis revealed R281C and T435N mutants to be temperature-sensitive. This characteristic may be important because most patients developed ketoacidosis during infections. Our data indicate that heterozygous carriers of OXCT1 mutations can develop severe ketoacidotic episodes in conjunction with ketogenic stresses.

AMOUNT OF RESIDUAL SILICONE OIL IN VITREOUS CAVITY IS SIGNIFICANTLY CORRELATED WITH AXIAL LENGTH.

PURPOSE: To determine the parameters of the eye that are significantly correlated with the amount of residual silicone oil remaining after most of it is removed by vitrectomy. METHODS: Nineteen eyes of 19 patients who had silicone oil removed were studied. The day after the surgery for silicone oil removal, B-scan ultrasonography was performed, and the residual silicone oil droplets were observed as hyperechoic particles in the ultrasonographic images. The images of the vitreous cavity were binarized, and the ratio of area of hyperechoic particles to the total vitreous area was quantified and named the silicone oil index (SOI). The correlations between SOI and clinical findings were determined. RESULTS: The SOI was significantly and positively correlated with the axial length (AL) and the preoperative intraocular pressure (AL, R = 0.676, P = 0.002; preoperative intraocular pressure, R = 0.771, P < 0.001). Partial correlation analysis showed that the AL remained significantly correlated with the SOI but the preoperative intraocular pressure was not (AL, R = 0.734, P = 0.001; preoperative intraocular pressure, R = 0.417, P = 0.096). None of the other clinical factors was significantly correlated with the SOI. CONCLUSION: Considering the significant correlation between the amount of residual silicone oil and the AL of the eye, myopic eyes should be carefully scrutinized for residual silicone oil.

Complete bone regeneration in hemophilic pseudotumor of the mandible.

Hemophilic pseudotumor (HP) is rare, seen in 1-2% of patients with hemophilia, and is extremely uncommon in the mandible. A 6-year-old boy with moderate hemophilia A presented to our hospital with left mandibular swelling. Based on clinical and radiological findings, a tentative diagnosis of HP was made. After factor VIII administration, the lesion was curetted and HP was confirmed on histopathology. The patient was treated with twice-weekly factor VIII until the lesion had completely resolved and bone had regenerated at 1 year. The best treatment for HP is not established; however, appropriate initial treatment and postoperative prophylaxis are effective.

Effectiveness of Medium-Chain Triglyceride Oil Therapy in Two Japanese Citrin-Deficient Siblings: Evaluation Using Oral Glucose Tolerance Tests.

Citrin deficiency, an inherited defect of the liver-type mitochondrial aspartate/glutamate carrier isoform (citrin), may cause impairment of glycolysis because of an increase in the cytosolic NADH/NAD+ ratio. We report a Japanese boy whose main complaint was recurrent hypoglycemic episodes. He was suspected as having citrin deficiency because of his peculiar preference for protein- and fat-rich food. His young sister also had a similar food preference. Both siblings were diagnosed with citrin deficiency by genetic analysis. The brother and sister underwent an oral glucose tolerance test (OGTT) at 10 and 7 yr of age, respectively. Blood glucose, ammonia, lactic acid, pyruvic acid, and insulin levels were monitored before starting the test, and then every 30 min. During this test, they maintained blood glucose levels until 180 min. At 210 min, they experienced vomiting, feeling ill, and decreased blood glucose levels (2.9 and 2.8 mmol/l in the brother and sister, respectively). The sister and brother recovered uneventfully by intravenous glucose injection. In a second OGTT, 4 months after medium-chain triglyceride (MCT) oil supplementation, they had no major symptoms and normal glucose levels were maintained, even after 240 min. Additionally, after MCT oil therapy, their food preference slightly changed as they started eating more carbohydrates. Our OGTT data suggest excess carbohydrate intake has adverse consequences in patients with citrin deficiency, including hypoglycemia after a few hours. MCT oil therapy may be effective in preventing such hypoglycemia and improving metabolic derangement, even during the so-called apparently healthy period.

Penetration of bevacizumab and ranibizumab through retinal pigment epithelial layer in vitro.

PURPOSE: To determine the permeability of bevacizumab and ranibizumab through highly-polarized retinal pigment epithelial (RPE) cells in vitro. METHODS: Highly-polarized RPE cells were cultured in the upper chamber of a Transwell culture system. Bevacizumab or ranibizumab was added to the upper chamber. After 3 hours, the concentrations of bevacizumab or ranibizumab were determined in the upper and lower chambers. The cytotoxicities of the two anti-vascular endothelial growth factor agents were determined histologically. The effects of inhibiting endocytosis by pharmacologic inhibitors were also evaluated. RESULTS: The concentration of ranibizumab was higher than that of bevacizumab in the lower chamber (P < 0.05). Vascular endothelial growth factor was found mainly in the lower chamber under normal conditions. However, the concentration of vascular endothelial growth factor in the lower chamber was significantly less when ranibizumab was added to the upper chamber than when bevacizumab was added. Histology showed no obvious changes in bevacizumab-exposed or ranibizumab-exposed RPE cells. Pretreatment with protein kinase C inhibitor staurosporine had significant negative effects on the permeability of bevacizumab and ranibizumab (P < 0.05). CONCLUSION: Ranibizumab is more permeable than bevacizumab through the highly-polarized RPE layer at clinically equivalent concentrations, and their permeability was partially protein kinase C-dependent. Ranibizumab might be more therapeutically effective than bevacizumab on choroidal neovascularization beneath the RPE layer.

Retinal morphologic changes and concentrations of cytokines in eyes with diabetic macular edema.

PURPOSE: To determine the relationship between the retinal morphologic changes and concentrations of intravitreal cytokines in eyes with diabetic macular edema. METHODS: A retrospective comparative study was performed. The preoperative optical coherence tomography images were evaluated to determine the presence of serous retinal detachments (SRDs), retinal cystic changes, and retinal swelling. The concentrations of vascular endothelial growth factor, interleukin (IL)-6, and IL-8 in vitreous samples collected during vitrectomy were determined. The correlations between optical coherence tomography parameters, other clinical factors, and the concentration of cytokines were calculated. RESULTS: Fifty-two eyes (52 patients) were investigated. An SRD was found in 19 of the 52 eyes (36.5%). Multivariate regression analysis showed that IL-6 was the only factor significantly associated with the presence of an SRD (P = 0.001; odds ratio, 1.268; 95% confidence interval, 1.105-1.452). The other morphologic changes, such as retinal cystic changes and retinal swellings, were not significantly associated with the concentrations of intravitreal cytokines. CONCLUSION: The significant association of SRD with intravitreal IL-6 indicates that inflammation may play an important role in the development of SRD in diabetic macular edema.

Individualized, spectral domain-optical coherence tomography-guided facedown posturing after macular hole surgery: minimizing treatment burden and maximizing outcome.

PURPOSE: To evaluate the individualized, optical coherence tomography-guided facedown posturing after macular hole (MH) surgery in minimizing the burden and maximizing outcome. METHODS: A retrospective comparative study. One hundred and seven consecutive eyes with an MH (<500 µm) received vitrectomy and gas tamponade. After surgery, optical coherence tomography examination was performed from 6 hours to postoperative Day 2. In Group A, with a pro re nata posturing protocol, the duration of facedown posturing was determined from the optical coherence tomography findings. Group A was subdivided as follows: Group A1, facedown posturing required postoperatively and Group A2, no posturing required. When MH closure was confirmed, facedown posturing (if any) was discontinued. If the MH did not close, additional posturing was advised. Group B was the control group, consisted of 42 consecutive eyes with traditional 7 days of posturing. RESULTS: After a single surgery, Group A had the MH closure rate of 96.2%, 95.8% in Group A1 and 97.1% in Group A2, whereas Group B had the MH closure rate of 95.2%. The average posturing period was 42 hours for Group A, 57 hours for Group A1 and 10 hours for Group A2 (P < 0.001). The MH size was correlated significantly with the closure time (R = 0.47, P = 0.005, Spearman correlation coefficient). CONCLUSION: A pro re nata posturing protocol achieves a high MH closure rate with a significant reduction of posturing time especially for pseudophakic eyes.

TNF-α disrupts morphologic and functional barrier properties of polarized retinal pigment epithelium.

Retinal pigment epithelial (RPE) cells form a blood-ocular barrier, and their polarized property is crucial for maintaining the barrier functions. Tumor necrosis factor alpha (TNF-α), a major pleotropic inflammatory cytokine that disrupts the barrier function and eventual angiogenesis, is expressed in the choroidal neovascularizations of age-related macular degeneration eyes. Thus, it most likely plays an important role in the progression of the disease. The purpose of this study was to compare the effects of TNF-α on the barrier function of polarized RPE cells. Non-polarized RPE cells were used as negative controls. Isolated porcine RPE cells were seeded on Transwell™ membranes. The polarization of the RPE cells was determined by their high transepithelial electrical resistance (TER >150 Ω cm(2)) and by their differential secretion of vascular endothelial growth factor (lower layer/upper layer >2.5X). Polarized RPE cells were incubated with 10 ng/ml of TNF-α and the TER was measured. TNF-α significantly decreased the TER of polarized RPE cells by 17.6 ± 2.7% (P < 0.001) of the control at 24 h and that of non-polarized RPE cells by 5.4 ± 6.5% (P = 0.401). The p38 mitogen-activated protein kinase (MAPK) inhibitor, SB203580, blocked the effects of TNF-α of decreasing the TER. Cell junction-related molecules, e.g., ZO-1, located between cells in control RPE cells, were disassembled by TNF-α, and this breakdown was suppressed by SB203580 in polarized RPEs. These results indicate that the breakdown of the RPE barrier function was caused exclusively by TNF-α in polarized RPEs, and TNF-α was acting through the p38 MAPK pathways. Investigations of polarized RPE cells should be more suitable for in vitro studies of the pathophysiology of retinochoroidal diseases.

Responsiveness of eyes with polypoidal choroidal vasculopathy with choroidal hyperpermeability to intravitreal ranibizumab.

BACKGROUND: To determine the role played by vascular endothelial growth factor (VEGF) in polypoidal choroidal vasculopathy (PCV) based on an interventional immunology theory. METHODS: Eyes with PCV were divided in a masked fashion into those with choroidal hyperpermeability (HP group) and those with normal choroidal permeability (NP group) based on the indocyanine green angiograms. The inter-rater agreement rate was evaluated using Fleiss' kappa. Patients were treated by intravitreal ranibizumab (IVB). The central choroidal thickness and central foveal thickness (CFT) at the baseline and 7 days after the treatment were measured by optical coherence tomography. RESULTS: Among the 57 consecutive eyes diagnosed with PCV, 42 eyes of 42 patients met the inclusion criteria (21 eyes/HP group vs 21 eyes /NP group). Central choroidal thickness in HP group was significantly thicker than that in the NP group (P < .001, Mann-Whitney U test). The inter-rater agreement was high with a Fleiss' kappa = 0.95, P < .0001. The percentage reduction in the CFT in HP group (14.0%) was significantly less than that in NP group (20.4%; P = .013, Mann-Whitney U test). CONCLUSIONS: Eyes with PCV that are associated with choroidal hyper-permeability may not be strongly associated with VEGF-related pathology, and may not respond favorably to anti-VEGF monotherapy.

Monitoring with wearable devices will clarify the association between indoor temperature and blood pressure.

The association of blood pressure and temperature is well known in seasonal observation, and low temperature in the winter season is often considered a cause of high blood pressure. The current evidence for short-term studies of temperature and blood pressure is based on the daily observation, however continuous monitoring with wearable devices will allow us to evaluate the rapid effect of cold temperature exposure on blood pressure. In a Japanese, prospective intervention study from 2014 to 2019 (the Smart Wellness Housing survey), approximately 90% of Japanese lived in cold houses (indoor temperature less than 18 °C). Importantly, the indoor temperature was associated with the increase of morning systolic blood pressure. We recently addressed the sympathetic nervous activation of individuals in both their houses and a highly insulated and airtight model house in the winter season using portable electrocardiography equipment. A few subjects showed a morning surge in sympathetic activity, which was more intense at their cold houses, which suggests the importance of the indoor environment in the management of early morning hypertension. In near future, real-time monitoring with wearable devices will provide important information for a better life-environment, leading to risk reduction of morning surge and cardiovascular events.

Detection of transient abnormal myelopoiesis blasts in a liver biopsy specimen by double-immunostaining for full-length GATA1 and CD42b.

BACKGROUND: Transient abnormal myelopoiesis (TAM) is characterized by leukocytosis with increased circulating megakaryoblasts that harbor N-terminal truncating mutations in the GATA1 gene. Approximately 10% of affected patients experience early death. OBSERVATIONS: A 2-month-old boy with Down syndrome was diagnosed with TAM and followed without treatment. Although the blasts in the peripheral blood disappeared, liver failure progressed. A pathological examination revealed liver fibrosis, and double-immunostaining for full-length GATA1 and CD42b identified megakaryocytes with a GATA1 mutation. CONCLUSIONS: This simple and cost-effective method can be applied in routine practice to detect TAM blasts during assessment in a TAM crisis.

Morning surge in sympathetic nervous activity in the indoor environment during the cold winter season.

We addressed to the sympathetic nervous activation of the same people in both their houses and a highly insulated and airtight model house (model house) during the cold winter season. Eight subjects (4 males and 4 females) stayed two nights at each house and were continuously monitored for sympathetic nerve system by calculating LF (low frequency)/HF (high frequency) in the analysis of heart rate variability using a wearable electrocardiography equipment. The room temperatures were kept constant at 20 °C or more in model house, but much lower in their houses. In all subjects, the sleeping duration is longer in model house compared with that in the participants' houses. Four subjects showed a morning surge in sympathetic activity that were more intense at their houses. This morning surge in sympathetic activity in a residential setting suggests the importance of the indoor environment in the management of early morning hypertension.

Immediate postnatal central hypothyroidism caused by maternal Graves' disease: Importance of early screening.

This report illustrates a case of central hypothyroidism in a newborn immediately after birth caused by maternal Graves' disease. Infants from mothers with Graves' disease require careful examination without waiting for neonatal screening results, even though the mother's thyroid function is normal at birth or the newborn does not have goiter.

NSAIDs inhibit neovascularization of choroid through HO-1-dependent pathway.

Intraocular neovascularization is the leading cause of severe visual loss and anti-vascular endothelial growth factor (VEGF) therapy is currently performed for choroidal neovascularization (CNV). Despite its potent anti-angiogenic effect, there are concerns about its long-term safety. Non-steroidal anti-inflammatory drugs (NSAIDs) are common therapeutic agents used for treating inflammatory diseases, and their anti-stress effects are attracting attention now. We studied the effects of topical NSAIDs on CNV, focusing on anti-stress proteins. Cultured retinal pigment epithelium (RPE) cells were treated with NSAIDs: bromfenac, indomethacin, or vehicle control. Transcription factor NF-E2-related factor 2 (Nrf2) and its downstream anti-oxidant protein heme oxygenase (HO)-1 were assessed using western blot and immunohistochemistry. As a result, NSAIDs induced translocation of Nrf2 into the nucleus and the robust expression of HO-1 in a dose- and time-dependent manner. Flow cytometric analysis revealed that bromfenac inhibited H(2)O(2)-induced apoptosis in cultured RPE cells. Next, we studied the effects of topical bromfenac on laser-induced CNV model in rat. The expressions of Nrf2 and HO-1, infiltrations of ED-1-positive macrophages at CNV lesions and size were analyzed. VEGF in the ocular fluid of these rats was also measured using enzyme-linked immunosorbent assay. Rats administered an inhibitor of HO-1 stannic mesoporphyrin (SnMP) were also studied. The results showed that topical bromfenac led to translocation of Nrf2 and induction of HO-1 in CNV lesions and that the number of infiltrating macrophages at the CNV lesion decreased. The sizes of CNV lesions were significantly smaller in bromfenac-treated rats than control CNV, and the effects were diminished by SnMP. VEGF increased in the ocular fluid after laser treatment and was inhibited by bromfenac and SnMP canceling these effects. NSAIDs inhibit CNV through the novel anti-stress protein HO-1-dependent pathway, indicating its potential therapeutic value for various intraocular angiogenic diseases including CNV.

Stromal cell-derived factor-1 is essential for photoreceptor cell protection in retinal detachment.

Stromal cell-derived factor-1 (SDF-1) causes chemotaxis of CXCR4-expressing bone marrow-derived cells. SDF-1 is involved in the pathogenesis of various vascular diseases, including those of the eye. However, the role of SDF-1 in neuronal diseases is not completely understood. Here, we show higher SDF-1 levels in the vitreous humor of patients with retinal detachment (RD) compared with normal patients. SDF-1 correlated positively with the duration as well as the extent of RD. Furthermore, SDF-1 correlated significantly with levels of interleukin-6 and interleukin-8, but not with vascular endothelial growth factor. Western blot analysis results showed significant SDF-1 up-regulation in detached rat retinas compared with normal animals. Immunohistochemistry data showed that SDF-1 was co-localized with the glial cells of the detached retina. SDF-1 blockade with a neutralizing antibody increased photoreceptor cell loss and macrophage accumulation in the subretinal space. The retinal precursor cell line R28 expressed CXCR4. SDF-1 rescued serum starvation-induced apoptosis in R28 cells and enhanced their ability to participate in wound closure in a scratch assay. Our results indicate a surprising, protective role for SDF-1 in RD. This effect may be mediated directly or indirectly through other cell types.