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.

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.

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/.

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.

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 .

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.

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.

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.

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.

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.

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.

Production of recombinant human granulocyte macrophage colony-stimulating factor from silkworm Bombyx mori Bm5 cells.

Using silkworm Bombyx mori Bm5 cells, we established a stable cell line expressing the human granulocyte macrophage colony-stimulating factor (hGM-CSF), which gets its name from the Bm5-hGM-CSF cell in which the glycoprotein of the hGM-CSF is secreted in the cell culture supernatant (CCS). It was demonstrated that secreted hGM-CSF had in vivo biological activity and the white blood cell (WBC) value increased two times that of the control. We expect to produce useful human recombinant glycoproteins from silkworm cultured cells for a low price and a large quantity.

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.

7-ketocholesterol induces endoplasmic reticulum stress in HT-29 cells.

7-Ketocholesterol (7-Kchol, oxidized cholesterol) is an important mediator of cell death in atherosclerosis mediated by up-regulated Nox 4 gene expression. In the current study using the human colon cancer HT-29 cell line, we have demonstrated that 7-Kchol promotes endoplasmic reticulum (ER) stress via gene up-regulation of ER chaperone and membrane kinases.

Expression of transferrin binding protein in the capillaries of the brain in the developing chick embryo.

Transferrin-binding protein (TfBP) has been shown to be a novel protein, structurally related to the chicken heat shock protein 108. The physiological function of this protein, however, has not yet been established. Antiserum to TfBP selectively stains transferrin- and iron-rich oligodendrocytes and choroidal epithelium in the adult and embryonic chick brain, suggesting a role for this protein in transferrin and iron storage in these cells. In this study, we further demonstrate TfBP-immunoreactivity (IR) in the blood vessels of the embryonic chick central nervous system. A strong TfBP-IR was present in blood vessels from E6, declined from E10 and was absent by E18. Thus, the expression of the TfBP in the blood vessels precedes its expression in the oligodendrocytes. At the subcellular level, TfBP-IR was confined to the cytoplasm of capillary pericytes while the Tf-receptor IR was associated with the capillary endothelium of the brain. The up-regulated expression of TfBP, together with the Tf-receptor of the brain capillaries, suggests that pericytes may be associated with the high iron uptake required for the metabolic demands of the developing brain.

Deferoxamine induces endoplasmic reticulum stress in PC12 cells.

Deferoxamine (DFA, N'-[5-(acetyl-hydroxy-amino)-pentyl]-N-[5-[3-(5-aminopentyl-hydroxy-carbamoyl) propanoylamino]pentyl]-N-hydroxy-butane diamide) is a chelating agent used to remove excess iron from the body and to reduce organ and tissue damage. DFA enhances both iron regulatory protein 1 (IRP1) expression and its endoplasmic reticulum (ER) membrane-binding activity, as occurs in hypoxia, an ER stress, in cultured cells. Here, we show that DFA promotes ER stress via an ER signal pathway.

Secretion of the antibacterial recombinant protein enbocin.

The insect baculovirus expression vector system (BEVS) is useful for the production of biologically active recombinant proteins. However, the overexpression of foreign proteins in this system often results in misfolded proteins and the formation of protein aggregates. To overcome this limitation, we have developed a versatile baculovirus expression and secretion system using the Bombyx mori protein disulfide isomerase (bPDI) as a fusion partner. bPDI gene fusion improved the secretion and antibacterial activity of recombinant enbocin proteins. Thus, bPDI gene fusion is a useful addition to the BEVS for the large-scale production of bioactive recombinant proteins.

Ischemia-responsive protein (irp94) gene expression in neurons.

An increased expression of the ischemia-responsive protein gene (irp94) was detected in a Mongolian gerbil brain after an ischemic injury, particularly in the cerebral cortex and hippocampus. In a rat phaeochromocytoma tumour cell line (PC12 cells), actinomycin D blocked the irp94 gene expression but cycloheximide did not. This indicates that irp94 gene expression is transcriptionally controlled. The half-life of irp94 mRNA was estimated to be approx. 5 h using 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). In addition, irp94 expression was enhanced by either endoplasmic reticulum (ER)-stress-inducible drugs or protease inhibitors. This suggests that irp94 gene expression is strongly associated with the unfolded protein response (UPR) in neurons.