Application of linear and machine learning models to genomic prediction of fatty acid composition in Japanese Black cattle.

We collected 3180 records of oleic acid (C18:1) and monounsaturated fatty acid (MUFA) measured using gas chromatography (GC) and 6960 records of C18:1 and MUFA measured using near-infrared spectroscopy (NIRS) in intermuscular fat samples of Japanese Black cattle. We compared genomic prediction performance for four linear models (genomic best linear unbiased prediction [GBLUP], kinship-adjusted multiple loci [KAML], BayesC, and BayesLASSO) and five machine learning models (Gaussian kernel [GK], deep kernel [DK], random forest [RF], extreme gradient boost [XGB], and convolutional neural network [CNN]). For GC-based C18:1 and MUFA, KAML showed the highest accuracies, followed by BayesC, XGB, DK, GK, and BayesLASSO, with more than 6% gain of accuracy by KAML over GBLUP. Meanwhile, DK had the highest prediction accuracy for NIRS-based C18:1 and MUFA, but the difference in accuracies between DK and KAML was slight. For all traits, accuracies of RF and CNN were lower than those of GBLUP. The KAML extends GBLUP methods, of which marker effects are weighted, and involves only additive genetic effects; whereas machine learning methods capture non-additive genetic effects. Thus, KAML is the most suitable method for breeding of fatty acid composition in Japanese Black cattle.

Splashed E-box and AP-1 motifs cooperatively drive regeneration response and shape regeneration abilities.

Injury triggers a genetic program that induces gene expression for regeneration. Recent studies have identified regeneration-response enhancers (RREs); however, it remains unclear whether a common mechanism operates in these RREs. We identified three RREs from the zebrafish fn1b promoter by searching for conserved sequences within the surrounding genomic regions of regeneration-induced genes and performed a transgenic assay for regeneration response. Two regions contained in the transposons displayed RRE activity when combined with the -0.7 kb fn1b promoter. Another non-transposon element functioned as a stand-alone enhancer in combination with a minimum promoter. By searching for transcription factor-binding motifs and validation by transgenic assays, we revealed that the cooperation of E-box and activator protein 1 motifs is necessary and sufficient for regenerative response. Such RREs respond to variety of tissue injuries, including those in the zebrafish heart and Xenopus limb buds. Our findings suggest that the fidelity of regeneration response is ensured by the two signals evoked by tissue injuries. It is speculated that a large pool of potential enhancers in the genome has helped shape the regenerative capacities during evolution.

Exploring the size of reference population for expected accuracy of genomic prediction using simulated and real data in Japanese Black cattle.

BACKGROUND: Size of reference population is a crucial factor affecting the accuracy of prediction of the genomic estimated breeding value (GEBV). There are few studies in beef cattle that have compared accuracies achieved using real data to that achieved with simulated data and deterministic predictions. Thus, extent to which traits of interest affect accuracy of genomic prediction in Japanese Black cattle remains obscure. This study aimed to explore the size of reference population for expected accuracy of genomic prediction for simulated and carcass traits in Japanese Black cattle using a large amount of samples. RESULTS: A simulation analysis showed that heritability and size of reference population substantially impacted the accuracy of GEBV, whereas the number of quantitative trait loci did not. The estimated numbers of independent chromosome segments (Me) and the related weighting factor (w) derived from simulation results and a maximum likelihood (ML) approach were 1900-3900 and 1, respectively. The expected accuracy for trait with heritability of 0.1-0.5 fitted well with empirical values when the reference population comprised > 5000 animals. The heritability for carcass traits was estimated to be 0.29-0.41 and the accuracy of GEBVs was relatively consistent with simulation results. When the reference population comprised 7000-11,000 animals, the accuracy of GEBV for carcass traits can range 0.73-0.79, which is comparable to estimated breeding value obtained in the progeny test. CONCLUSION: Our simulation analysis demonstrated that the expected accuracy of GEBV for a polygenic trait with low-to-moderate heritability could be practical in Japanese Black cattle population. For carcass traits, a total of 7000-11,000 animals can be a sufficient size of reference population for genomic prediction.

Multicenter retrospective analysis of clinicopathological features and prognosis of oral tongue squamous cell carcinoma in adolescent and young adult patients.

ABSTRACT: The aim of this study is to report the differences in clinicopathological features of oral tongue squamous cell carcinoma (OTSCC) and survival between adolescent and young adult (AYA) patients and elderly patients and to find the prognosticators. The medical records of 101 AYA patients and 175 control patients with OTSCC who underwent surgery were reviewed. Variables related to prognosis and their clinicopathological associations were analyzed. The 5-year overall survival (5y-OS) rates of AYA and control patients with stage I and II OTSCC were 94.4% and 89.6% (P = .353), respectively, and their 5-year disease-free survival (5y-DFS) rates were 82.0% and 76.6%, respectively (P = .476). The 5y-OS rates of patients with stages III and IV OTSCC were 83.3% and 66.7% (P = .333), respectively, and their 5y-DFS rates were 75.0% and 57.1% (P = .335), respectively. Logistic regression analysis revealed that there was no significant clinicopathological difference in AYA and control group. Furthermore, there was no significant difference in 5y-OS rates between patients who underwent elective neck dissection (END) and those who underwent therapeutic neck dissection (TND) in both group (P = 0.717 and 0.688). Overall, the present study revealed the clinicopathological features and prognosis of OTSCC were similar in AYA patients and elderly patients. Moreover, as there was no significant difference in OS between patients who underwent END and those who underwent TND in AYA and control groups, our results suggest that the indication for END in AYA patients with clinical N0 OTSCC is similar to that for elderly patients.

Detection of novel fusion genes by next-generation sequencing-based targeted RNA sequencing analysis in adenoid cystic carcinoma of head and neck.

OBJECTIVE: Adenoid cystic carcinoma (AdCC) is a rare, indolent salivary gland tumor that is reported to be driven by fusion genes. However, MYB/MYBL1-NFIB fusions have been detected in <60% of all AdCC cases and the oncogenic driver mutations in approximately 40% of AdCC remain unknown. Our aim was to identify novel gene fusions in AdCC. STUDY DESIGN: We investigated 20 AdCC cases using a targeted RNA sequencing panel to identify gene fusions and performed quantitative real-time reverse transcription polymerase chain reaction to assess MYB, MYBL1, and NFIB expression levels. RESULTS: A total of 36 fusion transcripts in 15 cases were detected and validated by Sanger sequencing. The MYB-NFIB and MYBL1-NFIB fusion genes were detected in 9 and 3 cases, respectively, in a mutually exclusive manner. Furthermore, novel gene fusions, namely, NFIB-EPB41L2, MAP7-NFIB, NFIB-MCMDC2, MYBL1-C8orf34, C8orf34-NFIB, and NFIB-CASC20, were identified. Among them, NFIB-EPB41L2 and NFIB-MCMDC2 are thought to activate MYB and MYBL1 expression, respectively, through the insertion of a genomic segment in proximity to MYB and MYBL1 genes, respectively. CONCLUSION: Six novel gene fusions other than MYB/MYBL1-NFIB were identified. The detection of novel fusion genes and investigation of the molecular mechanism will contribute to the development of novel molecular targeted therapies for this disease.

Fibroblast dedifferentiation as a determinant of successful regeneration.

Limb regeneration, while observed lifelong in salamanders, is restricted in post-metamorphic Xenopus laevis frogs. Whether this loss is due to systemic factors or an intrinsic incapability of cells to form competent stem cells has been unclear. Here, we use genetic fate mapping to establish that connective tissue (CT) cells form the post-metamorphic frog blastema, as in the case of axolotls. Using heterochronic transplantation into the limb bud and single-cell transcriptomic profiling, we show that axolotl CT cells dedifferentiate and integrate to form lineages, including cartilage. In contrast, frog blastema CT cells do not fully re-express the limb bud progenitor program, even when transplanted into the limb bud. Correspondingly, transplanted cells contribute to extraskeletal CT, but not to the developing cartilage. Furthermore, using single-cell RNA-seq analysis we find that embryonic and adult frog cartilage differentiation programs are molecularly distinct. This work defines intrinsic restrictions in CT dedifferentiation as a limitation in adult regeneration.

Characterization of genetically modified mice for phosphoglycerate mutase, a vitally-essential enzyme in glycolysis.

Glycolytic metabolism is closely involved in physiological homeostasis and pathophysiological states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been reported to exert certain physiological role in vitro, whereas its impact on glucose metabolism in vivo remains unclear. Here, we report the characterization of Pgam1 knockout mice. We observed that homozygous knockout mice of Pgam1 were embryonic lethal. Although we previously reported that both PGAM-1 and -2 affect global glycolytic profile of cancers in vitro, in vivo glucose parameters were less affected both in the heterozygous knockout of Pgam1 and in Pgam2 transgenic mice. Thus, the impact of PGAM on in vivo glucose metabolism is rather complex than expected before.

Secretory carcinoma around Stensen's duct misdiagnosed as salivary duct cyst.

Secretory carcinoma (SC) of the salivary gland was identified in 2010, and it is characterized by a specific ETV6 gene arrangement. The most common primary site for SC is the parotid gland; however, SC around the Stensen's duct is rare. Here we describe a rare case of a SC around the Stensen's duct that was initially misdiagnosed as a salivary duct cyst. A 59-year-old woman presented with a mass in the region of the left parotid papilla. Magnetic resonance imaging (MRI) revealed a well-circumscribed lesion and enhancement with a rim and an inner wall-like part that appeared in the late phase. Based on the initial clinical and imaging findings, a salivary duct cyst of the parotid gland was diagnosed. However, the lesion was histopathologically diagnosed as a SC based on immunohistochemical findings. The tumor cells showed diffuse positive staining for AE1/AE3, vimentin, and mammaglobin and focal positive staining for S-100 protein, SOX-10, and DOG-1. Fluorescence in-situ hybridization revealed ETV6 gene rearrangement in the tumor. In cases of cystic lesions around the Stensen's duct, clinicians should bear in mind that the possibility that they could be minor salivary gland cancers, such as SC.

Phosphoglycerate Mutase Cooperates with Chk1 Kinase to Regulate Glycolysis.

Dysregulated glycolysis, including the cancerous Warburg effect, is closely involved in pathological mechanisms of diseased states. Among glycolytic enzymes, phosphoglycerate mutase (PGAM) has been known to exert certain physiological impact in vitro, whereas its regulatory role on glycolysis remains unclear. Here, we identified that PGAM plays a key role in regulating glycolysis in cancer cells but not in standard cells. Cancer-prone phenotype by PGAM overexpression in vivo was associated with upregulated glycolytic features. PGAM interacts and cooperates with Chk1 to regulate the enhanced glycolysis in cancer cells, especially under oncogenic Ras expressing conditions. Genetic or chemical interference of the PGAM-Chk1 interaction, with intact PGAM activity, abrogated the maintenance of cancerous enhanced glycolysis. Thus, the nonenzymatic function of PGAM is essential for the Warburg effect that accompanies cancerous proliferation.

Robust and local positional information within a fin ray directs fin length during zebrafish regeneration.

It has been proposed that cells are regulated to form specific morphologies and sizes according to positional information. However, the entity and nature of positional information have not been fully understood yet. The zebrafish caudal fin has a characteristic V-shape; dorsal and ventral fin rays are longer than the central ones. This fin shape regenerates irrespective of the sites or shape of fin amputation. It is thought that reformation of tissue occurs according to positional information. In this study, we developed a novel transplantation procedure for grafting a whole fin ray to an ectopic position and examined whether the information that specifies fin length exists within each fin ray. Intriguingly, when long and short fin rays were swapped, they regenerated to form longer or shorter fin rays than the adjacent host fin rays, respectively. Further, the abnormal fin ray lengths were maintained for a long time, more than 5 months, and after further re-amputation. In contrast to intra-fin grafting, when fin ray grafting was performed between fish, cells in the grafts disappeared due to immune rejection, and the grafted fin rays adapted to the host position to form a normal fin. Together, our data suggest that the information that directs fin length does exist in cells within a single fin ray and that it has a robust property-it is stable for a long time and is hard to rewrite. Our study highlighted a novel positional information mechanism for directing regenerating fin length.

Heterogeneous fates and dynamic rearrangement of regenerative epidermis-derived cells during zebrafish fin regeneration.

The regenerative epidermis (RE) is a specialized tissue that plays an essential role in tissue regeneration. However, the fate of the RE during and after regeneration is unknown. In this study, we performed Cre-loxP-mediated cell fate tracking and revealed the fates of a major population of the RE cells that express fibronectin 1b (fn1b) during zebrafish fin regeneration. Our study showed that these RE cells are mainly recruited from the inter-ray epidermis, and that they follow heterogeneous cell fates. Early recruited cells contribute to initial wound healing and soon disappear by apoptosis, while the later recruited cells contribute to the regenerated epidermis. Intriguingly, many of these cells are also expelled from the regenerated tissue by a dynamic caudal movement of the epidermis over time, and in turn the loss of epidermal cells is replenished by a global self-replication of basal and suprabasal cells in fin. De-differentiation of non-basal epidermal cells into the basal epidermal cells did not occur during regeneration. Overall, our study reveals the heterogeneous fates of RE cells and a dynamic rearrangement of the epidermis during and after regeneration.

Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration.

Transplantation of Mesenchymal Cells Including the Blastema in Regenerating Zebrafish Fin.

Regeneration of fish fins and urodele limbs occurs via formation of the blastema, which is a mass of mesenchymal cells formed at the amputated site and is essential for regeneration. The blastema transplantation, a novel technique developed in our previous studies ( Shibata et al., 2016 ; Yoshinari et al., 2012 ) is a useful approach for tracking and manipulating the blastema cells during fish fin regeneration.

Supplemental feeding of captive neonatal koalas (Phascolarctos cinereus).

Koalas (Phascolarctos cinereus) are cautious animals, making supplemental feeding of neonates challenging because of disturbances to the normal routine. However, supplemental feeding is beneficial in improving juvenile nutrition using less formula than required for hand-rearing, and allowing maternal bonding to continue through suckling. In this study, two neonatal koalas, delivered by the same mother in 2 years, exhibited insufficient growth post-emergence from the pouch; supplemental feeding was therefore initiated. The amount of formula fed was determined according to the product instructions, and offspring weight was monitored. Slower than normal growth was not initially noticed in the first offspring. This caused delayed commencement of supplemental feeding. An attempt was made to counteract this by providing more formula for a longer period; however, this meant No. 1 was unable to eat enough eucalyptus when weaning. Supplemental feeding was started earlier for the second offspring than for the first, and was terminated at weaning; this juvenile showed a healthy body weight increase. Furthermore, it was able to eat eucalyptus leaves at an earlier stage than No. 1. Although No. 1 showed delayed growth, both koalas matured and are still living. This study showed that supplemental feeding is useful for koalas, if the mother will accept human intervention. The key factors for successful supplemental feeding of koalas identified by comparing the two feeding systems observed in this study are that: (1) it should be initiated as soon as insufficient growth is identified; and (2) it should be terminated before weaning age. Zoo Biol. 36:62-65, 2017. © 2016 Wiley Periodicals, Inc.

Ruthenium-Sulfonamide-Catalyzed Direct Dehydrative Condensation of Benzylic C-H Bonds with Aromatic Aldehydes.

The first catalytic dehydrative condensation of the benzylic C-H bonds of toluene and p-xylene with aromatic aldehydes is reported herein. This protocol provides highly atom-economical access to stilbene and p-distyrylbenzene derivatives, whereby water is the sole byproduct. The reaction is based on the deprotonation-functionalization of benzylic C-H bonds through η6-complexation of the arenes, which is realized for the first time using a catalytic amount of a transition metal activator. The key to the success of this method is the use of a sulfonamide anion as a catalyst component, which appears to facilitate not only the deprotonation of the benzylic C-H bonds but also the formation of a C-C bonds via an electrophilic tosylimine intermediate.

Fgf signalling controls diverse aspects of fin regeneration.

Studies have shown that fibroblast growth factor (Fgf) signalling is necessary for appendage regeneration, but its exact function and the ligands involved during regeneration have not yet been elucidated. Here, we performed comprehensive expression analyses and identified fgf20a and fgf3/10a as major Fgf ligands in the wound epidermis and blastema, respectively. To reveal the target cells and processes of Fgf signalling, we performed a transplantation experiment of mesenchymal cells that express the dominant-negative Fgf receptor 1 (dnfgfr1) under control of the heat-shock promoter. This mosaic knockdown analysis suggested that Fgf signalling is directly required for fin ray mesenchyme to form the blastema at the early pre-blastema stage and to activate the regenerative cell proliferation at a later post-blastema stage. These results raised the possibility that the early epidermal Fgf20a and the later blastemal Fgf3/10a could be responsible for these respective processes. We demonstrated by gain-of-function analyses that Fgf20a induces the expression of distal blastema marker junbl, and that Fgf3 promotes blastema cell proliferation. Our study highlights that Fgfs in the wound epidermis and blastema have distinct functions to regulate fin regeneration cooperatively.

Glycolipid dynamics in generation and differentiation of induced pluripotent stem cells.

Glycosphingolipids (GSLs) are glycoconjugates that function as mediators of cell adhesion and modulators of signal transduction. Some well-defined markers of undifferentiated human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) are glycoconjugates, such as SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. However, Comprehensive GSL profiles of hiPSCs have not yet been elucidated. The global images of GSLs from the parental cells, hiPSCs, and differentiated cells revealed that there are parental cell-independent specific glycolipids, including Globo H (fucosyl-Gb5Cer) and H type1 antigen (fucosyl-Lc4Cer) that are novel markers for undifferentiated hiPSCs. Interestingly, undifferentiated hiPSCs expressed H type 1 antigen, specific for blood type O, regardless of the cells' genotypes. Thus, in this study, we defined the dynamics of GSL remodeling during reprogramming from parental cell sets to iPSC sets and thence to iPSC-neural cells.

Long-term surveillance and treatment of subclinical cryptococcosis and nasal colonization by Cryptococcus neoformans and C. gattii species complex in captive koalas (Phascolarctes cinereus).

Cryptococcosis is an important systemic mycosis caused by members of the Cryptococcus neoformans species complex. This disease is potentially fatal in various animals, including koalas. We describe the long-term surveillance and treatment of subclinical cryptococcosis and nasal colonization of koalas by Cryptococcus neoformans and C. gattii. Of the 15 animals investigated through the use of samples obtained by nasal swabs, antigen titer measurements, and pathologic examination, C. neoformans was found associated with nine koalas and C. gattii with one animal. Nine koalas showed subclinical disease and one clinical infections and antigenemia. Treatment with fluconazole, itraconazole and amphotericin B upon detection of C. neoformans or C. gattii was not effective. The results of the present study showed that C. neoformans was the predominant species isolated from the nasal swab samples and the fungus might have naturally become associated with the koalas' nasal cavities at Kanazawa Zoological Gardens. The unclear treatment effectiveness might have been caused by a shorter treatment period that is routinely used and unstable itraconazole absorption. This investigation also underscores the need for identifying effective treatment regimens for subclinical cryptococcosis and efficient measures for eradicating C. neoformans and C. gattii in koalas.

Opposing role of condensin hinge against replication protein A in mitosis and interphase through promoting DNA annealing.

Condensin is required for chromosome dynamics and diverse DNA metabolism. How condensin works, however, is not well understood. Condensin contains two structural maintenance of chromosomes (SMC) subunits with the terminal globular domains connected to coiled-coil that is interrupted by the central hinge. Heterotrimeric non-SMC subunits regulate SMC. We identified a novel fission yeast SMC hinge mutant, cut14-Y1, which displayed defects in DNA damage repair and chromosome segregation. It contains an amino acid substitution at a conserved hinge residue of Cut14/SMC2, resulting in diminished DNA binding and annealing. A replication protein A mutant, ssb1-418, greatly alleviated the repair and mitotic defects of cut14-Y1. Ssb1 protein formed nucleolar foci in cut14-Y1 cells, but the number of foci was diminished in cut14-Y1 ssb1-418 double mutants. Consistent with the above results, Ssb1 protein bound to single-strand DNA was removed by condensin or the SMC dimer through DNA reannealing in vitro. Similarly, RNA hybridized to DNA may be removed by the SMC dimer. Thus, condensin may wind up DNA strands to unload chromosomal components after DNA repair and prior to mitosis. We show that 16 suppressor mutations of cut14-Y1 were all mapped within the hinge domain, which surrounded the original L543 mutation site.

Visual discrimination among patients with depression and schizophrenia and healthy individuals using semiquantitative color-coded fast spin-echo T1-weighted magnetic resonance imaging.

INTRODUCTION: Fast spin-echo (FSE) T1-weighted (T1W) magnetic resonance imaging (MRI) at 3T, which is sensitive to neuromelanin-related contrast, can quantitatively detect signal alterations in the locus ceruleus (LC) and the substantia nigra pars compacta (SNc) of depressive and schizophrenic patients; however, its qualitative diagnostic performance remains unknown. We investigated whether visual interpretation of semiquantitative color maps can be used for discriminating between depressive and schizophrenic patients and healthy individuals. METHODS: We retrospectively examined 23 patients with major depression, 23 patients with schizophrenia, and 23 age-matched healthy controls by using a FSE-T1W MRI technique. Semiquantitative color maps of sections through the LC and SNc were visually interpreted by nine raters using a continuous confidence rating scale for receiver operating characteristic (ROC) analysis. RESULTS: The area under the ROC curve (Az), which reflects the performance in differentiating between depressive patients and controls, was 0.88, and the sensitivity and specificity at the maximum likelihood were 76% and 83%, respectively. In contrast, the Az value, sensitivity, and specificity values between schizophrenics and controls and between depressives and schizophrenics were 0.66 and 0.69, 42% and 48%, and 82% and 84%, respectively. CONCLUSION: Semiquantitative, color-coded FSE-T1W MRI at 3T can be used for visually differentiating depressive patients from healthy individuals with a substantially high likelihood, but this technique cannot be applied to distinguish schizophrenic patients from the other two groups.