JTK: targeted diploid genome assembler.

MOTIVATION: Diploid assembly, or determining sequences of homologous chromosomes separately, is essential to elucidate genetic differences between haplotypes. One approach is to call and phase single nucleotide variants (SNVs) on a reference sequence. However, this approach becomes unstable on large segmental duplications (SDs) or structural variations (SVs) because the alignments of reads deriving from these regions tend to be unreliable. Another approach is to use highly accurate PacBio HiFi reads to output diploid assembly directly. Nonetheless, HiFi reads cannot phase homozygous regions longer than their length and require oxford nanopore technology (ONT) reads or Hi-C to produce a fully phased assembly. Is a single long-read sequencing technology sufficient to create an accurate diploid assembly? RESULTS: Here, we present JTK, a megabase-scale diploid genome assembler. It first randomly samples kilobase-scale sequences (called 'chunks') from the long reads, phases variants found on them, and produces two haplotypes. The novel idea of JTK is to utilize chunks to capture SNVs and SVs simultaneously. From 60-fold ONT reads on the HG002 and a Japanese sample, it fully assembled two haplotypes with approximately 99.9% accuracy on the histocompatibility complex (MHC) and the leukocyte receptor complex (LRC) regions, which was impossible by the reference-based approach. In addition, in the LRC region on a Japanese sample, JTK output an assembly of better contiguity than those built from high-coverage HiFi+Hi-C. In the coming age of pan-genomics, JTK would complement the reference-based phasing method to assemble the difficult-to-assemble but medically important regions. AVAILABILITY AND IMPLEMENTATION: JTK is available at https://github.com/ban-m/jtk, and the datasets are available at https://doi.org/10.5281/zenodo.7790310 or JGAS000580 in DDBJ.

A high-quality, haplotype-phased genome reconstruction reveals unexpected haplotype diversity in a pearl oyster.

Homologous chromosomes in the diploid genome are thought to contain equivalent genetic information, but this common concept has not been fully verified in animal genomes with high heterozygosity. Here we report a near-complete, haplotype-phased, genome assembly of the pearl oyster, Pinctada fucata, using hi-fidelity (HiFi) long reads and chromosome conformation capture data. This assembly includes 14 pairs of long scaffolds (>38 Mb) corresponding to chromosomes (2n = 28). The accuracy of the assembly, as measured by an analysis of k-mers, is estimated to be 99.99997%. Moreover, the haplotypes contain 95.2% and 95.9%, respectively, complete and single-copy BUSCO genes, demonstrating the high quality of the assembly. Transposons comprise 53.3% of the assembly and are a major contributor to structural variations. Despite overall collinearity between haplotypes, one of the chromosomal scaffolds contains megabase-scale non-syntenic regions, which necessarily have never been detected and resolved in conventional haplotype-merged assemblies. These regions encode expanded gene families of NACHT, DZIP3/hRUL138-like HEPN, and immunoglobulin domains, multiplying the immunity gene repertoire, which we hypothesize is important for the innate immune capability of pearl oysters. The pearl oyster genome provides insight into remarkable haplotype diversity in animals.

Training residents in medical incident report writing to improve incident investigation quality and efficiency enables accurate fact gathering.

We assessed whether training on writing readable and accurate medical incident reports (IRs) improves the quality of fact description. In this training, 124 residents created fictional IRs. We provided tips, including using When, Where, Who, What, Why, How. We compared the fictional IRs with and without tips, and the trainees' and non-trainees' IRs submitted in the first five months after training. Results indicated that the subject words in IRs were more clarified and the readability was improved. The fictional IRs using tips were more accurate, with increased descriptions of the patient's background, reporter's actions, team members' actions and conversations, safety check procedures, result of the error, and post-incident response. The reporter's actions, work procedures, and environment were more clarified in the trainees' IRs than in the non-trainees' IRs. This training may help analysts comprehend the sequence of and underlying factors for reporter's actions based on IRs.

Long-read metagenomic exploration of extrachromosomal mobile genetic elements in the human gut.

BACKGROUND: Elucidating the ecological and biological identity of extrachromosomal mobile genetic elements (eMGEs), such as plasmids and bacteriophages, in the human gut remains challenging due to their high complexity and diversity. RESULTS: Here, we show efficient identification of eMGEs as complete circular or linear contigs from PacBio long-read metagenomic data. De novo assembly of PacBio long reads from 12 faecal samples generated 82 eMGE contigs (2.5~666.7-kb), which were classified as 71 plasmids and 11 bacteriophages, including 58 novel plasmids and six bacteriophages, and complete genomes of five diverse crAssphages with terminal direct repeats. In a dataset of 413 gut metagenomes from five countries, many of the identified plasmids were highly abundant and prevalent. The ratio of gut plasmids by our plasmid data is more than twice that in the public database. Plasmids outnumbered bacterial chromosomes three to one on average in this metagenomic dataset. Host prediction suggested that Bacteroidetes-associated plasmids predominated, regardless of microbial abundance. The analysis found several plasmid-enriched functions, such as inorganic ion transport, while antibiotic resistance genes were harboured mostly in low-abundance Proteobacteria-associated plasmids. CONCLUSIONS: Overall, long-read metagenomics provided an efficient approach for unravelling the complete structure of human gut eMGEs, particularly plasmids.

Increased Hematocrit During Sodium-Glucose Cotransporter 2 Inhibitor Therapy Indicates Recovery of Tubulointerstitial Function in Diabetic Kidneys.

Sodium-glucose cotransporter 2 (SGLT2) inhibitors have been attracting attention for cardiovascular as well as antidiabetic effects since the results of the Empagliflozin Cardiovascular Outcome Event Trial in Type 2 Diabetes Mellitus Patients (EMPA-REG OUTCOME Trial) were reported. The hematocrit increases during treatment with SGLT2 inhibitors, which have a diuretic effect but do not cause sufficient hemoconcentration to increase the risk of cerebral infarction. Elevation of the hematocrit during SGLT2 inhibitor therapy is presumed to involve enhancement of erythropoiesis in addition to hemoconcentration. In patients with diabetes, the erythropoietin level increases after initiation of treatment with the SGLT2 inhibitor dapagliflozin and reaches a plateau in 2 - 4 weeks. The reticulocyte count increases simultaneously, followed by elevation of hemoglobin and hematocrit. In patients with diabetes, the proximal tubules are overtaxed by excessive glucose reabsorption and the increased oxygen requirement causes tubulointerstitial hypoxia. Consequently, erythropoietin production is impaired because "neural crest-derived" fibroblasts surrounding the damaged renal tubules undergo transformation into dysfunctional fibroblasts. SGLT2 inhibitors reduce the workload of the proximal tubules and improve tubulointerstitial hypoxia, allowing fibroblasts to resume normal erythropoietin production. These drugs represent a new class of diuretics that have a renoprotective effect by improving tubulointerstitial hypoxia, which is the final common pathway to end-stage renal disease. In patients with diabetes, elevation of hematocrit may be a surrogate marker for recovery from reversible tubulointerstitial injury.

The Mechanism of False in Vitro Elevation of Uric Acid Level in Mouse Blood.

Thirty minutes incubation at room temperature elevates the uric acid (UA) level of mouse blood in a test tube, and has previously been reported as "false in vitro elevation of the uric acid level." However the UA level of human blood does not elevate using the same incubation. We clarified the mechanism of the false in vitro UA elevation using mice with highly active hypoxanthine phosphoribosyl transferase (Hprt) of B6-ChrXC(MSM), a consomic mouse strain with the chromosome portion of Mus musculus morocinus in the Hprt gene site, or mice with a targeted deletion of the urate oxidase gene (Uox) (Uox-knockout (KO)). The plasma levels of UA, hypoxanthine, and xanthine, determined by HPLC, were compared with those of C57BL/6J laboratory mice used as controls. The uric acid level of Uox-KO mice was approximately 10 times higher than that of control, did not elevated after incubation in the test tube. With allopurinol, the hypoxanthine levels of B6-ChrXC(MSM) and Uox-KO were significantly lower than that of controls. Without allopurinol, the UA and xanthine levels of B6-ChrXC(MSM) were significantly lower than those of C57BL/6J controls. Even with allopurinol, the UA and xanthine levels were still significantly lower than that of controls. In conclusion, "false in vitro elevation of uric acid level" seems to be caused by low levels of erythrocyte HPRT activity and the low plasma uric acid level of laboratory mice.

Construction of a model for predicting creatinine clearance in Japanese patients treated with Cisplatin therapy.

BACKGROUND/AIM: There exist various useful predictive models, such as the Cockcroft-Gault model, for estimating creatinine clearance (CLcr). However, the prediction of renal function is difficult in patients with cancer treated with cisplatin. Therefore, we attempted to construct a new model for predicting CLcr in such patients. PATIENTS AND METHODS: Japanese patients with head and neck cancer who had received cisplatin-based chemotherapy were used as subjects. A multiple regression equation was constructed as a model for predicting CLcr values based on background and laboratory data. RESULTS: A model for predicting CLcr, which included body surface area, serum creatinine and albumin, was constructed. The model exhibited good performance prior to cisplatin therapy. In addition, it performed better than previously reported models after cisplatin therapy. CONCLUSION: The predictive model constructed in the present study displayed excellent potential and was useful for estimating the renal function of patients treated with cisplatin therapy.

Circulatory dynamics of the cauda equina in lumbar canal stenosis using dynamic contrast-enhanced magnetic resonance imaging.

BACKGROUND CONTEXT: There has been no study regarding the cauda equina circulation of patients with neurogenic intermittent claudication (NIC) in lumbar spinal canal stenosis (LSCS) using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). PURPOSE: The mechanism responsible for the onset of NIC was investigated using DCE-MRI to examine changes in cauda equina blood flow in patients with LSCS. STUDY DESIGN: This was a retrospective longitudinal registry and magnetic resonance imaging study. PATIENT SAMPLE: The subjects consisted of 23 patients who had LSCS associated with NIC (stenosis group). Ten asymptomatic volunteers who did not have NIC served as controls (control group). In the LSCS group, the cross-sectional area of the dural sac was <75 mm2 at the site of most severe stenosis. These patients were further divided into single and double stenosis subgroups. OUTCOME MEASURES: The main measures we used were the signal intensity (S-I) ratio and the shape and size of the time intensity (T-I) curves. We compared these between the stenosis and control groups. METHODS: At first, plain T1-weighted MR images were obtained and the lumbar dural sac cross-sectional area was measured using a digitizer. For DCE-MRI, sagittal T1-weighted images of the same slice were acquired continuously for 10 minutes after administration of gadolinium as an intravenous bolus to observe the distribution of contrast medium (gadolinium) in the cauda equina. To objectively evaluate changes in contrast enhancement of the cauda equina at the site of canal stenosis, regions of interest were established. The signal intensity (SI) ratio was calculated to compare the signal intensities before and after contrast enhancement, and time-intensity curves were prepared to investigate changes over time. RESULTS: The static imaging findings and the changes of gadolinium uptake showed striking differences between the study and control patients. In the stenosis group, abnormal intrathecal enhancement showed around the site of stenosis on enhanced MR imaging. The SI ratio at the site of canal stenosis had a slower increase in the arterial phase when compared with that in the control group and remained high in the venous phase for up to 10 minutes. Finally, abnormal intrathecal enhancement was visible around the site of stenosis on enhanced MR imaging in all patients. CONCLUSIONS: These clinical data indicate that cauda equina nerve roots in the LSCS patients are pathologic even when symptoms are not elicited in the supine position, suggesting that intraradicular venous congestion and edema themselves do not influence the existence of radicular symptoms.

Preventive effects of Chlorella on skeletal muscle atrophy in muscle-specific mitochondrial aldehyde dehydrogenase 2 activity-deficient mice.

BACKGROUND: Oxidative stress is involved in age-related muscle atrophy, such as sarcopenia. Since Chlorella, a unicellular green alga, contains various antioxidant substances, we used a mouse model of enhanced oxidative stress to investigate whether Chlorella could prevent muscle atrophy. METHODS: Aldehyde dehydrogenase 2 (ALDH2) is an anti-oxidative enzyme that detoxifies reactive aldehydes derived from lipid peroxides such as 4-hydroxy-2-nonenal (4-HNE). We therefore used transgenic mice expressing a dominant-negative form of ALDH2 (ALDH2*2 Tg mice) to selectively decrease ALDH2 activity in the muscles. To evaluate the effect of Chlorella, the mice were fed a Chlorella-supplemented diet (CSD) for 6 months. RESULTS: ALDH2*2 Tg mice exhibited small body size, muscle atrophy, decreased fat content, osteopenia, and kyphosis, accompanied by increased muscular 4-HNE levels. The CSD helped in recovery of body weight, enhanced oxidative stress, and increased levels of a muscle impairment marker, creatine phosphokinase (CPK) induced by ALDH2*2. Furthermore, histological and histochemical analyses revealed that the consumption of the CSD improved skeletal muscle atrophy and the activity of the mitochondrial cytochrome c oxidase. CONCLUSIONS: This study suggests that long-term consumption of Chlorella has the potential to prevent age-related muscle atrophy.

Basal-Supported Oral Therapy with Sitagliptin Counteracts Rebound Hyperglycemia Caused by GLP-1 Tachyphylaxis.

Introduction. Treatment with a glucagon-like peptide 1 (GLP-1) analog fails in some patients due to rebound hyperglycemia caused by tachyphylaxis (GLP-1 tachyphylaxis). We investigated the efficacy of basal-supported oral therapy (BOT) with insulin glargine and sitagliptin for counteracting GLP-1 tachyphylaxis. Materials and Methods. The subjects were 12 men and 3 women aged 59.9 ± 10.0 years who had been treated with GLP-1 analogs. All of them had developed rebound hyperglycemia caused by GLP-1 tachyphylaxis. Their GLP-1 analog-based therapy was switched to BOT with insulin glargine plus sitagliptin and other medications. The primary outcomes were whether switching of therapy was associated with a change of hemoglobin A1c (HbA1c) and whether weight gain occurred. Results. Baseline HbA1c was 8.0 ± 0.9%. It decreased to 7.3 ± 0.9% at 3 months after switching (P < 0.01) and to 7.2 ± 0.9% at 4 months (P < 0.05). Weight gain was 1.1 kg after 1 month (P < 0.01) and 2.3 kg after 5 months (P < 0.01). Conclusion. Switching to BOT with insulin glargine and sitagliptin improved glycemic control. The significant decrease of HbA1c demonstrated that this combination can counteract deterioration of glycemic control due to rebound hyperglycemia secondary to GLP-1 tachyphylaxis. However, weight gain remains a problem.

[Triage DOA screening in a case of methamphetamine and its analogue poisoning].

CASE REPORT: A 20-year-old woman presented with the chest pain, nausea, respiratory strange feeling, and a large quantity of sweating. On the stimulant zone of 8 groups of drugs of Triage DOA screening it showed an equivocal positivity while all of the other zones gave negative results. She denied taking drugs. No injection scar was found. And she was then hospitalized because little was known about her symptoms. When the unconscious patient was discovered at rest room inside hospital the next day, she was transferred to emergency and critical care center. In the same screening test positivity on the stimulant zone was observed, and furthermore both amphetamine and methamphetamine were detected by GCMS analysis. For 4 days positivity on the stimulant zone lasted. From the fact of disturbance of consciousness, restlessness, excitation and tachycardia, respiration disorder, and the pupil dilatation drug poisoning was deeply suspected. DISCUSSION: While the stimulant zone of Triage DOA showed the equivocal positivity when 7 hours has elapsed until she became aware of abnormality and hospitalized, in the same screening of 30 hours later positivity was verified clearly. Several problems derived from the detection method, pharmacokinetic factors and pharmacodynamic aspect were discussed as for the difference of the results detected.

Sitagliptin counteracts seasonal fluctuation of glycemic control.

AIM: To assess the effect of sitagliptin therapy on seasonal fluctuation of glycemic control in Japanese type 2 diabetic patients. METHODS: Participating patients (age: 29-80 years) had been treated with conventional oral antidiabetic agents and/or diet and exercise therapy for over 6 mo. From December 2009, 35 patients were additionally prescribed oral sitagliptin starting from 50 mg once daily, while 19 patients taking α-glucosidase inhibitors were switched to sitagliptin. Twenty-four patients who refused sitagliptin formed the control group. Changes of mean monthly hemoglobin A(1c) (HbA(1c)) during the "winter holiday season" were compared between groups using Student's t-test (2008-2009 vs 2009-2010). Statistical significance was accepted at P < 0.05. Multivariate analysis was performed to assess whether sitagliptin use was associated with deterioration or improvement of glycemic control. RESULTS: Both add-on sitagliptin and switching from α-glucosidase inhibitors to sitagliptin prevented the seasonal deterioration of glycemic control and tended to improve HbA(1c). Multivariate analysis revealed that both adding and switching to sitagliptin were negatively correlated with deterioration of glycemic control. In 44 patients who continued sitagliptin therapy for another year, elevation of HbA(1c) was suppressed without adverse effects. CONCLUSION: Sitagliptin is a suitable oral agent for preventing deterioration of glycemic control during the winter holiday season.

A new preventive strategy for hypoglycemia incorporating added food diet in patients with type 2 diabetes who received sitagliptin therapy.

There has been concern as to whether dipeptidyl peptidase-4 (DPP-4) inhibitors can be used safely in patients with relatively good glycemic control. This study, approved by the institutional review board of Hanzoumon Diabetes City Atlas Clinic, examined whether DPP-4 inhibitor sitagliptin could safely achieve good glycemic control without severe hypoglycemia by employing the "added food" concept. The subjects were 60 patients (46 men and 14 women) with type 2 diabetes who started sitagliptin therapy during a 1-month period from December 15, 2009 to January 15, 2010. They were recommended to have added food between meals to prevent hypoglycemia, while maintaining the same daily calorie intake. HbA(1c) decreased from 7.1 ± 1.2% to 6.5 ± 0.6% after 6 months of sitagliptin treatment (p < 0.001). In patients with a baseline HbA(1c) <7%, it decreased from 6.5 ± 0.3% to 6.1 ± 0.4% (p < 0.001). Systolic blood pressure was significantly reduced from 127.7 ± 17.0 to 122.7 ± 17.9 mmHg in the patients with a baseline HbA(1c) < 7% (p = 0.018). However, body weight increased by approximately 900 g and high-density lipoprotein cholesterol decreased significantly from 1.57 ± 0.46 to 1.43 ± 0.35 mmol/L (p < 0.01) in the patients concomitantly receiving sulfonylureas with sitagliptin. Excellent glycemic control was achieved by sitagliptin treatment together with the added food concept. However, combined use of sitagliptin with sulfonylureas requires attention to weight gain and the lipid profile. Further clinical studies will elucidate whether sitagliptin can decrease cardiovascular events as well as normalizing blood glucose and lowering the blood pressure.

Sertoli cells proliferate in adult rats with prenatal exposure to 3,3',4,4',5-pentachlorobiphenyl.

Sertoli cells play a critical role in spermatogenesis, and in adults, they are terminally differentiated with loss of proliferative activity. This study revealed Sertoli cell proliferation in 17-week-old Sprague-Dawley rats whose dams had been intragastrically administered 250 ng of 3,3',4,4',5-pentachlorobiphenyl/kg on days 13-19 postconception. Immunohistochemical evidence of proliferating cell nuclear antigen (PCNA) expression and electron microscope observation of mitotic figures confirmed the proliferation. Because the serum follicle stimulating hormone (FSH), luteinizing hormone (LH) and testosterone concentrations were similar to those of vehicle-treated rats, a direct endocrine cause for the observed effects was unlikely.

Cyclin D1/cdk4, estrogen receptors α and ß, in N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric carcinogenesis: immunohistochemical study.

Hyperproliferative cell growth due to cyclin D1/cdk4, marker of cellular proliferation, is considered to be regulated by the expression of estrogen receptors (ERs). We investigated the immunohistochemical expression of cyclin D1/cdk4 and ERs in N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced rat gastric carcinogenesis. The gastric cancer incidence and expression of cyclin D1/ckd4 in gastric carcinogenesis were significantly higher in males than females. Although the ERα expression index was similar in both sexes, the ERß expression in preneoplastic hyperplastic lesions as well as gastric cancers was significantly higher in females than in males. The present study revealed a gender difference in MNNG-induced rat gastric carcinogenesis that seemed to involve the sex difference in cyclin D1/cdk4 expression, and ERß expression became evident at the preneoplastic promotion stage in gastric carcinogenesis.

Impact of concomitant diabetes and chronic kidney disease on preload-induced changes in left ventricular diastolic filling in hypertensive patients.

OBJECTIVES: Concomitant diabetes and/or chronic kidney disease (CKD) in hypertensive patients may portend additive deleterious effects on active left ventricular relaxation. We investigated the effect of a passive leg lifting (PLL) maneuver, a means of increasing preload, on left ventricular filling to assess the relationship of concomitant diabetes mellitus (DM) and/or CKD with diastolic function in hypertensive patients. METHODS: A total of 155 asymptomatic essential hypertensive patients underwent Doppler echocardiography to compare the echocardiographic indices at baseline and during PLL. In 51 patients, the effect of physiological saline infusion was also examined. RESULTS: The changes in echocardiographic indices, including deceleration time of early diastolic filling (EDT) and the ratio of transmitral early left ventricular filling velocity to early diastolic Doppler tissue imaging of the mitral annulus (E/E') by saline infusion showed a good correlation with those induced by PLL (Bland-Altman plot and linear regression). We next divided the total participants into four groups according to the presence/absence of diabetes and/or CKD [DM(-)/CKD(-); n = 48, DM(+)/CKD(-); n = 25, DM(-)/CKD(+); n = 43, and DM(+)/CKD(+); n = 39)] and found that the changes in EDT (F = 15.92, P < 0.01) as well as those in E/E' (F = 8.87, P < 0.01) were significantly different among the subgroups. Multiple logistic regression analysis revealed that these complications were independent predictors of EDT less than 150 ms [DM, odds ratio (OR): 2.82; CKD, OR: 2.18, P < 0.05, respectively] as well as E/E' ratio at least 15.0 during PLL (DM, OR: 4.78; CKD, OR: 3.32, P < 0.05, respectively). CONCLUSION: This simple preloading test unmasks latent progression of left ventricular dysfunction in essential hypertension; that is, these complications potentially cause deterioration of left ventricular compliance and preload reserve even in the early stages of diastolic dysfunction.

Interleukin-6 as an independent predictor of future cardiovascular events in high-risk Japanese patients: comparison with C-reactive protein.

Inflammation is associated with the development of atherosclerotic vascular lesions and some inflammatory parameters are used as cardiovascular (CV) risk markers. The present study was designed to assess the predictive power of interleukin (IL)-6 for future CV events. In 121 Japanese patients with multiple CV risk factors and/or disease, serum concentrations of IL-6 and high sensitive C-reactive protein (hs-CRP) were measured. During follow-up periods (mean, 2.9 years) after the baseline assessment, 50 patients newly experienced CV events such as stroke/transient ischemic attack (n=10), heart failure hospitalization (n=6), acute coronary syndrome (n=7), and revascularization for coronary artery disease (n=15) and peripheral arterial disease (n=12). The serum level of IL-6, but not hs-CRP, was significantly higher in patients who had CV events than in event-free subjects (3.9±2.6 and 3.0±2.2 pg/mL, P=0.04). When the patients were divided into three groups by tertiles of basal levels of IL-6 (<1.85, 1.85-3.77, and ≥3.77 pg/mL), cumulative event-free rates by the Kaplan-Meier method were decreased according to the increase in basal IL-6 levels (65%, 50%, and 19% in the lowest, middle, and highest tertiles of IL-6, respectively; log-rank test, P=0.002). By univariate Cox regression analysis, previous CV disease, creatinine clearance, and serum IL-6 levels were significantly associated with CV events during follow-up. Among these possible predictors, the highest tertile of IL-6 was only an independent determinant for the morbidity in the multivariate analysis (hazard ratio 2.80 vs. lowest tertile, P=0.006). These findings indicate that IL-6 is a powerful independent predictor of future CV events in high-risk Japanese patients, suggesting its predictive value is superior to that of hs-CRP.