A novel tracking and analysis system for time-lapse cellular imaging of Schizosaccharomyces pombe.

The importance of the parent-progeny relationship tracking technique in single-cell analysis has grown with the passage of time. In this study, fundamental image-processing techniques were combined to develop software capable of inferring cell cycle alterations in fission yeast cells, which exhibit equipartition during division. These methods, exclusively relying on bright-field images as input, could track parent-progeny relationships after cell division by assessing the temporal morphological transformation of these cells. In the application of this technique, the software was employed for calculating intracellular fluorescent dots during every stage of the cell cycle, using a yeast strain expressing EGFP-fused Swi6, which binds to chromatin. The results obtained with this software were consistent with those of previous studies. This software facilitated single-cell-level tracking of parent-progeny relationships in cells exhibiting equipartition during division and enabled the monitoring of spatial fluctuations in a cell cycle-dependent protein. This method, expediting the analysis of extensive datasets, may also empower large-scale screening experiments that cannot be conducted manually.

COL1A2 Barcoding: Bone Species Identification via Shotgun Proteomics.

Species identification of fragmentary bones remains a challenging task in archeology and forensics. A species identification method for such fragmentary bones that has recently attracted interest is the use of bone collagen proteins. Here, we describe a method similar to DNA barcoding that reads collagen protein sequences in bone and automatically determines the species by performing sequence database searches. The method is almost identical to conventional shotgun proteomics analysis of bone samples, except that the database used by the SEQUEST search engine consisted only of entries for collagen type 1 alpha 2 (COL1A2) proteins from various vertebrates. Accordingly, the COL1A2 peptides that differ in sequence among species act as species marker peptides. In SEQUEST-based shotgun proteomics, the protein entries that contain more marker peptide sequences are assigned higher scores; therefore, the highest-scoring protein entry will be the COL1A2 entry for the species from which the analyzed bone was derived. We tested our method using bone samples from 30 vertebrate species and found that all species were correctly identified. In conclusion, COL1A2 can be used as a bone protein barcode and can be read through shotgun proteomics, allowing for automatic bone species identification. Data are available via ProteomeXchange with the identifier PXD045402.

Gene profiles and mutations in the development of cataracts in the ICR rat model of hereditary cataracts.

Cataracts are opacifications of the lens that cause loss of visual acuity and ultimately of eyesight. Age-related cataract develops in most elderly people, but the mechanisms of cataract onset are incompletely understood. The Ihara Cataract Rat (ICR) is an animal model of hereditary cataracts showing cortical opacity that commonly develops prematurely. We identified putative mechanisms of cataract onset in the ICR rat model by measuring gene expression changes before and after cortical cataract development and conducting point mutation analysis. Genes differentially expressed between 4-week-old animals without cortical cataracts and 8-10-week-old animals with cortical cataracts were selected from microarray analysis. Three connections were identified by STRING analysis: (i) Epithelial-Mesenchymal Transition (EMT), including Col1a2, and Pik3r1. (ii) Lens homeostasis, including Aqp5, and Cpm. (iii) Lipid metabolism, including Scd1, Srebf1, and Pnpla3. Subsequently, mutation points were selected by comparing ICR rats with 12 different rats that do not develop cataracts. The apolipoprotein Apoc3 was mutated in ICR rats. Analyses of gene expression changes and point and mutations suggested that abnormalities in EMT or lipid metabolism could contribute to cataract development in ICR rats.

A new approach for forensic analysis of saliva-containing body fluid mixtures based on SNPs and methylation patterns of nearby CpGs.

Saliva samples obtained from crime scenes often contain body fluids from other people, which makes it difficult to not only interpret the obtained DNA profiles, but also interpret saliva identification test results. α-amylase activity, an indicator of most saliva identification methods, can be slightly detected in other body fluids. This study aimed to overcome these difficulties. Here, we identified 13 saliva-specific methylated regions and five saliva-specific unmethylated regions neighboring common single nucleotide polymorphisms (SNPs) by array-based genome-wide methylation analysis of pooled saliva, blood, semen, or vaginal swab samples. Bisulfite sequencing by massively parallel sequencing (MPS) technology was then performed using individual body fluid samples to evaluate the saliva-specificity of each CpG of the three regions selected from the identified candidates. Although no single CpG demonstrated complete saliva-specificity, we found that the reads that were simultaneously (un)methylated at the selected neighboring two to three CpGs of each region were highly specific for saliva DNA. Based on these findings, we then designed MPS-based bisulfite sequencing assays for each region to analyze the selected CpGs and SNP(s) on the same read. These assays could identify the saliva of a target person from body fluid mixtures of known contributors (individual-specific saliva identification) by calculating the ratios of simultaneous (un)methylation at the selected CpGs within the reads containing SNP alleles unique to the target person. Moreover, these assays could indicate the SNP types of saliva DNA (saliva-specific genotyping) from body fluid mixtures by analyzing the alleles of the reads simultaneously (un)methylated at the selected CpGs, while careful attention should be paid to interpret the results of heterologous genotypes. Although further regions should be identified, especially for saliva-specific individual identification, the CpG-SNP approach may be an effective method to interpret the complicated results obtained from saliva-containing body fluid mixtures.

A vertebrate-specific qPCR assay as an endogenous internal control for robust species identification.

The identification of vertebrate species is important in numerous fields including archaeology, ecology, as well as food and forensic sciences. Real-time quantitative PCR (qPCR) assays specific for one vertebrate species are promising approaches for species identification, although there are several drawbacks such as difficulty determining whether the detected DNA is authentic or a contaminant. Here, we describe a qPCR assay specific for vertebrate mitochondrial DNA (mtDNA) which can overcome these drawbacks. Since we found that mitochondrial 16S rRNA contains regions that are perfectly (not highly) conserved across virtually all vertebrates, but are variable in invertebrates, we were able to design a vertebrate-specific qPCR assay by placing primers/probe within these regions. The specificity and accuracy of this assay were validated with representative vertebrate and invertebrate samples. This assay detected DNA from all vertebrate samples, but not from any invertebrate samples. In addition, this assay was able to quantify vertebrate mtDNAs as accurately as previously reported species-specific qPCR assays. The results demonstrated it is feasible to quantify vertebrate mtDNA specifically and accurately in a sample. This means that it is possible to determine the ratio of specific vertebrate species mtDNA to total vertebrate mtDNA in a sample. In conjunction with this assay as an endogenous internal control, species-specific qPCR assays will allow for the robust identification of vertebrate species.

Automatic landmark annotation in 3D surface scans of skulls: Methodological proposal and reliability study.

BACKGROUND AND OBJECTIVES: Craniometric landmarks are essential in many biomedical applications, such as morphometric analysis or forensic identification. The process of locating landmarks is usually a manual and slow task, highly influenced by fatigue, skills and the experience of the practitioner. Localization errors are propagated and magnified in subsequent steps, which can result in incorrect measurements or assumptions. Thereby, standardization, reliability and reproducibility lay the foundations for the necessary accuracy in subsequent measurements or anatomical analysis. In this paper, we present an automatic method to annotate 3D surface skull models taking into account anatomical and geometrical features. METHODS: The proposed method follows a hybrid structure where a deformable template is used to initialize the landmark positions. Then, a refinement stage is applied using prior anatomical knowledge to ensure a correct placement. Our proposal is validated over thirty 3D skull scans of male Caucasians, acquired by hand-held surface scanning, and a set of 58 craniometric landmarks. A statistical analysis was carried out to analyze the inter- and intra-observer variability of manual annotations and the automatic results, along with a visual assessment of the final results. RESULTS: Inter-observer errors show significant differences, which are reflected in the expert consensus used as reference. The average localization error was 2.19±1.5 mm when comparing the automatic landmarks to the reference location. The subsequent visual analysis confirmed the reliability of the refinement method for most landmarks. CONCLUSIONS: Repeated manual annotations show a high variability depending on both skills and expertise of the observer, and landmarks' location and characteristics. In contrast, the automatic method provides an accurate, robust and reproducible alternative to the tedious and error-prone task of manual landmarking.

Three-dimensional shape variation and sexual dimorphism of the face, nose, and mouth of Japanese individuals.

Three-dimensional (3D) shape variations of the face and facial parts in Japanese adults were examined to collect basic data to be used for facial comparison in forensics. In total, 1000 3D facial scans (500 males, 500 females) of Japanese individuals were re-meshed into anatomically homologous shape models and analyzed by principal component analysis (PCA) after Procrustes superimposition. Facial parts (the nose and the mouth) were segmented from homologous face models and analyzed by PCA, too. Among all kinds of objects (the face, the nose, and the mouth), the most predominant shape variation represented by the first principal component (PC1) was the height-width proportion. The second largest variation (PC2) in the face and the nose was depth; for the mouth, it was the relative protrusion of the upper and lower lips. We interpreted predominant shape variations represented by the first five principal components (PCs) in each object. Asymmetric shape variations were observed within these PCs for the nose and the mouth. Sexual dimorphism of the face and the facial parts was also examined by testing the significance of sex-linked differences in PC scores. A significant difference was found between males and females for many PCs. Sexual dimorphism was examined also by emphasizing the shape difference between average male and female faces. Our results revealed predominant 3D shape variations and sexual dimorphism of the face and facial parts. The results may be informative for performing facial comparison in police investigations, an increasingly used technique.

Development of a DNA methylation-based semen-specific SNP typing method: A new approach for genotyping from a mixture of body fluids.

Genotyping from samples containing different types of body fluids is a major difficulty in forensic investigations. Recently, CpG sites that are specifically methylated or unmethylated in different types of body fluids have been reported as novel markers for body fluid identification. In this study, we hypothesized that the simultaneous analysis of CpGs and neighboring polymorphic sites on the same molecule could be useful for individual DNA typing from mixed samples. We performed a proof-of-concept study of this approach by searching the genome-wide methylation dataset deposited at the National Center for Biotechnology Information Gene Expression Omnibus repository for semen-specific CpG markers adjacent to common single nucleotide polymorphisms. From the identified candidates, we selected 5 regions on different chromosomes and validated the presence of semen-specific methylation or unmethylation in each region by pyrosequencing analyses. By combining methylation-specific polymerase chain reaction and pyrosequencing technology, we developed a semen-specific DNA typing method for two semen-specific methylated regions and one semen-specific unmethylated region. Finally, the method successfully identified semen-derived alleles from mixed stains, indicating that this methylation-based approach can be applicable to actual forensic samples. Since existing separation techniques physically isolate cells derived from each type of body fluid, this approach may be useful when existing methods cannot be performed due to the degradation of samples.

Remission of Refractory Ascites and Discontinuation of Hemodialysis after Additional Rituximab to Long-term Glucocorticoid Therapy in a Patient with TAFRO Syndrome.

Thrombocytopenia, ascites, myelofibrosis, renal dysfunction, and organomegaly (TAFRO) syndrome is a newly recognized but rare disease, and its treatment has not yet been established. We reported a 50-year-old woman with TAFRO syndrome diagnosed 2 years after the initial symptoms of a fever, fatigue, epigastric pain, edema, ascites, lymphadenopathy, thrombocytopenia and renal insufficiency. The patient showed refractory ascites and required hemodialysis under corticosteroid mono-therapy for suspected immune-mediated disease but was successfully treated with additive rituximab, resulting in improvement in her laboratory data, the withdrawal of hemodialysis and the disappearance of ascites. This case underscores the therapeutic utility of rituximab in patients with corticosteroid-resistant TAFRO syndrome, even long after the onset of the disease.

Discontinuation of Hemodialysis in a Patient with Anti-GBM Disease by the Treatment with Corticosteroids and Plasmapheresis despite Several Predictors for Dialysis-Dependence.

A 26-year-old man highly suspected of having antiglomerular basement membrane (GBM) disease was treated with corticosteroid pulse therapy 9 days after initial infection-like symptoms with high procalcitonin value. The patient required hemodialysis the next day of the treatment due to oliguria. In addition to corticosteroid therapy, plasmapheresis was introduced and the patient could discontinue hemodialysis 43 days after the treatment. Kidney biopsy after initiation of hemodialysis confirmed anti-GBM disease with 86.3% crescent formation. Physician should keep in mind that active anti-GBM disease shows even high procalcitonin value in the absence of infection. To pursue recovery of renal function, the challenge of the immediate and persistent treatment with high-dose corticosteroids plus plasmapheresis for highly suspected anti-GBM disease is vitally important despite the presence of reported predictors for dialysis-dependence including oliguria and requiring hemodialysis at presentation.

Analysis of novel Sir3 binding regions in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the HMR, HML, telomere and rDNA regions are silenced. Silencing at the rDNA region requires Sir2, and silencing at the HMR, HML and telomere regions requires binding of a protein complex, consisting of Sir2, Sir3 and Sir4, that mediates repression of gene expression. Here, several novel Sir3 binding domains, termed CN domains (Chromosomal Novel Sir3 binding region), were identified using chromatin immunoprecipitation (ChIP) on chip analysis of S. cerevisiae chromosomes. Furthermore, analysis of G1-arrested cells demonstrated that Sir3 binding was elevated in G1-arrested cells compared with logarithmically growing asynchronous cells, and that Sir3 binding varied with the cell cycle. In addition to 14 CN regions identified from analysis of logarithmically growing asynchronous cells (CN1-14), 11 CN regions were identified from G1-arrested cells (CN15-25). Gene expression at some CN regions did not differ between WT and sir3Δ strains. Sir3 at conventional heterochromatic regions is thought to be recruited to chromosomes by Sir2 and Sir4; however, in this study, Sir3 binding occurred at some CN regions even in sir2Δ and sir4Δ backgrounds. Taken together, our results suggest that Sir3 exhibits novel binding parameters and gene regulatory functions at the CN binding domains.

Stimulation of V1a receptor increases renal uric acid clearance via urate transporters: insight into pathogenesis of hypouricemia in SIADH.

BACKGROUND: Hypouricemia is pathognomonic in syndrome of inappropriate secretion of antidiuretic hormone (SIADH) but the underlying mechanism remains unclear. Based on the previous studies, we hypothesized that V1a receptor may play a principal role in inducing hypouricemia in SIADH and examined uric acid metabolism using a rat model. METHODS: Terlipressin (25 ng/h), a selective V1a agonist, was subcutaneously infused to 7-week-old male Wistar rats (n = 9). Control rats were infused with normal saline (n = 9). The rats were sacrificed to obtain kidney tissues 3 days after treatment. In addition to electrolyte metabolism, changes in expressions of the urate transporters including URAT1 (SLC22A12), GLUT9 (SLC2A9), ABCG2 and NPT1 (SLC17A1) were examined by western blotting and immunohistochemistry. RESULTS: In the terlipressin-treated rats, serum uric acid (UA) significantly decreased and the excretion of urinary UA significantly increased, resulting in marked increase in fractional excretion of UA. Although no change in the expression of URAT1, GLUT9 expression significantly decreased whereas the expressions of ABCG2 and NPT1 significantly increased in the terlipressin group. The results of immunohistochemistry corroborated with those of the western blotting. Aquaporin 2 expression did not change in the medulla, suggesting the independence of V2 receptor stimulation. CONCLUSION: Stimulation of V1a receptor induces the downregulation of GLUT9, reabsorption urate transporter, together with the upregulation of ABCG2 and NPT1, secretion urate transporters, all changes of which clearly lead to increase in renal UA clearance. Hypouricemia seen in SIADH is attributable to V1a receptor stimulation.

Acute Kidney Injury Facilitates Hypocalcemia by Exacerbating the Hyperphosphatemic Effect of Muscle Damage in Rhabdomyolysis.

BACKGROUND/AIMS: Hypocalcemia is an important complication of rhabdomyolysis for which several pathogenic factors, including acute kidney injury (AKI), have been proposed. To gain insight regarding the hypocalcemic roles of AKI in rhabdomyolysis, we retrospectively examined patients with rhabdomyolysis. METHODS: Of 28,387 patients admitted to the Department of Internal Medicine, 51 patients met the inclusion criteria for the study. Serum calcium was analyzed based on laboratory data including indicators of AKI, serum creatine kinase (CK) and serum inorganic phosphate (iP). RESULTS: Twenty-two patients (43%) had hypocalcemia. Compared with patients without hypocalcemia, they had a higher prevalence of AKI (82 vs. 55%; p = 0.046), higher levels of peak CK (39,100 ± 50,600 vs. 9,800 ± 11,900 IU/l; p = 0.003) and higher levels of peak iP (1.77 ± 1.10 vs. 1.10 ± 0.35 mmol/l; p = 0.007). Indicators of AKI were correlated with peak CK and peak iP and were not significant variables in the regression analysis for hypocalcemia. Peak CK and peak iP were not correlated with each other. Impaired phosphate use by muscle contributed to the increased iP. CONCLUSION: These findings indicate that muscle damage is the primary hypocalcemic factor in rhabdomyolysis. AKI facilitated hypocalcemia by exacerbating the hyperphosphatemic effects of muscle damage. Aggressive hydration, which could increase oxygen supply and subsequently repair phosphate use in muscle, might reduce the incidence of hypocalcemia in rhabdomyolysis.

Three-dimensional analyses of aging-induced alterations in facial shape: a longitudinal study of 171 Japanese males.

We sought to generate data to facilitate forensic facial comparisons. Specifically, we conducted a longitudinal study of alterations in face shape induced by aging. We obtained two three-dimensional facial shape measurements in 171 Japanese males at intervals of approximately 10 years. With this data, we created a homologous model consisting of 10,741 data points for each face based on 33 anatomical landmarks. We averaged the movements of corresponding data points between the two homologous models for each individual and used this data to predict up to 30 years of face aging in an average Japanese male. We clearly identified aging-induced shape changes, such as drooping and denting of the facial folds, drooping of the upper lip, and projection of the lower eyelid, in the virtually aged model. A quantitative comparison of aging-induced shape alterations among three age groups (individuals in their 20's, 30's, and 40-50's) showed that these alterations accelerated more quickly as age increased. Using our predictive model, we conducted a preliminary study focused on facial shape alterations induced by reductions in body weight. Our findings indicated that our proposed method would also be valid for this purpose.

Combination of ACE inhibitor with nicorandil provides further protection in chronic kidney disease.

An inhibition in the renin-angiotensin system (RAS) is one of the most widely used therapies to treat chronic kidney disease. However, its effect is occasionally not sufficient and additional treatments may be required. Recently, we reported that nicorandil exhibited renoprotective effects in a mouse model of diabetic nephropathy. Here we examined if nicorandil can provide an additive protection on enalapril in chronic kidney disease. Single treatment with either enalapril or nicorandil significantly ameliorated glomerular and tubulointerstitial injury in the rat remnant kidney while the combination of these two compounds provided additive effects. In addition, an increase in oxidative stress in remnant kidney was also blocked by either enalapril or nicorandil while the combination of the drugs was more potent. A mechanism was likely due for nicorandil to preventing manganase superoxide dismutase (MnSOD) and sirtuin (Sirt)3 from being reduced in injured kidneys. A study with cultured podocytes indicated that the antioxidative effect could be mediated through sulfonylurea receptor (SUR) in the mitochondrial KATP channel since blocking SUR with glibenclamide reduced MnSOD and Sirt3 expression in podocytes. In conclusion, nicorandil may synergize with enalapril to provide superior protection in chronic kidney disease.

An evaluation of the effect of microwave irradiation on bone decalcification aimed to DNA extraction.

An effect of intermittent microwave irradiation on decalcification of compact bone followed by DNA extraction was verified. In order to perform quantitative analysis regarding the degree of decalcification, Cubic bone specimens were prepared from bovine metacarpal bone and micro-focus X-ray CT imaging was applied to measure precise volume of decalcified area in the cubes. Microwave irradiation was performed under strict control of temperature using commercially available experimental device which is designed for advancing tissue fixation, decalcification, and antigen-antibody reaction by intermittent microwave. The integrity of the DNA obtained from irradiated specimen was also examined by PCR analysis. The results of morphological analysis with CT imaging showed that microwave irradiation has a positive effect on decalcification though that effect is not so drastic. The results obtained from PCR analysis showed that microwave irradiation decrease amplifiable DNA, suggesting that we should be careful to use microwave for the purpose of bone DNA extraction.