Identification and characterization of structural variants related to meat quality in pigs using chromosome-level genome assemblies.

BACKGROUND: Many studies have been performed to identify various genomic loci and genes associated with the meat quality in pigs. However, the full genetic architecture of the trait still remains unclear in part because of the lack of accurate identification of related structural variations (SVs) which resulted from the shortage of target breeds, the limitations of sequencing data, and the incompleteness of genome assemblies. The recent generation of a new pig breed with superior meat quality, called Nanchukmacdon, and its chromosome-level genome assembly (the NCMD assembly) has provided new opportunities. RESULTS: By applying assembly-based SV calling approaches to various genome assemblies of pigs including Nanchukmacdon, the impact of SVs on meat quality was investigated. Especially, by checking the commonality of SVs with other pig breeds, a total of 13,819 Nanchukmacdon-specific SVs (NSVs) were identified, which have a potential effect on the unique meat quality of Nanchukmacdon. The regulatory potentials of NSVs for the expression of nearby genes were further examined using transcriptome- and epigenome-based analyses in different tissues. CONCLUSIONS: Whole-genome comparisons based on chromosome-level genome assemblies have led to the discovery of SVs affecting meat quality in pigs, and their regulatory potentials were analyzed. The identified NSVs will provide new insights regarding genetic architectures underlying the meat quality in pigs. Finally, this study confirms the utility of chromosome-level genome assemblies and multi-omics analysis to enhance the understanding of unique phenotypes.

Regulation of Dihydropyrimidinase-like 3 Gene Expression by MicroRNAs in PC12 Cells with Induced Ischaemia and Hypothermia.

Although hypothermic treatment has been reported to have some beneficial effects on ischaemia at the clinical level, the mechanism of ischaemia suppression by hypothermia remains unclear due to a lack of mechanism understanding and insufficient data. The aim of this study was to isolate and characterize microRNAs specifically expressed in ischaemia-hypothermia for the dihydropyrimidinase-like 3 (Dpysl3) gene. PC12 cells were induced with CoCl2 for chemical ischaemia and incubated at 32 ℃ for hypothermia. In ischaemia-hypothermia, four types of microRNAs (miR-106b-5p, miR-194-5p, miR-326-5p, and miR-497-5p) were highly related to the Dpysl3 gene based on exosomal microRNA analysis. Dpysl3 gene expression was up-regulated by miR-497-5p but down-regulated by miR-106b-5p, miR-194-5p and miR-326-5p. Our results suggest that these four microRNAs are involved in the regulation of Dpysl3 gene expression. These findings provide valuable clues that exosomal microRNAs could be used as therapeutic targets for effective treatment of ischaemia.

A chromosome-level genome assembly of the Korean crossbred pig Nanchukmacdon (Sus scrofa).

As plentiful high-quality genome assemblies have been accumulated, reference-guided genome assembly can be a good approach to reconstruct a high-quality assembly. Here, we present a chromosome-level genome assembly of the Korean crossbred pig called Nanchukmacdon (the NCMD assembly) using the reference-guided assembly approach with short and long reads. The NCMD assembly contains 20 chromosome-level scaffolds with a total size of 2.38 Gbp (N50: 138.77 Mbp). Its BUSCO score is 93.1%, which is comparable to the pig reference assembly, and a total of 20,588 protein-coding genes, 8,651 non-coding genes, and 996.14 Mbp of repetitive elements are annotated. The NCMD assembly was also used to close many gaps in the pig reference assembly. This NCMD assembly and annotation provide foundational resources for the genomic analyses of pig and related species.

msPIPE: a pipeline for the analysis and visualization of whole-genome bisulfite sequencing data.

BACKGROUND: DNA methylation is an important epigenetic modification that is known to regulate gene expression. Whole-genome bisulfite sequencing (WGBS) is a powerful method for studying cytosine methylation in a whole genome. However, it is difficult to obtain methylation profiles using the WGBS raw reads and is necessary to be proficient in all types of bioinformatic tools for the study of DNA methylation. In addition, recent end-to-end pipelines for DNA methylation analyses are not sufficient for addressing those difficulties. RESULTS: Here we present msPIPE, a pipeline for DNA methylation analyses with WGBS data seamlessly connecting all the required tasks ranging from data pre-processing to multiple downstream DNA methylation analyses. The msPIPE can generate various methylation profiles to analyze methylation patterns in the given sample, including statistical summaries and methylation levels. Also, the methylation levels in the functional regions of a genome are computed with proper annotation. The results of methylation profiles, hypomethylation, and differential methylation analysis are plotted in publication-quality figures. The msPIPE can be easily and conveniently used with a Docker image, which includes all dependent packages and software related to DNA methylation analyses. CONCLUSION: msPIPE is a new end-to-end pipeline designed for methylation calling, profiling, and various types of downstream DNA methylation analyses, leading to the creation of publication-quality figures. msPIPE allows researchers to process and analyze the WGBS data in an easy and convenient way. It is available at https://github.com/jkimlab/msPIPE and https://hub.docker.com/r/jkimlab/mspipe .

Bi-allelic loss-of-function variants in TMEM147 cause moderate to profound intellectual disability with facial dysmorphism and pseudo-Pelger-Huët anomaly.

The transmembrane protein TMEM147 has a dual function: first at the nuclear envelope, where it anchors lamin B receptor (LBR) to the inner membrane, and second at the endoplasmic reticulum (ER), where it facilitates the translation of nascent polypeptides within the ribosome-bound TMCO1 translocon complex. Through international data sharing, we identified 23 individuals from 15 unrelated families with bi-allelic TMEM147 loss-of-function variants, including splice-site, nonsense, frameshift, and missense variants. These affected children displayed congruent clinical features including coarse facies, developmental delay, intellectual disability, and behavioral problems. In silico structural analyses predicted disruptive consequences of the identified amino acid substitutions on translocon complex assembly and/or function, and in vitro analyses documented accelerated protein degradation via the autophagy-lysosomal-mediated pathway. Furthermore, TMEM147-deficient cells showed CKAP4 (CLIMP-63) and RTN4 (NOGO) upregulation with a concomitant reorientation of the ER, which was also witnessed in primary fibroblast cell culture. LBR mislocalization and nuclear segmentation was observed in primary fibroblast cells. Abnormal nuclear segmentation and chromatin compaction were also observed in approximately 20% of neutrophils, indicating the presence of a pseudo-Pelger-Huët anomaly. Finally, co-expression analysis revealed significant correlation with neurodevelopmental genes in the brain, further supporting a role of TMEM147 in neurodevelopment. Our findings provide clinical, genetic, and functional evidence that bi-allelic loss-of-function variants in TMEM147 cause syndromic intellectual disability due to ER-translocon and nuclear organization dysfunction.

DLEB: a web application for building deep learning models in biological research.

Deep learning has been applied for solving many biological problems, and it has shown outstanding performance. Applying deep learning in research requires knowledge of deep learning theories and programming skills, but researchers have developed diverse deep learning platforms to allow users to build deep learning models without programming. Despite these efforts, it is still difficult for biologists to use deep learning because of limitations of the existing platforms. Therefore, a new platform is necessary that can solve these challenges for biologists. To alleviate this situation, we developed a user-friendly and easy-to-use web application called DLEB (Deep Learning Editor for Biologists) that allows for building deep learning models specialized for biologists. DLEB helps researchers (i) design deep learning models easily and (ii) generate corresponding Python code to run directly in their machines. DLEB provides other useful features for biologists, such as recommending deep learning models for specific learning tasks and data, pre-processing of input biological data, and availability of various template models and example biological datasets for model training. DLEB can serve as a highly valuable platform for easily applying deep learning to solve many important biological problems. DLEB is freely available at http://dleb.konkuk.ac.kr/.

Induction of apoptosis through the up-regulation of endoplasmic reticulum stress sensors by 5-hydroxy-7- (4"-hydroxy-3"-methoxyphenyl)-1-phenyl-3-heptanone.

The diarylheptanoid, 5-hydroxy-7-(4"-hydroxy-3"-metho-xyphenyl)-1-phenyl-3-heptanone (HPH), is isolated from rhizomes of Alpinia officinarum. There is no reported biological function for this compound other than the inhibition of pancreatic lipase. Cell viability, the expression of endoplasmic reticulum (ER) stress genes, the activation of ER stress sensors, and the induction of apoptosis and autophagy were confirmed following HPH treatment of PC12 cells. No cytotoxicity was observed when the cells were treated with 50 µg/ml HPH, but 40% cell death was observed using MTT assays with 100 µg/ml HPH. Although HPH did not change the expression of the ER chaperones PDI, binding BiP, and calnexin, it upregulated the expression of genes for the ER stress sensors ATF6, eIF2α, and PERK. HPH also induced apoptosis via the activation of ATF6 fragmentation, the phosphorylation of eIF2α, and XBP1 mRNA splicing. Eventually, the results of this study demonstrated that HPH induces apoptosis through upregulation of gene expression of ER stress sensors, which may provide a basis for the development of new drugs using HPH.

Impact of Endoplasmic Reticulum Stress Sensors on Pectolinarin Induced Apoptosis.

Pectolinarin, [5,7-Dihydroxy 4',6-dimethoxyflavone 7-rutinoside, 7-[[6-O-(6-Deoxy-α-L-mannopyranosyl)-ß-D-glucopyranosyl] oxy]-5-hydroxy-6-methoxy-2-(4-ethoxyphenyl)-4H-1-benzopyran-4-one], has been stated one of the major compounds in Cirsium nipponicum (Maxim.) Makino. It is characterized by biological functions of hepatoprotective, anti-inflammatory and antiobesity activities. In this research, it was explained that pectolinarin causes apoptosis in PC12 cells conducted by DNA fragmentation and formation on apoptotic bodies through the activation of ER stress sensors (ATF6 fragmentation and eIF2α phosphorylation). The result of treating the PC12 cells with 50 µM pectolinarin for 24 h has come to increase ATF6 mRNA expression up to 1.6 times, PERK expression up to 1.7 times and IRE1 expression up to 1.4 times, respectively, compared to those of the control. ATF6 fragmentation by pectolinarin treatment was increased about 2 times compared with its control, and phosphorylation of eIF2α was increased 2.5 times. The results proposed that the perception of the molecular mechanisms underlying pectolinarin-caused apoptosis may be useful in new natural medicinal products and health supplements for the apoptosis-related diseases.

Population analysis of the Korean native duck using whole-genome sequencing data.

BACKGROUND: Advances in next-generation sequencing technologies have provided an opportunity to perform population-level comparative genomic analysis to discover unique genomic characteristics of domesticated animals. Duck is one of the most popular domesticated waterfowls, which is economically important as a source of meat, eggs, and feathers. The objective of this study is to perform population and functional analyses of Korean native duck, which has a distinct meat flavor and texture phenotype, using whole-genome sequencing data. To study the distinct genomic features of Korean native duck, we conducted population-level genomic analysis of 20 Korean native ducks together with 15 other duck breeds. RESULTS: A total of 15.56 million single nucleotide polymorphisms were detected in Korean native duck. Based on the unique existence of non-synonymous single nucleotide polymorphisms in Korean native duck, a total of 103 genes related to the unique genomic characteristics of Korean native duck were identified in comparison with 15 other duck breeds, and their functions were investigated. The nucleotide diversity and population structures among the used duck breeds were then compared, and their phylogenetic relationship was analyzed. Finally, highly differentiated genomic regions among Korean native duck and other duck breeds were identified, and functions of genes in those regions were examined. CONCLUSIONS: This is the first study to compare the population of Korean native duck with those of other duck breeds by using whole-genome sequencing data. Our findings can be used to expand our knowledge of genomic characteristics of Korean native duck, and broaden our understanding of duck breeds.

Molecular cloning of troponin i from the two-spotted cricket, Gryllus bimaculatus, and its expression pattern during starvation / Clonagem molecular da Troponina I do grilo africano, Gryllus bimaculatus, e seu padrão de expressão durante a inanição

There is few information about troponin gene expression by starvation in insect skeletal muscle and digestive tracts. The objective of this study was to perform molecular cloning of troponin I from the two-spotted cricket, Gryllus bimaculatus (GrybiTnI) and determine its expression patterns in three different skeletal muscles and digestive tracts during starvation. GrybiTnI was translated into a protein encoding 198 amino acids with a theoretical isoelectric point of 9.78 and a molecular weight of 23671.46 Da. The GrybiTnI has both the TnC-binding site and actin/TnC-binding site shown in the typical TnI amino acid sequences. Homology analysis revealed that GrybiTnI exhibited high similarity at the amino acid level to those of other insects already reported; 89~77% identity with those of other insects. Expression of GrybiTnI by starvation did not change in dorsal wing flight muscle and dorsal ventral flight muscle, but showed up-expression in dorsal longitudinal flight muscle. In the digestive tracts, the up-expression of GrybiTnI by starvation was observed only in the hindgut but not in the rest parts including Malpighian tubules. Re-feeding following starvation restored those expressions about the level before starvation in the dorsal longitudinal flight muscle and hindgut. In conclusion, troponin modulates gene expression not only to muscle elements but also to physiological changes such as strains.

Existe pouca informação sobre a expressão gênica da troponina por inanição no músculo esquelético de insetos e no trato digestório. O objetivo deste estudo foi realizar clonagem molecular da troponina I do grilo africano, Gryllus bimaculatus (GrybiTnI) e determinar seus padrões de expressão em três diferentes músculos esqueléticos e tratos digestivos durante a inanição. GrybiTnI foi traduzido em uma proteína codificando 198 aminoácidos com um ponto isoelétrico teórico de 9,78 e um peso molecular de 23671,46 Da. O GrybiTnI tem o local de ligação a TnC e o local de ligação a actina/TnC mostrado nas sequências de aminoácidos TnI típicas. A análise de homologia revelou que GrybiTnI exibiu alta similaridade no nível de aminoácidos em relação àqueles de outros insetos já relatados; 89~77% de identidade com os de outros insetos. A expressão de GrybiTnI pela inanição não se alterou no músculo de vôo da asa dorsal e no músculo de vôo ventral dorsal, mas mostrou expressão positiva no músculo de vôo longitudinal dorsal. Nos tratos digestivos, a expressão positiva de GrybiTnI por inanição foi observada apenas no intestino grosso, mas não nas partes de repouso, incluindo os túbulos de Malpighi. A realimentação após a inanição restaurou as expressões aproximadamente ao nível antes da inanição no músculo de vôo longitudinal dorsal e no intestino grosso. Em conclusão, a troponina modula a expressão gênica não apenas em elementos musculares, mas também em alterações fisiológicas, como as cepas.

Luteolin-induced apoptosis through activation of endoplasmic reticulum stress sensors in pheochromocytoma cells.

Luteolin [2-(3,4-dihydroxyphenyl)-5,7-dihydroxy-4-chromenone] is an active flavonoid compound from Lonicera japonica (Caprifoliaceae). Luteolin inhibits tumor cell proliferation, inflammatory and oxidative stress better, when compared with other flavonoids. In the present study, it was demonstrated that luteolin induces typical apoptosis in PC12 cells (derived from a pheochromocytoma of the rat adrenal medulla) accompanied by DNA fragmentation and formation of apoptotic bodies. In addition, luteolin regulates expression of the endoplasmic reticulum (ER) chaperone binding immunoglobulin protein, activating ER stress sensors (eukaryotic initiation factor 2α phosphorylation and X­box binding protein 1 mRNA splicing) and induced autophagy. The results indicated that luteolin induces the upregulation of the unfolded protein response pathway through the ER stress sensors, which helps as an influential regulator for the apoptosis pathway in PC12 cells. The results suggested that the understanding of the molecular mechanisms underlying luteolin­induced apoptosis may be useful in cancer therapeutics, chemoprevention and neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease.

Effect of burdock extracts upon inflammatory mediator production.

We investigated the burdock extract on the inhibitions of NO generation, COX-2 expression, and the generations of IL-6 and TNF-α , to find out its anti-inflammatory effect in this study. After the treatment of the burdock extract in the cells, we measured the amount of NO generated in the inflammatory cells developed by LPS and UVB, and confirmed the developments of inflammatory mediators by RT-PCR. Upon the results on the NO generation after the development of inflammation by LPS in Raw 264.7 cell, we found approximately 50% of inhibitory effect at 200 µg/ml concentrations of the burdock extract. It was confirmed that the expression levels of TNF-α, COX-2, and IL-6 were declined to the levels of control by LPS and UVB stimulated inflammation in HaCat cell. This means the anti-inflammatory effect of the burdock extract.

Endoplasmic reticulum stress impairment in the spinal dorsal horn of a neuropathic pain model.

Endoplasmic reticulum (ER) stress has been implicated in neurodegenerative diseases, but its role in neuropathic pain remains unclear. In this study, we examined the ER stress and the unfolded protein response (UPR) activation in a L5 spinal nerve ligation (SNL)-induced rat neuropathic pain model. SNL-induced neuropathic pain was assessed behaviorally using the CatWalk system, and histologically with microglial activation in the dorsal spinal horn. L5 SNL induced BIP upregulation in the neuron of superficial laminae of dorsal spinal horn. It also increased the level of ATF6 and intracellular localization into the nuclei in the neurons. Moreover, spliced XBP1 was also markedly elevated in the ipsilateral spinal dorsal horn. The PERK-elF2 pathway was activated in astrocytes of the spinal dorsal horn in the SNL model. In addition, electron microscopy revealed the presence of swollen cisternae in the dorsal spinal cord after SNL. Additionally, inhibition of the ATF6 pathway by intrathecal treatment with ATF6 siRNA reduced pain behaviors and BIP expression in the dorsal horn. The results suggest that ER stress might be involved in the induction and maintenance of neuropathic pain. Furthermore, a disturbance in UPR signaling may render the spinal neurons vulnerable to peripheral nerve injury or neuropathic pain stimuli.

RhoGDI2 expression in astrocytes after an excitotoxic lesion in the mouse hippocampus.

The Rho GDP-dissociation inhibitor (RhoGDI) originally downregulates Rho family GTPases by preventing nucleotide exchange and membrane association. Although RhoGDI2 functions as a metastasis regulator, little is known in glial cells under neuropathological conditions. We monitored RhoGDI2 expression in the mouse brain after administering a kainic acid(KA)-induced excitotoxic lesion. In control, RhoGDI2 immunoreactivity (IR) was evident in the neuronal layer of the hippocampus. However, RhoGDI2 IR was increased in astrocytes markedly throughout the hippocampus at day 3 post-treatment with KA. To further investigate the molecular mechanism of RhoGDI2-induced cellular migration, primary astrocytes were transfected with the flag-tagged RhoGDI2 cDNA. Cell migration assay revealed that RhoGDI2 cDNA transfection inhibits astrocyte migration. Overexpression of RhoGDI2 leads to inhibit protein kinase B (PKB) activation and cdc42 and cAMP-responsive element-binding protein (CREB) phosphorylation. In conclusion, our results suggested for the first time that RhoGDI2 is required for PKB and CREB activation and cdc42 expression in astrocyte migration after KA-mediated excitotoxic lesion in mouse brain.

Low-dose radiation suppresses Pokemon expression under hypoxic conditions.

Our previous data demonstrated that CoCl2-induced hypoxia controls endoplasmic reticulum (ER) stress-associated and other intracellular factors. One of them, the transcription factor Pokemon, was differentially regulated by low-dose radiation (LDR). There are limited data regarding how this transcription factor is involved in expression of the unfolded protein response (UPR) under hypoxic conditions. The purpose of this study was to obtain clues on how Pokemon is involved in the UPR. Pokemon was selected as a differentially expressed gene under hypoxic conditions; however, its regulation was clearly repressed by LDR. It was also demonstrated that both expression of ER chaperones and ER stress sensors were affected by hypoxic conditions, and the same results were obtained when cells in which Pokemon was up- or down-regulated were used. The current state of UPR and LDR research associated with the Pokemon pathway offers an important opportunity to understand the oncogenesis, senescence, and differentiation of cells, as well as to facilitate introduction of new therapeutic radiopharmaceuticals.

Naloxone induces endoplasmic reticulum stress in PC12 cells.

Naloxone is an opioid inverse agonist used in the treatment of opiate overdose, with well known pharmacology. In the present study, we determined the effects of naloxone on the unfolded protein response (UPR) in PC12 cells. Data from a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that naloxone may accelerate PC12 cell apoptosis in a dose-dependent manner. We also demonstrated that naloxone upregulated gene expression of endoplasmic reticulum (ER) chaperones, including binding immunoglobulin protein (Bip), calnexin, ER protein 29 (ERp29) and protein disulfide isomerase (PDI), and ER stress sensors, including activating transcription factor 6 (ATF6), inositol-requiring enzyme 1 (IRE1) and protein kinase-like ER kinase (PERK). In addition, naloxone also induced typical ER stress phenomena, including ART6 proteolytic cleavage, eIF2α phosphorylation and XBP1 mRNA splicing. Furthermore, naloxone upregulated gene expression of ER chaperones and ER stress sensors in in vivo experiments. To the best of our knowledge, these results are the first to indicate that naloxone induces ER stress in vitro and in vivo.

Astrocytic phosphorylation of PDK1 on Tyr9 following an excitotoxic lesion in the mouse hippocampus.

3-phosphoinositide-dependent kinase-1 (PDK1) is suggested to play important roles in the regulation of synaptic plasticity and neuronal cell survival in the mature CNS. Although few studies have investigated the roles of PDK1, little is known about PDK1 changes in glial cells under neuropathological conditions. In current report, phosphorylation of PDK1 was monitored specially on tyrosine residues, following the induction of an excitotoxic lesion in rat brain by using kainic acid administration. In injured hippocampal CA3 region, Tyr9 phosphorylation of PDK1 was increased from 4h until 3 day post-injection. Double immunohistochemistry further evaluated that these phosphorylated forms of PDK1 were localized in astrocytes not other cells. Overexpression of unphosphorylatable mutant, PDK1-Y9F leads to inhibit Protein kinase B (PKB/Akt) activation and cAMP responsive element binding protein (CREB) phosphorylation. In conclusion, our results suggested for the first time that tyrosine phosphorylation of PDK1 is required for PKB and CREB activation in KA-mediated excitotoxic lesion in mouse brain.

Lidocaine induces endoplasmic reticulum stress-associated apoptosis in vitro and in vivo.

We demonstrated that upregulation of both gene expression of endoplasmic reticulum (ER) stress chaperones (BiP, calnexin, calreticulin, and PDI) and ER stress sensors (ATF6, IRE1 and PERK) was induced by lidocaine, a local anesthetic, in PC12 cells. In addition to gene regulation, lidocaine also induced typical ER stress phenomena such as ART6 proteolytic cleavage, eIF2 alpha phosphorylation, and XBP1 mRNA splicing. In in vivo experiments, while lidocaine downregulated gene expression of antiapoptotic factors (Bcl-2 and Bcl-xl), pro-apoptotic factor (Bak and Bax) gene expression was upregulated. Furthermore, lidocaine induced apoptosis, as measured histochemically, and upregulated PARP1, a DNA damage repair enzyme. These results are the first to show that lidocaine induces apoptosis through ER stress in vitro and in vivo.

Silkworm hemolymph down-regulates the expression of endoplasmic reticulum chaperones under radiation-irradiation.

We demonstrated that up-regulation of gene expression of endoplasmic reticulum (ER) chaperones (BiP, calnexin, calreticulin, ERp29) and ER membrane kinases (IRE1, PERK, ATF6) was induced by radiation in neuronal PC12 cells. However, addition of silkworm, Bombyx mori, hemolymph to irradiated cells resulted in an obvious decrease in expression of these genes, compared with a single radiation treatment. In contrast, one of the ER chaperones, "ischemia-responsive protein 94 kDa" (irp94), was up-regulated by radiation. However, addition of silkworm hemolymph resulted in no change in the expression of irp94, with an expression pattern that differed from that of ER chaperones. Based on these results, we propose that silkworm hemolymph contains factors that regulate a decrease in the expression of ER chaperones under radiation-irradiation conditions, with the exception of irp94, which is not down-regulated. We suggest that this difference in the molecular character of irp94 may provide a clue to the biological functions associated with ER stress pathways, particularly the effects of radiation.