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Neural tube defects (NTDs) are characterized by the failure of neural tube closure during embryogenesis and are considered the most common and severe central nervous system anomalies during early development. Recent microRNA (miRNA) expression profiling studies have revealed that the dysregulation of several miRNAs plays an important role in retinoic acid (RA)-induced NTDs. However, the molecular functions of these miRNAs in NTDs remain largely unidentified. Here, we show that miR-10a-5p is significantly upregulated in RA-induced NTDs and results in reduced cell growth due to cell cycle arrest and dysregulation of cell differentiation. Moreover, the cell adhesion molecule L1-like ( Chl1) is identified as a direct target of miR-10a-5p in neural stem cells (NSCs) in vitro , and its expression is reduced in RA-induced NTDs. siRNA-mediated knockdown of intracellular Chl1 affects cell proliferation and differentiation similar to those of miR-10a-5p overexpression, which further leads to the inhibition of the expressions of downstream ERK1/2 MAPK signaling pathway proteins. These cellular responses are abrogated by either increased expression of the direct target of miR-10a-5p ( Chl1 ) or an ERK agonist such as honokiol. Overall, our study demonstrates that miR-10a-5p plays a major role in the process of NSC growth and differentiation by directly targeting Chl1 , which in turn induces the downregulation of the ERK1/2 cascade, suggesting that miR-10a-5p and Chl1 are critical for NTD formation in the development of embryos.

Bombyx mori triose-phosphate transporter protein (BmTPT) is a member of the solute carrier (SLC) family. Its main function is to transport triose phosphate between intracellular and extracellular. In this study, BmTPT was cloned and characterised from the fat body of the silkworm Bombyx mori, resulting in an open reading frame (ORF) with a full length of 936 bp, which can encode 311 amino acid residues and has eight transmembrane structural domains. BmTPT was distributed throughout the cell and deposited the most in the nucleus, and is expressed in all tissues of Bombyx mori. Bombyx mori nucleopolyhedrovirus (BmNPV) infection significantly up-regulated BmTPT expression in immune tissue fat bodies. In addition, overexpression of BmTPT significantly inhibited BmNPV infection and markedly reduced the expression of enzymes related to the cellular glycolytic pathway; on the contrary, down-regulation of BmTPT expression by RNA interference resulted in robust replication of BmNPV and a significant increase in the expression of enzymes related to the cellular glycolytic pathway. This is the first report that BmTPT has antiviral effect in silkworm, and also could result in a lack of energy and raw materials for BmNPV replication and infection through down-regulation of the cellular glycolytic pathway., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Although the NiS exhibits the most widely adjustable metal-to-insulator (MIT) properties among the chalcogenides, the mechanisms, with respect to the regulations in their critical temperatures ( T MIT ), are yet unclear. Herein, we demonstrate the overlooked role associated with the structurally tetragonal distortion in elevating the T MIT of NiS; this is in distinct contrast to the previously expected hybridization and bandwidth regulations that usually reduces T MIT . Compared to the perspective of structure distortions, the orbital hybridization and band regulation of NiS are ∼19 times more effective adjustment in T MIT . As a result, the respective abruptions in both the electrical and thermal resistive switches across the T MIT of NiS can be better preserved in the low-temperature range (<273 K), shedding light on their optimum usage at cryogenic temperatures.

The Asian corn borer (Ostrinia furnacalis) is an important agricultural pest causing serious damage to economic crops, such as corn and sorghum. The gut is the first line of defense against pathogens that enter through the mouth. Staphylococcus aureus was used to infect the O. furnacalis midgut to understand the midgut immune mechanism against exogenous pathogens to provide new ideas and methods for the prevention and control of O. furnacalis. A sequencing platform was used for genome assembly and gene expression. The unigene sequences were annotated and functionally classified by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. Significant differences were found in the induced expression profiles before and after infection. Some differentially expressed genes have important relations with lipid metabolism and immune mechanism, suggesting that they play an important role in the innate immune response of O. furnacalis. Furthermore, quantitative real-time polymerase chain reaction assay was used to identify the key genes involved in the signaling pathway, and the expression patterns of these key genes were confirmed. The results could help study the innate immune system of lepidopteran insects and provide theoretical support for the control of related pests and the protection of beneficial insects., (© 2023 Wiley Periodicals LLC.)

Heat current in ferromagnets can generate a transverse electric voltage perpendicular to magnetization, known as anomalous Nernst effect (ANE). ANE originates intrinsically from the combination of large Berry curvature and density of states near the Fermi energy. It shows technical advantages over the conventional longitudinal Seebeck effect in converting waste heat to electricity due to its unique transverse geometry. However, materials showing giant ANE remain to be explored. Herein,  a large ANE thermopower of S yx ≈ 2 µV K -1 at room temperature in ferromagnetic Fe 3 Pt epitaxial films is reported, which also show a giant transverse thermoelectric conductivity of α yx ≈ 4 A K -1  m -1 and a remarkable coercive field of 1300 Oe. The theoretical analysis reveals that the strong spin-orbit interaction in addition to the hybridization between Pt 5d and Fe 3d electrons leads to a series of distinct energy gaps and large Berry curvature in the Brillouin zone, which is the key for the large ANE. These results highlight the important roles of both Berry curvature and spin-orbit coupling in achieving large ANE at zero magnetic field, providing pathways to explore materials with giant transverse thermoelectric effect without an external magnetic field., (© 2023 Wiley-VCH GmbH.)

Introduction: We have reported that high total homocysteine and the coexistence of inadequate thyroid hormones in maternal serum increase the risk of fetal neural tube defects (NTDs). Placental iodothyronine deiodinases (DIOs: DIO1, DIO2, and DIO3) play a role in regulating the conversions between different forms of maternal thyroid hormones. This study hypothesized that single nucleotide polymorphisms (SNPs) in placental DIOs genes could be related to NTDs., Methods: We performed a case-control study from 2007 to 2009 that included pregnant women from Lüliang, Shanxi Province, China. Nine distinct SNPs in DIOs genes were analyzed, and placental samples were obtained from 83 pregnant women with NTD fetuses and 90 pregnant women with normal fetuses. The nine SNPs were analyzed using the Cochran-Armitage test and the Fisher's exact test., Results: There were no statistically significant differences between case and control in the nine SNPs of DIOs (p &gt; 0.05)., Conclusions: The results of this study suggested that SNPs of DIO genes in the placenta among pregnant women have no statistically significant difference between the two groups, suggesting that other factors might be involved in metabolism of maternal thyroid hormone provided to fetuses, such as epigenetic modification of methylation and homocysteinylation and genomic imprinting in the placenta. Further functional studies on placenta samples are necessary., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Peptidoglycan recognition proteins (PGRPs) recognize invading microbes via detecting peptidoglycans from microbial cell walls. PGRPs are highly conserved from insects to vertebrates and all play roles during the immune defensive response. Ten putative PGRPs have been identified through transcriptome analysis in the Asian corn borer, Ostrinia furnacalis (Guenée). Whereas, the biochemical functions of most of them have not yet been elucidated. In this study, we found PGRP6 messenger RNA exhibited extremely high expression levels in the midgut, and its transcript level increased dramatically upon bacterial infection. Moreover, the enzyme-linked immunosorbent assay indicated recombinant PGRP6 exhibited a strong binding affinity to peptidoglycans from Micrococcus luteus and Bacillus subtilis, which could agglutinate M. luteus and yeast Pichia pastoris. Additionally, we demonstrated that PGRP6 was involved in the pathway of antimicrobial peptides synthesis, but could not enhance encapsulation and melanization of hemocytes. Overall, our results indicated that O. furnacalis PGRP6 serves as a pattern recognition receptor and detects peptidoglycans from microbes to initiate the immune response., (© 2022 Wiley Periodicals LLC.)

C-type lectins (CTLs) are widely distributed in mammals, insects, and plants, which act as pattern recognition receptors (PRRs) to recognize pathogens and initiate immune responses. In this study, we identified a C-type lectin gene called BmIML-2 from the silkworm Bombyx mori . Its open reading frame (ORF) encodes 314 amino acids, which contain dual tandem C-type lectin-like domain (CTLD). BmIML-2 is highly expressed in the fat body and is significantly induced at 24 h after BmNPV infection. Moreover, overexpression of BmIML-2 dramatically inhibited the proliferation of BmNPV, and knockdown assay via siRNA further validated the inhibition of BmIML-2 on viral proliferation. In addition, transcript level detection of apoptosis-related genes and observation of apoptosis bodies implied that overexpression of BmIML-2 promoted BmNPV-induced apoptosis. Immunofluorescence analysis indicated that BmIML-2 distributed throughout the cytoplasm and was slightly concentrated in the cell membrane. Taken together, our results suggest that BmIML-2 could inhibit in the proliferation of BmNPV by facilitating cell apoptosis.

The silkworm, Bombyx mori, is an important model organism of lepidopteran insects, and its testis is a main male reproductive organ and spermatogenesis place. Studying the testis helps to understand the mechanisms of genetic sterility of lepidopteran insects and to achieve sterile insect technique (SIT) for pest control. Herein, we performed a comparative transcriptome analysis of testes between three biological replicates of the GMS mutant and wild strain 898WB, respectively. In total, 1872 up-regulated genes and 1823 down-regulated genes were identified in the testis of the GMS mutant. Several genes contribute significantly to spermatogenesis and testis development, such as "serine/threonine protein kinase", "organic cation transporter protein", "tyrosine protein kinase", "lncRNAs" and "immune-associated genes". The KEGG pathway analysis shows that the DEGs were annotated to 123 pathways, and 10 pathways were significantly enriched, such as "metabolic pathway", "biosynthesis of amino acids", and "phagosome-lysosome pathway", which are associated with testis development and spermatogenesis. The results of the qPCR expression were consistent with the RNA-seq data, which shows that the RNA-seq results were accurate. The DEGs of the testes between GMS mutant and 898WB were screened by RNA-Seq technology, which provides a reliable reference to understand the molecule mechanism of male sterility of the GMS mutant., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Recognition of invading foreign exogenous pathogen is the first step to initiate the innate immune response of insects, which accomplished by the pattern recognition receptors (PRRs). Peptidoglycan recognition proteins (PGRPs) serve as an important type of PRRs, which activate immune response by detecting peptidoglycan of microbial cell wall. In this study, we have cloned the full-length cDNA of PGRP gene called PGRP-S1 from the Diaphania pyloalis (Walker). The open reading frame (ORF) of D. pyloalis PGRP-S1 encodes 211 amino acids which containing a secretion signal peptide and a canonical PGRP domain. Multisequence alignment revealed that PGRP-S1 possess the amino acid residues responsible for zinc binding and amidase activity. D. pyloalis PGRP-S1 exhibited the highest transcript level in fat body and followed in head. The mRNA concentration dramatically increased after an injection of Escherichia coli or Micrococcus luteus. Purified recombinant PGRP-S1 exhibit binding ability to peptidoglycans from Staphylococcus aureus or Bacillus subtilis and cause intensive agglutination of E. coli, M. luteus or S. aureus in the presence of zinc ions. Furthermore, phenoloxidase activity significantly increased when the plasma from larvae was incubated with recombinant PGPR-S1 and peptidoglycans from B. subtilis or M. luteus simultaneously. These results implied that PGRP-S1 was a member involving the prophenoloxidase activation pathway. Overall, our results indicated that D. pyloalis PGRP-S1 serve as a PRR to participate in the recognition of foreign pathogen and prophenoloxidase pathway stimulation., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: This study tested the hypothesis that abnormal maternal metabolism of both homocysteine and thyroid hormone network in pregnant women is associated with neural tube defects (NTDs) in a part of China with high NTD prevalence., Methods: A case-control study was performed between 2007 and 2009 in Lüliang Mountains, Shanxi Province. This study included 83 pregnant women who had fetuses with NTDs (cases) and 90 pregnant women with normal fetuses (controls). In addition, a cell model to illustrate the epidemiological findings was established., Results: Fetuses of mother who had both high total homocysteine (tHcy) and inadequate free thyroxine were 3 times more at risk of developing NTDs (adjusted odds ratio = 3.5; 95 % confidence interval = 1.2-10.4; cases vs. controls) using multivariate logistic regression models. Furthermore, biological interaction between metabolisms of Hcy and thyroid hormones was demonstrated in vitro. In homocysteine thiolactone of a metabolite of Hcy-treated mouse embryonic neural stem NE4C cells, genes (Bmp7, Ctnnb1, Notch 1, Gli2, and Rxra) related to both neural tube closure and thyroid hormone network were shown to be regulated by H3K79 homocysteinylation, which increased their expression levels., Conclusions: The effect of maternal serum high tHcy on risk of developing NTDs is depended on maternal serum level of thyroxine. Meanwhile, a higher level of tHcy might also affect both maternal metabolism of thyroid hormone and neural tube closure in embryogenesis through homocysteinylation of histones., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

The progress of epigenetic research has led to the discovery and confirmation of age-related markers based on DNA methylation. These DNA methylation indices are called "epigenetic clock/age". The concept of "epigenetic clock/age" and the establishment of its evaluation system are helpful to solve some of the long-standing problems in the field of life and medicine. When facing the current global aging, it is of great significance to refer to the comprehensive health parameters to determine the biological age and life span of an individual, and thus to design a plan to slow down the process of life cycle. This paper has summarized the concept and development of "epigenetic clock/age" in recent years.

Insect innate immunity is initiated by the special recognition and binding of the foreign pathogens, which is accomplished by the pattern recognition receptors (PRRs). As an important type of PRRs, C-type lectins (CTLs) play various roles in insect innate immunity, including pathogen recognition, stimulation of prophenoloxidase, regulation of cellular immunity and so on. In this study, we have cloned the full-length cDNA of a CTL gene named CTL-S6 from the silkworm, Bombyx mori. The open reading frame (ORF) of B. mori CTL-S6 encodes 378 amino acids, which contain a secretion signal peptide. The mRNA of CTL-S6 exhibited the highest transcriptional level in the midgut. Its transcriptional level increased dramatically in fat body and hemocytes upon Escherichia coli or Micrococcus luteus challenge. Purified recombinant CTL-S6 could bind to bacterial cell wall components, including peptidoglycan (PGN, from Bacillus subtilis) and lipopolysaccharide (LPS, from E. coli 0111:B4), and recombinant CTL-S6 was involved in the encapsulation and melanization of hemocytes. Furthermore, the addition of recombinant CTL-S6 to the hemolymph of silkworm resulted in a significant increase in phenoloxidase activity. Overall, our results indicated that B. mori CTL-S6 may serve as a PRR for the recognition of foreign pathogens, prophenoloxidase pathway stimulation and involvement in the innate immunity., (© The Author(s) 2021. Published by Oxford University Press on behalf of Entomological Society of America.)

To provide alternative methods of analyzing amino acids without liquid chromatography, 19 F NMR-based simultaneous and individual detection methods for amino acids using o -phthalaldehyde (OPA)-based 19 F labeling have been developed. Since the chemical shifts of almost all 19 F-labeled amino acids differ from each other, and they can be discriminated on the 19 F NMR spectrum, simultaneous detection of amino acids has been successfully demonstrated. The discrimination pattern of the peak identical to that of the 19 F-labeled amino acids was largely dependent on the chemical structure of the thiols having 19 F nuclei, strongly suggesting that there is a large potential for clearer discrimination of amino acids by optimizing the thiol structure and/or combined use of thiols.

Aims: To investigate the diagnostic value of lung-specific X protein (LUNX) messenger ribonucleic acid (mRNA) expression in peripheral blood of patients with nonsmall cell lung cancer (NSCLC) in micrometastasis., Materials and Methods: Peripheral blood samples of 112 patients with NSCLC were collected, and the expression of LUNX, cytokeratin 19 (CK19), and carcinoembryonic antigen (CEA) mRNA was measured by reverse transcription-polymerase chain reaction (RT-PCR)., Results: The expression of LUNX, CK19, and CEA mRNA was increased in peripheral blood of patients with NSCLC compared with that of patients with benign lung disease (P < 0.05), and the sensitivity of LUNX mRNA was higher than that of CK19 and CEA mRNA (P < 0.05). LUNX-positive expression was also associated with lymph node metastasis, tumor-node-metastasis (TNM) staging, and reduced 5-year survival rate of patients in our cohort (P < 0.05). Further, the 5-year survival improved for those LUNX-positive patients who became LUNX negative following adjuvant chemotherapy compared to those who remain LUNX positive (P < 0.05). Multivariate analysis showed that lymph node metastasis, TNM stage, and LUNX mRNA expression in peripheral blood were independent prognostic factors., Conclusion: The detection of LUNX expression in peripheral blood of patients with NSCLC by RT-PCR is a highly specific and sensitive detection method for tumor micrometastasis that may be used for molecular diagnosis of tumor micrometastasis. LUNX mRNA expression in peripheral blood is an independent factor affecting prognosis of NSCLC and thus may reliably predict NSCLC prognosis and guide appropriate adjuvant chemotherapy treatment., Competing Interests: None

Background: Neural tube defects (NTDs) are severe, common birth defects that result from failure of normal neural tube closure during early embryogenesis. Accumulating strong evidence indicates that genetic factors contribute to NTDs etiology, among them, HOX genes play a key role in neural tube closure. Although abnormal HOX gene expression can lead to NTDs, the underlying pathological mechanisms have not fully been understood., Method: We detected that H3K27me3 and expression of the Hox genes in a retinoic acid (RA) induced mouse NTDs model on E8.5, E9.5 and E10.5 using RNA-sequencing and chromatin immunoprecipitation sequencing assays. Furthermore, we quantified 10 Hox genes using NanoString nCounter in brain tissue of fetuses with 39 NTDs patients including anencephaly, spina bifida, hydrocephaly and encephalocele., Results: Here, our results showed differential expression in 26 genes with a > 20-fold change in the level of expression, including 10 upregulated Hox genes. RT-qPCR revealed that these 10 Hox genes were all upregulated in RA-induced mouse NTDs as well as RA-treated embryonic stem cells (ESCs). Using ChIP-seq assays, we demonstrate that a decrease in H3K27me3 level upregulates the expression of Hox cluster A-D in RA-induced mouse NTDs model on E10.5. Interestingly, RA treatment led to attenuation of H3K27me3 due to cooperate between UTX and Suz12, affecting Hox gene regulation. Further analysis, in human anencephaly cases, upregulation of 10 HOX genes was observed, along with aberrant levels of H3K27me3. Notably, HOXB4, HOXC4 and HOXD1 expression was negatively correlated with H3K27me3 levels., Conclusion: Our results indicate that abnormal HOX gene expression induced by aberrant H3K27me3 levels may be a risk factor for NTDs and highlight the need for further analysis of genome-wide epigenetic modification in NTDs.

Half-metallic Heusler alloys are attracting considerable attention because of their unique half-metallic band structures, which exhibit high spin polarization and yield huge magnetoresistance ratios. Besides serving as ferromagnetic electrodes, Heusler alloys also have the potential to host spin-charge conversion. Here, we report on the spin-charge conversion effect in the prototypical Heusler alloy NiMnSb. An unusual charge signal was observed with a sign change at low temperature, which can be manipulated by film thickness and ordering structure. It is found that the spin-charge conversion has two contributions. First, the interfacial contribution causes a negative voltage signal, which is almost constant versus temperature. The second contribution is temperature dependent because it is dominated by minority states due to thermally excited magnons in the bulk part of the film. This work provides a pathway for the manipulation of spin-charge conversion in ferromagnetic metals by interface-bulk engineering for spintronic devices., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

In superconductors, a topological configuration of the superconducting order parameter called a superconducting vortex carries magnetization. Such a magnetic topological object behaves like a minute particle generating a magnetic flux. Since the flux is localized with a nanometer scale, the vortex provides a nano-scale probe for local magnetic fields. Here we show that information of magnetic stripes in insulators can be read out by using vortices in an adjacent superconductor film as a probe. The orientation and width of magnetic micro stripes are both transcribed into resistance change of the superconductor through the modulation of vortex mobility affected by local magnetization. By changing the direction of external magnetic fields, zero-field resistance changes continuously according to the stripe orientation, and its modulation magnitude reaches up to 100%. The width of the stripes can also be estimated from the oscillatory magnetoresistance. Our results demonstrate a new possibility for non-volatile analog memory devices based on topological objects.

The natural colorful cuticles of insects play important roles in many physiological processes. Pigmentation is a physiological process with a complex regulatory network whose regulatory mechanism remains unclear. Bombyx mori pigmentation mutants are ideal materials for research on pigmentation mechanisms. The purple quail-like (q-l p ) and brown quail-like (q-l b ) mutants originated from plain silkworm breeds 932VR and 0223JH respectively exhibit similar cuticle pigmentation to that of the quail mutant. The q-l p mutant also presents a developmental abnormality. In this study, genes controlling q-l p and q-l b mutants were located on chromosome 8 by positional cloning. Then the neuropeptide gene orcokinin (OK) was identified to be the major gene responsible for two quail-like mutants. The B. mori orcokinin gene (BommoOK) produces two transcripts, BommoOKA and BommoOKB, by alternative splicing. The CRISPR/Cas9 system and orcokinin peptides injection were used for further functional verification. We show a novel function of BommoOKA in inhibiting pigmentation, and one mature peptide of orcokinin A, OKA&#95;type2, is the key factor in pigmentation inhibition. These results provide a reference for studying the function of orcokinin and are of theoretical importance for studying the regulatory mechanism of pigmentation., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Interaction analysis in vivo greatly promotes the analyses and understanding of biological functions. The interaction between DNA and peptides or proteins is very important in terms of readout and amplifying information from genomic DNA. In this study, we designed and synthesized a photo-cross-linkable amino acid, l-3-cyanovinlycarbazole amino acid (l-CNVA), to double-stranded DNA. Reversible photo-cross-linking between DNA and peptides containing CNVA, having 3-cyanovinylcarbazole moieties capable of photo-cross-linking to nucleic acids, was demonstrated. As a result, it was shown that the GCN4 peptide, containing CNVA, can be photo-cross-linked to DNA, and its adduct was photo-split into the original peptide and DNA with 312 nm-irradiation. This is the first report that reversibly manipulates photo-crosslinking between double stranded DNA and peptides. In addition, this reversible photo-cross-linking, using l-CNVA, is faster and with higher yield than that using diazirine and psoralen.

Bombyx mori nuclear polyhedrosis virus (BmNPV) is the most damaging virus for the production of silkworm cocoons. Antivirus research continues to be an important aspect of the silkworm industry. Two-dimensional electrophoresis and mass spectrometry have been applied for analyzing the midgut proteome of BmNPV-infected silkworms. In recent years, the isobaric tags for relative and absolute quantitation (iTRAQ) method has frequently been used when studying interaction between BmNPV and Bombyx mori, and useful information has been obtained. In this study, midgut proteins of BmNPV-infected silkworms were extracted from silkworm variety NIL·LVR with anti-BmNPV activity at 48 h, and proteome analysis was carried out using the label-free method. 2196 proteins were identified. Among them, there were 85 differentially expressed proteins, 45 upregulated proteins (immune-activated proteins), 28 downregulated proteins, and six proteins were specific for the BmNPV group and another six specific for control group. Many of the immune-activated proteins have been reported to have innate immune functions, and the downregulated proteins are involved in apoptosis or abnormal cell viability. In conclusion, this study provides evidence for host defense against BmNPV infection by both innate immunity and apoptosis, revealing the potential function of the midgut after oral infection of BmNPV in Bombyx mori. SIGNIFICANCE: Bombyx mori nuclear polyhedrosis virus (BmNPV) has a great impact on the sericulture industry. However, the mechanism of resistance to BmNPV has not been fully elucidated. The silkworm midgut is not only the major organ for food digestion and nutrient absorption but also an immune organ serving as the first line of defense against microbial invasion and proliferation. Here we combined label-free quantitative proteomic, bioinformatics, quantitative real-time PCR and SDS-PAGE analyses and found that BmNPV invasion causes complex protein alterations in the larval midgut of NIL·LVR with anti-BmNPV activity. The results showed that many upregulated differentially expressed proteins have been reported to have innate immune functions and the downregulation proteins are involved in apoptosis or abnormal cell viability. These findings provide evidence for host defense against BmNPV infection by both innate immunity and apoptosis, and reveals the potential function of the midgut after infection of BmNPV in Bombyx mori., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Folate deficiency in early development leads to disturbance in multiple processes, including neurogenesis during which fibroblast growth factor (FGF) pathway is one of the crucial pathways. Whether folic acid (FA) directly affects FGF pathways to influence neurodevelopment and the possible mechanism remains unclear. In this study, we presented evidence that in human FA-insufficient encephalocele, the FGF pathway was interfered. Furthermore, in Brachyury knockout mice devoid of such T-box transcription factors regulating embryonic neuromesodermal bipotency and a key component of FGF pathway, change in expression of Brachyury downstream targets, activator Fgf8 and suppressor dual specificity phosphatase 6 was detected, along with the reduction in expression of other key FGF pathway genes. By using a FA-deficient cell model, we further demonstrated that decrease in Brachyury expression was through alteration in hypermethylation at the Brachyury promoter region under FA deficiency conditions, and suppression of Brachyury promoted the inactivation of the FGF pathway. Correspondingly, FA supplementation partially reverses the effects seen in FA-deficient embryoid bodies. Lastly, in mice with maternal folate-deficient diets, aberrant FGF pathway activity was found in fetal brain dysplasia. Taken together, our findings highlight the effect of FA on FGF pathways during neurogenesis, and the mechanism may be due to the low expression of Brachyury gene via hypermethylation under FA-insufficient conditions.-Chang, S., Lu, X., Wang, S., Wang, Z., Huo, J., Huang, J., Shangguan, S., Li, S., Zou, J., Bao, Y., Guo, J., Wang, F., Niu, B., Zhang, T., Qiu, Z., Wu, J., Wang, L. The effect of folic acid deficiency on FGF pathway via Brachyury regulation in neural tube defects.

Neural tube defects (NTDs) are serious congenital malformations. Excessive maternal homocysteine (Hcy) increases the risk of NTDs, while its mechanism remains elusive. Here we report the role of histone homocysteinylation in neural tube closure (NTC). A total of 39 histone homocysteinylation sites are identified in samples from human embryonic brain tissue using mass spectrometry. Elevated levels of histone KHcy and H3K79Hcy are detected at increased cellular Hcy levels in human fetal brains. Using ChIP-seq and RNA-seq assays, we demonstrate that an increase in H3K79Hcy level down-regulates the expression of selected NTC-related genes including Cecr2, Smarca4, and Dnmt3b. In human NTDs brain tissues, decrease in expression of CECR2, SMARCA4, and DNMT3B is also detected along with high levels of Hcy and H3K79Hcy. Our results suggest that higher levels of Hcy contribute to the onset of NTDs through up-regulation of histone H3K79Hcy, leading to abnormal expressions of selected NTC-related genes.

Aldose reductase (AR) is a rate-limiting enzyme in the polyol pathway and is also the key enzyme involved in diabetic complications. The silkworm purple quail-like mutant (q-l p ) exhibits pigmented dots on its epidermis. The q-l p mutant also shows developmental abnormalities and decreased vitality. In this study, fat bodies from the q-l p mutant and the wildtype 932VR strain were subjected to two-dimensional gel electrophoresis (2-DE) analysis, and the Bombyx mori AR (BmAR) protein was found to be significantly downregulated in the q-l p mutant. The expression of BmAR at the mRNA level was also significantly downregulated, as verified through quantitative reverse transcription PCR (qRT-PCR). Knockdown of the expression of BmAR via RNAi resulted in a reduction of silkworm weight. The sorbitol level in q-l p was significantly lower than in the wildtype. These results suggested that the BmAR gene is closely related to the development of the q-l p mutant. Investigation of the cause of BmAR downregulation in the q-l p mutant could contribute to revealing the function of AR in insects and offers a new method of identifying AR inhibitors for the treatment of diabetic complications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Colossal magnetoresistance (CMR) refers to a large change in electrical conductivity induced by a magnetic field in the vicinity of a metal-insulator transition and has inspired extensive studies for decades 1,2 . Here we demonstrate an analogous spin effect near the Néel temperature, T N  = 296 K, of the antiferromagnetic insulator Cr 2 O 3 . Using a yttrium iron garnet YIG/Cr 2 O 3 /Pt trilayer, we injected a spin current from the YIG into the Cr 2 O 3 layer and collected, via the inverse spin Hall effect, the spin signal transmitted into the heavy metal Pt. We observed a two orders of magnitude difference in the transmitted spin current within 14 K of the Néel temperature. This transition between spin conducting and non-conducting states was also modulated by a magnetic field in isothermal conditions. This effect, which we term spin colossal magnetoresistance (SCMR), has the potential to simplify the design of fundamental spintronics components, for instance, by enabling the realization of spin-current switches or spin-current-based memories.

Information transport and processing by pure magnonic spin currents in insulators is a promising alternative to conventional charge-current-driven spintronic devices. The absence of Joule heating and reduced spin wave damping in insulating ferromagnets have been suggested for implementing efficient logic devices. After the successful demonstration of a majority gate based on the superposition of spin waves, further components are required to perform complex logic operations. Here, we report on magnetization orientation-dependent spin current detection signals in collinear magnetic multilayers inspired by the functionality of a conventional spin valve. In Y 3 Fe 5 O 12 |CoO|Co, we find that the detection amplitude of spin currents emitted by ferromagnetic resonance spin pumping depends on the relative alignment of the Y 3 Fe 5 O 12 and Co magnetization. This yields a spin valve-like behavior with an amplitude change of 120% in our systems. We demonstrate the reliability of the effect and identify its origin by both temperature-dependent and power-dependent measurements.

Disturbed epigenetic modifications have been linked to the pathogenesis of Neural Tube Defects (NTDs) in those with folate deficiency during pregnancy. However, evidence is lacking to delineate the critical region in epigenome regulated by parental folic acid and mechanisms by which folate deficiency affects normal embryogenesis. Our data from clinical samples revealed the presence of aberrant DNA methylation in GNAS imprinting cluster in NTD samples with low folate concentrations. Results from mouse models indicated that the establishment of GNAS imprinting was influenced by both maternal and paternal folate-deficient diets. Such aberrant GNAS imprinting was present prior to the gametogenesis period. Imprinting in Exon1A/GNAS gDMR was abolished in both spermatozoa and oocytes upon treating with a parental folate-deficient diet (3.6% in spermatozoa, 9.8% in oocytes). Interestingly, loss of imprinting in the GNAS gene cluster altered chromatin structure to an overwhelmingly open structure (58.48% in the folate-free medium group vs. 39.51% in the folate-normal medium group; P < 0.05), and led to a disturbed expression of genes in this region. Furthermore, an elevated cyclic AMP levels was observed in folate acid deficiency group. Our results imply that GNAS imprinting plays major roles in folic acid metabolism regulation during embryogenesis. Aberrant GNAS imprinting is an attribute to NTDs, providing a new perspective for explaining the molecular mechanisms by which folate supplementation in human pregnancy provides protection from NTDs., Competing Interests: CONFLICTS OF INTEREST None of the authors declared a conflicts of interest.

MicroRNAs (miRNAs) have emerged as key players in host-pathogen interaction and many virus-encoded miRNAs have been identified (computationally and/or experimentally) in a variety of organisms. A novel Bombyx mori nucleopolyhedrosis virus (BmNPV)-encoded miRNA miR-415 was previously identified through high-throughput sequencing. In this study, a BmNPV-miR-415 expression vector was constructed and transfected into BmN cells. The differentially expressed protein target of rapamycin isoform 2 (TOR2) was observed through two-dimensional gel electrophoresis and mass spectrometry. Results showed that TOR2 is not directly a target gene of BmNPV-miR-415, but its expression is up-regulated by BmNPV-miR-415 via Bmo-miR-5738, which could be induced by BmNPV.

When a suspension of polarizable particles is subjected to a gradient AC electric field, the particles exhibit collective motion due to an interaction between the dipole induced in the particles and the spatial gradient of the electric field; this is known as dielectrophoresis. In the present study, the collective dynamics of suspended particles in a parallel-plate electric chamber was investigated by simulating numerically the trajectories of individual particles under the action of combined dielectrophoretic and dipole-dipole interparticle forces. The particles were transported by the dielectrophoretic forces toward the grounded electrodes. Before long, when the particles approached the site of the minimum field strength, attractive/repulsive interparticle forces became dominant and acted among the particles attempting to form a column-like cluster, having the particles distribution in concentric circles in its cross-section, in line with the centerline of the grounded electrodes. Our results also well reproduced the transient particle aggregation that was observed experimentally., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A new purple quail-like (q-lp) mutant found from the plain silkworm strain 932VR has pigment dots on the epidermis similar to the pigment mutant quail (q). In addition, q-lp mutant larvae are inactive, consume little and grow slowly, with a high death rate and other developmental abnormalities. Pigmentation of the silkworm epidermis consists of melanin, ommochrome and pteridine. Silkworm development is regulated by ecdysone and juvenile hormone. In this study, we performed RNA-Seq on the epidermis of the q-lp mutant in the 4th instar during molting, with 932VR serving as the control. The results showed 515 differentially expressed genes, of which 234 were upregulated and 281 downregulated in q-lp. BLASTGO analysis indicated that the downregulated genes mainly encode protein-binding proteins, membrane components, oxidation/reduction enzymes, and proteolytic enzymes, whereas the upregulated genes largely encode cuticle structural constituents, membrane components, transport related proteins, and protein-binding proteins. Quantitative reverse transcription PCR was used to verify the accuracy of the RNA-Seq data, focusing on key genes for biosynthesis of the three pigments and chitin as well as genes encoding cuticular proteins and several related nuclear receptors, which are thought to play key roles in the q-lp mutant. We drew three conclusions based on the results: 1) melanin, ommochrome and pteridine pigments are all increased in the q-lp mutant; 2) more cuticle proteins are expressed in q-lp than in 932VR, and the number of upregulated cuticular genes is significantly greater than downregulated genes; 3) the downstream pathway regulated by ecdysone is blocked in the q-lp mutant. Our research findings lay the foundation for further research on the developmental changes responsible for the q-lp mutant.

Spin Hall magnetoresistance (SMR) has been investigated in Pt/NiO/YIG structures in a wide range of temperature and NiO thickness. The SMR shows a negative sign below a temperature that increases with the NiO thickness. This is contrary to a conventional SMR theory picture applied to the Pt/YIG bilayer, which always predicts a positive SMR. The negative SMR is found to persist even when NiO blocks the spin transmission between Pt and YIG, indicating it is governed by the spin current response of the NiO layer. We explain the negative SMR by the NiO "spin flop" coupled with YIG, which can be overridden at higher temperature by positive SMR contribution from YIG. This highlights the role of magnetic structure in antiferromagnets for transport of pure spin current in multilayers.

The biogenesis of ribosomes in vivo is an essential process for cellular functions. Transcription of ribosomal RNA (rRNA) genes is the rate-limiting step in ribosome biogenesis controlled by environmental conditions. Here, we investigated the role of folate antagonist on changes of DNA double-strand breaks (DSBs) landscape in mouse embryonic stem cells. A significant DSB enhancement was detected in the genome of these cells and a large majority of these DSBs were found in rRNA genes. Furthermore, spontaneous DSBs in cells under folate deficiency conditions were located exclusively within the rRNA gene units, representing a H3K4me1 hallmark. Enrichment H3K4me1 at the hot spots of DSB regions enhanced the recruitment of upstream binding factor (UBF) to rRNA genes, resulting in the increment of rRNA genes transcription. Supplement of folate resulted in a restored UBF binding across DNA breakage sites of rRNA genes, and normal rRNA gene transcription. In samples from neural tube defects (NTDs) with low folate level, up-regulation of rRNA gene transcription was observed, along with aberrant UBF level. Our results present a new view by which alterations in folate levels affects DNA breakage through epigenetic control leading to the regulation of rRNA gene transcription during the early stage of development., (© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Sharp structures in the magnetic field-dependent spin Seebeck effect (SSE) voltages of Pt/Y&#95;{3}Fe&#95;{5}O&#95;{12} at low temperatures are attributed to the magnon-phonon interaction. Experimental results are well reproduced by a Boltzmann theory that includes magnetoelastic coupling. The SSE anomalies coincide with magnetic fields tuned to the threshold of magnon-polaron formation. The effect gives insight into the relative quality of the lattice and magnetization dynamics.

Spin fluctuation and transition have always been one of the central topics of magnetism and condensed matter science. Experimentally, the spin fluctuation is found transcribed onto scattering intensity in the neutron-scattering process, which is represented by dynamical magnetic susceptibility and maximized at phase transitions. Importantly, a neutron carries spin without electric charge, and therefore it can bring spin into a sample without being disturbed by electric energy. However, large facilities such as a nuclear reactor are necessary. Here we show that spin pumping, frequently used in nanoscale spintronic devices, provides a desktop microprobe for spin transition; spin current is a flux of spin without an electric charge and its transport reflects spin excitation. We demonstrate detection of antiferromagnetic transition in ultra-thin CoO films via frequency-dependent spin-current transmission measurements, which provides a versatile probe for phase transition in an electric manner in minute devices.

Applying magnetic fields has been the method of choice to magnetize non-magnetic materials, but they are difficult to focus. The magneto-electric effect and voltage-induced magnetization generate magnetization by applied electric fields, but only in special compounds or heterostructures. Here we demonstrate that a simple metal such as gold can be magnetized by a temperature gradient or magnetic resonance when in contact with a magnetic insulator by observing an anomalous Hall-like effect, which directly proves the breakdown of time-reversal symmetry. Such Hall measurements give experimental access to the spectral spin Hall conductance of the host metal, which is closely related to other spin caloritronics phenomena such as the spin Nernst effect and serves as a reference for theoretical calculation.

In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2nd instar (nm2). Genetic analysis indicated that the nm2 mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, nm2 was located in a region approximately 275 kb on the 5th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the BmCPG10 gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene's structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The BmCPG10 mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The BmCPG10 mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of nm2 larvae was lower than that of the wild-type larvae. The nm2 mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7-dehydrocholesterol (7dC), but the rescued nm2 only developed to the 4th instar and subsequently died. The moulting time of silkworms could be delayed by BmCPG10 RNAi. Thus, we speculated that the mutation of BmCPG10 was responsible for the silkworm mutant that did not moult in the 2nd instar.

Magnons are the elementary excitations of a magnetically ordered system. In ferromagnets, only a single band of low-energy magnons needs to be considered, but in ferrimagnets the situation is more complex owing to different magnetic sublattices involved. In this case, low lying optical modes exist that can affect the dynamical response. Here we show that the spin Seebeck effect (SSE) is sensitive to the complexities of the magnon spectrum. The SSE is caused by thermally excited spin dynamics that are converted to a voltage by the inverse spin Hall effect at the interface to a heavy metal contact. By investigating the temperature dependence of the SSE in the ferrimagnet gadolinium iron garnet, with a magnetic compensation point near room temperature, we demonstrate that higher-energy exchange magnons play a key role in the SSE.

Bisphenol A (BPA), as an emerging environmental pollutant, is potentially harmful to plant growth. Chlorophyll (Chl) is critical in photosynthesis that provides matter and energy for plant growth. How BPA affects the chlorophyll content remains largely unknown. Here, the effects of BPA on Chl synthesis in soybean seedlings were investigated. Exposure to 1.5 mg/L BPA decreased the 5-aminolevulinic acid (ALA) content and increased protoporphyrin IX (Proto IX), magnesium protoporphyrin, and protochlorophyll contents and 5-aminolaevulinic acid dehydratase, porphobilinogen deaminase, uroporphyrinogen III synthase, uroporphyrinogen III decarboxylase, and protoporphyrinogen oxidase activities. Exposure to 17.2 and 50.0 mg/L BPA exerted the opposite effects on these four intermediates and five enzymes. Following the withdrawal of BPA exposure, the aforementioned parameters gradually recovered, except magnesium protoporphyrin content in exposure to 50.0 mg/L BPA. Our findings revealed that exposure to low-concentration BPA increased the Chl content in soybean seedlings through improving Chl synthesis, especially the conversion from ALA to Proto IX, whereas exposure to high-concentration BPA decreased the Chl content through inhibiting Chl synthesis, especially the conversion from ALA to Proto IX. The dual effects of BPA were largely reversed following the withdrawal of BPA exposure.

RNAi technology is taking strong position among the key therapeutic modalities, with dozens of siRNA-based programs entering and successfully progressing through clinical stages of drug development. To further explore potentials of RNAi technology as therapeutics, we engineered and tested VEGFR2 siRNA molecules specifically targeted to tumors through covalently conjugated cyclo(Arg-Gly-Asp-d-Phe-Lys[PEG-MAL]) (cRGD) peptide, known to bind αvβ3 integrin receptors. cRGD-siRNAs were demonstrated to specifically enter and silence targeted genes in cultured αvβ3 positive human cells (HUVEC). Microinjection of zebrafish blastocysts with VEGFR2 cRGD-siRNA resulted in specific inhibition of blood vessel growth. In tumor-bearing mice, intravenously injected cRGD-siRNA molecules generated no innate immune response and bio-distributed to tumor tissues. Continuous systemic delivery of two different VEGFR2 cRGD-siRNAs resulted in down-regulation of corresponding mRNA (55 and 45%) and protein (65 and 45%) in tumors, as well as in overall reduction of tumor volume (90 and 70%). These findings demonstrate strong potential of cRGD-siRNA molecules as anti-tumor therapy., (© The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Congenital hydrocephalus is heterogeneous in its etiology, and in addition to a genetic component, has been shown to be caused by environmental factors. Until now, however, no methylation alterations of target genes have been connected with congenital hydrocephalus in humans. Frizzled 3(FZD3) is a planar cell polarity (PCP) gene required for PCP signaling. Partial restoration of frizzled 3 activities in FZD3 mutant mice results in hydrocephalus. To analyze the possible roles of epigenetic modifications of the FZD3 gene in congenital hydrocephalus pathogenesis, DNA methylation in the promoter region of FZD3 was assayed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Gene expression and chromatin accessibility were also determined to assess the role of methylation alterations. Our study found methylation levels of the FZD3 gene were increased in congenital hydrocephalus, especially in males (10.57 ± 3.90 vs. 7.08 ± 0.94, p=0.001). Hypermethylation of FZD3 increased congenital hydrocephalus risk, with an odds ratio of 10.125 (p=0.003). Aberrant methylation modification of FZD3 altered both chromatin structure in this region and FZD3 expression levels. Totally, aberrant methylation modification of the FZD3 gene increases the risk of congenital hydrocephalus by altering chromatin structure and disturbing gene expression., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Two novel body marking mutants were discovered during silkworm (Bombyx mori) breeding. The mutants have no obvious eye-spots compared with normal marking (+) individuals, but their star spots and semilunar markings on dorsal sides are normal, and there are dots and lines with longitudinal wave markings on dorsal sides of the 6th to 7th abdominal segments which consist quail markings in between star spots and semilunar markings. The whole body markings are very similar to that of quail mutant (q); thus these mutants are named as quail-like mutants (q-l). Young larvae of one mutant are in brown color, and develop normally. Their cocoons are regular and uniform in size. Thus, this mutant is designated as brown quail-like (q-lb). Another mutant's larvae are in light purple skin; thus this mutant is named as purple quail-like (q-lp). They take little amount of mulberry leaves, and are weak and develop slowly and unevenly. Their larval bodies and cocoons are small. Genetic analysis revealed that both q-lb and q-lp were recessive genes, and they were allelic, with q-lb recessive to q-lp. These genes are different from quail mutant (q) and located on the chromosome 8 after tested by the morphological markers, P3(2), p(2), Ze(3), L(4), re(5), E(6), q(7), I-a(9), ms(12), ch(13), oa(14), cts(16), mln(18), msn(19), rb(21) and so(26) and SSR markers.

Previous studies have shown that the 3' UTR of BmVMP23 was destroyed by an inserted fragment, and the BmVMP23 expression was downregulated in the lethal egg of "Ming" (l-e(m)). In this study, we found a miRNA (bmo-miR-1a-3p) that matches the 3' UTR sequence of BmVMP23 perfectly. The relationship between bmo-miR-1a-3p and the BmVMP23 expression was examined by the co-transfection in vitro. The luciferase assay result showed that luc expression was strongly repressed. This result inferred that bmo-miR-1a-3p may downregulate BmVMP23 expression via complementary interaction with the target sites at the 3' UTR., (Copyright © 2013 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive "Ming" lethal egg mutants (l-e(m)) lose water and become concaved around 1h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e(m) mutant to 360 kb on chromosome 10 using 2287 F(2) individuals. Using expression analysis and RNA interference, the best l-e(m) candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~1.9 kb region from the 3' untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a >100 kb DNA fragment in the l-e(m) mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e(m) mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e(m) mutant. These results indicate that BmEP80 is responsible for the l-e(m) mutation., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Bisphenol A (BPA) is a representatively endocrine disruptor, which shows a highly toxic effect on life system. Its potential toxicity on plants that are the primary producers in earth's ecosystem is not well documented. Here, the effects of BPA on the growth, photosynthesis, content of chlorophyll (Chl), initial fluorescence (F(0)), maximal photochemical efficiency (F(v)/F(m)), effective quantum yield of photosystem II (Φ(PSII)) and photosynthetic electron transport rate (ETR) in soybean seedlings were investigated by using gas exchange measurement system and chlorophyll fluorometer to understand the toxic effect of BPA on plants. It was found that when soybean seedlings were treated with BPA at the low concentration (1.5 mg L(-1)), the growth indices (the plant height, fresh and dry weights of stems, fresh and dry weights of leaves, leaf area) were increased obviously compared with those of the control, which was not related with the photosynthesis, the content of Chl and the chlorophyll fluorescence. When soybean seedlings were treated with BPA at the high concentrations (7.0, 12.0, 17.2 and 50.0 mg L(-1)), the growth indices, net photosynthetic rate, the content of chlorophyll, F(v)/F(m), Φ(PSII), ETR were decreased, while F(0) was increased, compared with those of the control. Obviously, BPA at the high concentrations showed the toxic effect on soybean seedlings. The results from correlation analysis indicated that the inhibition in the growth of soybean seedlings treated with BPA was related to the decrease in photosynthesis because of the decrease in the content of chlorophyll and the change in chlorophyll fluorescence., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Hatching enzyme (HE) is an enzyme that digests an egg envelop at the time of embryo hatching. Previously, we have reported a kind of Bombyx mori hatching enzyme-like gene (BmHEL). In this paper, the full length of another BmHEL cDNA sequence (BmHELII, GenBank ID: JN627443) was cloned from bluish-silkworm-eggs. The cDNA was 977 bp in length with an open reading frame of 885 bp which encodes a polypeptide of 294 amino acids including a putative signal peptide of 16 amino acid residues and a mature protein of 278 amino acids. The deduced BmHELII had a predicted molecular mass of 33.62 kDa, isoelectric point of 5.44 and two conserved signature sequences of astacin family. Bioinformatic analysis results showed that the deduced protease domain amino acid sequence of BmHELII had 29.5-87.0% identities to that of HE identified in the other species. The BmHELII gene structure was 6-exon-5-intron, and the promoter region harbored some basal promoter elements and some embryo development related transcription factor binding sites. Semi-quantitative RT-PCR analysis revealed that the relative level of BmHELII transcripts at different stages during egg incubation increased with the development of embryos and reached to a maximum just before hatching, hence declined gradually after hatching. The spatio-temporal expression pattern of BmHELII basically resembled that of hatching enzyme gene. Moreover, the BmHELII transcript was detected in testis of the silkworm, and semi-quantitative RT-PCR analysis showed that it kept at the high level in testis of silkworm from larvae to moth, which suggested that BmHELII might take part in the development of sperm. These results will be helpful to provide a molecular basis for understanding the mechanism underlying silkworm hatching as well as spermatogenesis., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Preferentially expressed antigen in melanoma (PRAME) and prostate-specific membrane antigen (PSMA) are tumor-associated antigens implicated in cellular differentiation, genetic stability, and angiogenesis. MKC1106-PP is an immunotherapeutic regimen cotargeting PRAME and PSMA, comprised of a recombinant plasmid (pPRA-PSM encoding fragments derived from both antigens) and 2 peptides (E-PRA and E-PSM derived from PRAME and PSMA, respectively). This multicenter study evaluated MKC1106-PP with a fixed plasmid dose and 2 different peptide doses, administered by intralymph node injection in a prime-boost sequence in human leukocyte antigen-A*0201 and tumor-antigen-positive patients with progressing metastatic solid tumors who had failed standard therapy. Immune monitoring was done by tetramer and enzymatic-linked immune spot analysis. The treatment was well tolerated, with no significant differences in safety, immune response, and clinical outcome relative to peptide doses. Fifteen of 24 evaluable patients showed an immune response, as defined by the expansion of PRAME-specific or PSMA-specific T cells in the blood. There were no partial or complete responses by the Response Evaluation Criteria in Solid Tumors. Seven patients showed stable disease (SD) for 6 months or longer, or prostate specific antigen decline: 4 of 10 with prostate carcinoma, 2 of 2 with renal clear cell carcinoma, and 1 of 10 with metastatic melanoma. In addition, there was an association between the induction and persistence of antigen-specific T cells in blood above baseline levels and disease control, defined as SD for 6 months or longer. These results support further development of MKC1106-PP in specific clinical indications.

Purpose: The goal of this study was to test the safety and activity of a therapeutic vaccine, MKC1106-MT, in patients with metastatic melanoma., Experimental Design: MKC1106-MT comprises a plasmid (pMEL-TYR) and two peptides (E-MEL and E-TYR), corresponding to Melan A and tyrosinase, administered by intra-lymph node injection in a prime-boost sequence. All 18 patients were HLA-A*0201 positive and received a fixed priming dose of plasmid and a low or a high peptide dose. Enumeration of antigen-specific T cells was done prior to and throughout the treatment. Patients who did not exhibit disease progression remained on study and could receive up to eight cycles of treatment., Results: The MKC1106-MT regimen was well tolerated and resulted in an overall immune response rate of 50%. The treatment showed disease control, defined as stable disease that lasted for 8 weeks or more in 6 of 18 (33%) of the patients: 14% and 46% in the low and high peptide dose, respectively. Interestingly, four patients, all with tumor burden largely confined to lymph nodes and Melan A-specific T cells at baseline, showed durable disease control associated with radiologic evidence of tumor regression. There was no noticeable correlation between the expansion of antigen-specific T cells in blood and the clinical outcome; yet, there was evidence of active tumor-infiltrating lymphocytes (TIL) in two regressing lesions., Conclusions: MKC1106-MT showed immunogenicity and evidence of disease control in a defined patient population. These findings support further development of this investigational agent and the concept of therapeutic vaccination in metastatic melanoma., (©2011 AACR.)

Developing new vaccination strategies and optimizing current vaccines through heterologous prime-boost carries the promise of integrating the benefits of different yet synergistic vectors. It has been widely thought that the increased immunity afforded by heterologous prime-boost vaccination is mainly due to the minimization of immune responses to the carrier vectors, which allows a progressive build up of immunity against defined epitopes and the subsequent induction of broader immune responses against pathogens. Focusing on CD8+ T cells, we put forward a different yet complementary hypothesis based primarily on the systematic analysis of DNA vaccines as priming agents. This hypothesis relies on the finding that during the initiation of immune response, acquisition of co-inhibitory receptors such as programmed cell death-1 (PD-1) is determined by the pattern of antigen exposure in conjunction with Toll-like receptor (TLR)-dependent stimulation, critically affecting the magnitude and profile of secondary immunity. This hypothesis, based upon the acquisition and co-regulation of pivotal inhibitory receptors by CD8+ T cells, offers a rationale for gene-based immunization as an effective priming strategy and, in addition, outlines a new dimension to immune homeostasis during immune reaction to pathogens. Finally, this model implies that new and optimized immunization approaches for cancer and certain viral infections must induce highly efficacious T cells, refractory to a broad range of immune-inhibiting mechanisms, rather than solely or primarily focusing on the generation of large pools of vaccine-specific lymphocytes.

Elevated Programmed Death-1 (PD-1) expression can inhibit T cell activity and is a potential barrier to achieving persisting and optimal immunity via therapeutic vaccination. Using a direct lymph node-targeted vaccination procedure that enabled uncoupling of synthetic peptide (signal 1, TCR-mediated) and adjuvant (signal 2, non-TCR-mediated), we evaluated the impact of varied doses of Toll-like receptor (TLR)-9 ligand CpG oligodeoxynucleotide (ODN) adjuvant on epitope-specific CD8(+) T cell-associated PD-1 expression. Peptide vaccination without adjuvant yielded CD8(+) T cells with significantly elevated PD-1 expression. This conferred impaired function ex vivo, but was reversible by antibody-mediated PD-1 blockade. By comparison, peptide vaccination with escalating doses of CpG ODN adjuvant yielded higher magnitudes of CD8(+) T cells with progressively lower PD-1 expression and greater ex vivo function. CpG ODN adjuvant in context of titrated peptide doses for vaccination yielded the lowest overall PD-1 expression levels, demonstrating that fine-tuning both TCR-independent (adjuvant dose) and -dependent (antigen dose) stimuli can synergize to co-regulate PD-1 expression on epitope-specific CD8(+) T cells. These data hint at strategies to elicit PD-1(low) CD8(+) T cells using TLR-9 ligand adjuvants, and also shed light on the PD-1-regulated homeostasis of CD8(+) T cells.