Relish regulates innate immunity via mediating ATG5 activity in Antheraea pernyi.

In innate immunity, autophagy is an important molecular mechanism that plays a critical role in the animal defense system. Given the importance of anti-microbial autophagy in the innate immune processes, the relationship between anti-microbial autophagy and LPS-induced innate immunity in A. pernyi was investigated. Quantitative RT-PCR analysis revealed that autophagy-related genes (ATG6, ATG5, and ATG12) were induced following LPS injection. LPS treatment in the Relish knockdown larvae reduced the expression of autophagy-related genes, especially ATG5. Furthermore, ATG5 depletion decreased the innate immune effect, while its over-expression with ATG12 was induced after the LPS challenge. The dual-luciferase assay revealed that Relish could regulate ATG5 expression by binding directly to the promoter of the ATG5 gene. Overall, our findings show that Relish regulates the ATG5 transcription to eliminate Gram-negative bacteria by anti-microbial autophagy, implying a strong connection between autophagy and innate immunity in immunologic homeostasis.

Quercus acutissima Carruth. root extract triggers apoptosis, autophagy and inhibits cell viability in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The bark of Quercus acutissima Carruth. (QA) has long been used by Chinese people to treat noncancerous growths and cancerous ailments. It was traditionally used by Chinese folk to inhibit tumor proliferation in cancerous treatment, but the specific mechanism remain to be elucidated. AIM OF THE STUDY: This study investigated the anticancer activities of QA root extract and its regulatory pathways in two human breast cancer cell lines (MCF-7 and SUM159). MATERIALS AND METHODS: Dried QA root barks were extracted by ethanol and used to treat human breast cancer MCF-7 and SUM159 cells with varying concentrations. The CCK-8 assay, Hoechst 33342 staining assay and wound healing assay were used to detect the cell proliferation, apoptotic cell morphology, and cell migration in each group, respectively. Caspase 3 activity assay kit was used to determine caspase 3 activity. Western blot was used to measure proteins expression level in apoptosis and autophagy pathways (Bcl-W, caspase 3, Beclin1, LC3 and Atg5). LC-MS was performed to determine the chemical components in QA root extract. RESULTS: CCK-8 assay showed that QA root extract significantly inhibited cell viability and proliferation in breast cancer cells by a concentration-dependent manner. Cell wound healing assay indicated that it had high suppression ability on cell migration both in MCF-7 and SUM159 cells. QA root extract treatment induced the morphological and nuclear structural changes in breast cancer cells including rounded appearance and shrunken nucleus with several nuclear body fragments. Western blot indicated that QA root extract induced mitochondria-mediated apoptosis by up-regulating caspase 3 and down-regulating Bcl-W. Moreover, QA root extract up-regulated Beclin1 and Atg5, and activated LC3 in two human breast cancer cell lines. LC-MS results showed that QA root extract contains high content of bioactive compounds like coumarins and derivatives, prenol lipids, flavonoids and tannins. CONCLUSIONS: QA root extract inhibited cell proliferation and migration in MCF-7 and SUM159 cells, and it also induced cell morphology changes and regulated mitochondria-mediated apoptotic cell death and autophagic cell death.

The anticancer activity of root extract from Quercus acutissima Carruth. Via regulating apoptosis and autophagy in breast cancer cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The bark of Quercus acutissima Carruth. (QA) has long been used by Chinese people to treat noncancerous growths and cancerous ailments. It was traditionally used by Chinese folk to inhibit tumor proliferation in cancerous treatment, but the specific mechanism remain to be elucidated. AIM OF THE STUDY: Breast cancer is the most common form of cancer in women and the leading cause of mortality around the globe. This study investigated the anticancer activities of QA root extract and its regulatory pathways in two human breast cancer cell lines (MCF-7 and SUM159). MATERIALS AND METHODS: Dried QA root barks were extracted by ethanol and used to treat human breast cancer MCF-7 and SUM159 cells with varying concentrations. The CCK-8 assay, Hoechst 33342 staining assay and wound healing assay were used to detect the cell proliferation, apoptotic cell morphology, and cell migration in each group, respectively. Caspase 3 activity assay kit was used to determine caspase 3 activity. Western blot was used to measure proteins expression level in apoptosis and autophagy pathways (Bcl-W, caspase 3, Beclin1, LC3 and Atg5). RESULTS: CCK-8 assay showed that QA root extract significantly inhibited cell viability and proliferation in breast cancer cells by a hormone receptor independent manner. Cell wound healing assay indicated that it had high suppression ability on cell migration both in MCF-7 and SUM159 cells. QA root extract treatment induced the morphological and nuclear structural changes in breast cancer cells including rounded appearance and shrunken nucleus with several nuclear body fragments. Western blot indicated that QA root extract induced mitochondria-mediated apoptosis by up-regulating caspase 3 and down-regulating Bcl-W. Moreover, QA root extract up-regulated Beclin1 and Atg5, and activated LC3 in two human breast cancer cell lines. CONCLUSIONS: QA root extract inhibited cell proliferation and migration in MCF-7 and SUM159 cells, and it also induced cell morphology changes and regulated mitochondria-mediated apoptotic cell death and autophagic cell death.

Complete mitochondrial genome of Hypomecis punctinalis Scopoli, 1763 and its phylogenetic position within family Geometridae.

Hypomecis punctinalis Scopoli, 1763 belongs to the Lepidopteran family Geometridae. We sequenced the complete mitochondrial genome (mitogenome) of H. punctinalis. The mitogenome is 15,648 bp long and contains a typical set of genes (13 protein-coding genes (PCGs), 22 tRNA genes, and two rRNA genes) and a 484 bp AT-rich region. All PCGs start with ATN codons and stop at TAA codon except for cox1 using CGA as initiation codon and nad4 and nad5 using incomplete termination codon T. Within the mitogenome, 17 intergenic spacers and seven overlaps are founded. The intergenic nucleotides are 294 bp in total and two longest intervals locate between trnGln and nad2 as well as trnCys and trnTyr . The overlap nucleotides are 47 bp in total and the maximum overlap lies between cox2 and trnLys . The AT-rich region of the mitogenome contains an 'ATAGA + polyT' motif, three copies of 30-bp-repeat and a short polyA tail. The phylogenetic tree shows the relationships of four subfamilies of Geometridae are (((Ennominae + Geometrinae)+Larentiinae)+Sterrhinae)) and the relationships within subfamily Ennominae are ((((Erannis+Biston)+(Jankowskia+(Hypomecis+(Apocheima+Milionia))))+Ectropis)+Abraxas)+Phthonandria)+Celenna).

Complete mitochondrial genome of Lasiommata deidamia and its phylogenetic implication to subfamily Satyrinae (Lepidoptera: Nymphalidae).

Lasiommata deidamia Eversmann taxonomically belongs to lepidopteran family Nymphalidae Rafinesque, 1815. The Complete mitochondrial genome (mitogenome) of the insect had been sequenced, with 15,244 bp of total length that has 81.12% AT content and contains a typical set of genes (13 protein-coding genes (PCGs), 22 tRNA genes and 2 rRNA genes) and a 417 bp AT-rich region. Another, 11 intergenic spacers (139 bp in total) and 16 overlaps (175 bp in total) have been founded. The longest interval is located between trnGln and nad2 while the maximum overlap is between trnHis and nad4. All PCG genes are started with the ATN codons and stop at TAA codons except cox1 which uses CGA as the initiation codon. No tandem repeat has been found in the AT-rich region. The phylogenetic tree inferred with Bayesian Inference based on all the 13 protein sequences of 45 mitogeomes reveals the phylogenetic relationships of the taxa in the subfamily Styrinae is (((Satyrini + Ypthimini) + (Amathusiini + Elymniini)) + Melanitini) and that within tribe Satyrini is ((((Lethina + Parargina) + Mycalesina) + Coenonymphina) +(Satyrina + (Melanargiina + Maniolina))).

Complete mitogenome of Parum colligata (Lepidoptera: Sphingidae) and its phylogenetic position within the Sphingidae.

In this study, the complete mitochondrial DNA sequence of Parum colligata (Lepidoptera: Sphingidae: Smerinthinae) was sequenced firstly. The mitogenome is 15,288 bp in size, containing 13 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), two ribosomal RNA genes (rRNAs), and an A+T-rich region. In the mitogenome, Ile, Leu2, and Phe are the most frequently used codon families, while codons GCG, TGC, GGC, CTG, AGG, and ACG are absent. The A+T-rich region is 358 bp in length including a motif 'ATAGA', an 18 bp poly-T stretch, three copies of a 12 bp 'TATATATATATA', and a short poly-A element. The nucleotides sequence of A+T-rich region is closer to Sphinginae than Macroglossinae. Phylogenetic analyses, based on the PCGs by using Maximum Likelihood (ML) and Bayesian Inference (BI) methods, generated consistent results that Smerinthinae was clustered together with Sphinginae to be the sister groups rather than Macroglossinae.

Mitochondrial genome characteristics of Somena scintillans (Lepidoptera: Erebidae) and comparation with other Noctuoidea insects.

In this study, mitogenome of Somena scintillans (Lepidoptera: Erebidae) were sequenced and compared with other Noctuoidea species. The mitogenome is 15,410 base pairs in length. All 13 protein-coding genes (PCGs) are initiated by ATN codons except cox1 with CGA and all of PCGs terminate with TAA except nad4 with TAG. The codons ACG and CGC are absent. All the tRNA genes could be folded into the typical cloverleaf secondary structure except the trnS1 which not only loses dihydrouridine (DHU) arm but also mutates its anticodon into TCT. In the AT-rich region of the mitogenome the motif 'ATAGA' mutates to 'ATATA' and two copies of 161 bp-tandem repeats and two 'TA' short tandem repeats are founded. Phylogenetic analyses showed that S. scintillans is clustered into subfamily Lymatriinae. The phylogenetic relationships within Noctuoidea is (((Nolidae + (Euteliidae + Noctuidae)) + Erebidae) + Notodontidae).

Characterization of the complete mitochondrial DNA of Theretra japonica and its phylogenetic position within the Sphingidae (Lepidoptera, Sphingidae).

In the present study, the complete mitogenome of Theretra japonica was sequenced and compared with other sequenced mitogenomes of Sphingidae species. The mitogenome of T. japonica, containing 37 genes (13 protein-coding genes, 22 tRNA genes, and two rRNA genes) and a region rich in adenine and thymine (AT-rich region), is a circular molecule with 15,399 base pairs (bp) in length. The order and orientation of the genes in the mitogenome are similar to those of other sequenced mitogenomes of Sphingidae species. All 13 protein-coding genes (PCGs) are initiated by ATN codons except for the cytochrome C oxidase subunit 1 gene (cox1) which is initiated by the codon CGA as observed in other lepidopteran insects. Cytochrome C oxidase subunit 2 gene (cox2) has the incomplete termination codon T and NADH dehydrogenase subunit 1 gene (nad1) terminates with TAG while the remainder terminates with TAA. Additionally, the codon distributions of the 13 PCGs revealed that Ile and Leu2 are the most frequently used codon families and codons CGG, CGC, CCG, CAG, and AGG are absent. The 431 bp AT-rich region includes the motif ATAGA followed by a 23 bp poly-T stretch, short tandem repeats (STRs) of TC and TA, two copies of a 28 bp repeat 'ATTAAATTAATAAATTAA TATATTAATA' and a poly-A element. Phylogenetic analyses within Sphingidae confirmed that T. japonica belongs to the Macroglossinae and showed that the phylogenetic relationship of T. japonica is closer to Ampelophaga rubiginosa than Daphnis nerii. Phylogenetic analyses within Theretra demonstrate that T. japonica, T. jugurtha, T. suffusa, and T. capensis are clustered into one clade.

Mitochondrial genome characteristics of two Sphingidae insects (Psilogramma increta and Macroglossum stellatarum) and implications for their phylogeny.

In this study, complete mitogenomes of P. increta and M. stellatarum (Lepidoptera: Sphingidae) were sequenced and compared with other Sphingidae species. The mitogenomes containing 37 genes and a AT rich region are circular molecules with 15,252 and 15,290 base pairs in length respectively. Except cox1 all 13 protein-coding genes (PCGs) are initiated by ATN codons. Most of PCGs terminate with TAA except nad5 and cox1 in P. increta and nad5 and cox2 in M. stellatarum. Ile and Leu2 are the most frequently used codon families in both species and codons CGC, CCG, TCG and ACG are absent in P. increta while in M. stellatarum CGC, CCG, CTG, AGG are absent. All the tRNA genes could be folded into the typical cloverleaf secondary structure except the trnS1 of P. increta which lost dihydrouridine (DHU) stem. The AT-rich region of both insects includes the motif ATAGA followed by a 18-19bp polyT stretch and 2-3 short tandem repeats (STRs) of TA, and a poly-A element. Phylogenetic analyses showed that the phylogenetic relationships are (((Sphinx morio+Manduca sexta)+(P. increta+Notonagemia analis scribae))+(Agrius convolvuli)+(M. stellatarum+(Ampelophaga rubiginosa+Daphnis nerii)).

Complete mitochondrial genome of Cnidocampa flavescens (Lepidoptera: Limacodidae).

Cnidocampa flavescens, lives in Nepal, Bhutan, China, Far East of Russia, Korea, and Japan, belongs to the Lepidoptera family Limacodidae. In this study, we describe the genomic features of the mitogenome sequences of the insects. The mitogenome of C. flavescens is 15,406 bp long consisting a typical set of genes (13 protein-coding genes, 22 tRNA genes, and 2 rRNA genes) and one major 415 bp non-coding A + T-rich region. All PCGs of C. flavescens start with ATN codons and end with TAA codons. The gene arrangement of C. flavescens mitogenome is same to Monema flavescens while the intergenic spacers and overlaps are different. The 415 bp A + T-rich region contains a conserved ATAGA motif followed a poly-T stretch.

Complete mitochondrial genome of Histia rhodope Cramer (Lepidoptera: Zygaenidae).

Histia rhodope Cramer, found in India, Chinese, Burma, Indonesia and other Southeast Asian regions, belongs to Lepidoptera family Zygaenidae. In this study, we describe the genomic features of the mitogenome sequences of these insects. The mitogenome of Histia rhodope Cramer is 15,205 bp long consisting a typical set of genes (13 protein-coding genes, 22 tRNA genes and two rRNA genes) and one major 376 bp non-coding A + T-rich region. All PCGs of Histia rhodope Cramer start with ATN codons except cox1 which start with CGA codon and all PCGs stop at TAA codons. Phylogenetic analysis demonstrates that Histia rhodope Cramer and Rhodopsona rbiginosa are clustered together into a monophyletic group Zygaenidae. Zygaenidae is phylogenetically closer to Limacodiae than Tortricidea.

The complete mitochondrial genome of the Chinese Daphnia pulex (Cladocera, Daphniidae).

Daphnia pulex has played an important role in fresh-water ecosystems. In this study, the complete mitochondrial genome of Daphnia pulex from Chaohu, China was sequenced for the first time. It was accomplished using long-PCR methods and a primer-walking sequencing strategy with genus-specific primers. The mitogenome was found to be 15,306 bp in length. It contained 13 protein-coding genes, two rRNA genes, 22 tRNA genes and a typical control region. This research revealed an overall A+T content of 64.50%. All of the 22 typical animal tRNA genes had a classical clover-leaf structure except for trnS1, in which its DHU arm simply formed a loop. The lengths of small and large rRNA were 744 bp and 1,313 bp, respectively. The A+T-rich region was 723 bp in length, which is longer than that from the North American species (689 bp). In terms of structure and composition, many similarities were found between the Chinese and North American Daphnia pulex.

The complete mitochondrial DNA genome of Chinese Daphnia carinata (Clasocera: Daphniidae).

In this paper, we determined the complete mitochondrial genome of Chinese Daphnia carinata for the first time by the long and accurate polymerase chain reaction and primer-walking methods. It was 15,245 bp in length, with an A + T content of 70.35%, containing 37 typical animal mitochondrial genes and an A + T-rich region. The COI gene started with ACTA. All the 22 typical tRNA genes had a classical cloverleaf structure except for trnS1, in which the D-stem pairings in the DHU arm were absent.

[Sequence analysis of the connexin 26 genes from a deafness family with A1555G mutation in Huaiyin].

OBJECTIVE: To ascertain whether connexin 26 (Cx26) gene was a nuclear modifier gene in an extensive family with matrilineal nonsyndromic deafness associated with A1555G mutation in Huaiyin, China. METHODS: Following PCR-restriction fragment length polymorphism (PCR-RFLP) with ApaI restriction enzyme, Cx26 genes from 26 cases, with A1555G mitochondrial mutations in this family, and 62 controls (including 2 patrilineal relatives, 10 spouse controls and 50 unrelated controls), were sequenced. RESULTS: Compared with the reference sequence of Cx26 gene, totally four kinds of nucleotide changes,79G -->A, 109G-->A, 341G-->A and 235delC, were detected in a heterozygous form. However, the former three were previously reported polymorphisms, and only the 235delC was a previously described recessive mutation associated with most autosomal nonsyndromic sensorineural hearing loss in Japan and China. Further study showed that the heterozygous 235delC mutation existed in both one individual with mild hearing loss and two individuals with normal hearing. Clinical characterization showed that 235delC mutation did not seem to modify the deafness phenotype due to the A1555G mutation. Moreover, this 235delC mutation was deduced to derive from a married-in control. Finally, there were no co-segregation between the phenotypes of hearing loss and the genotypes for Cx26 genes based on the four kinds of nucleotide changes. CONCLUSIONS: The heterozygous 235delC mutation of the Cx26 gene may not modulate the severity of hearing loss associated with A1555G mutation and Cx26 gene is unlikely to be a modifier gene for hearing loss due to A1555G mitochondrial mutation in this Chinese family.

Sequence analysis of the mitochondrial genome from a large family with maternally inherited nonsyndromic deafness.

OBJECTIVE: To ascertain whether other variations coexist with 1555(A--> G) mutation in the mitochondrial DNA and may aggravate the severity of hearing loss or increase the penetrance of 1555(A--> G) mutation in a large family with maternally inherited nonsyndromic deafness in Huaiyin, Jiangsu province. METHODS: PCR-restriction fragment length polymorphism (PCR-RFLP) was used to screen both the nt1555 and the nt7445 of the mitochondrial DNA from 27 maternal members in the core family; and then the mitochondrial genomes from two maternal members, and the 12S rRNA genes MTRNR1 and tRNA-Ser(UCN) gene MTTS1 from the others, were amplified by PCR-RFLP and were sequenced. RESULTS: 1555(A--> G) mutation in the mitochondrial DNA was reverified to be one of the major factors which cause maternally inherited nonsyndromic deafness and the cosegregation of 955-960(insC) and 1555(A--> G) was present in this family. Moreover, 7449 (insG), a novel homoplasmic mutation in the tRNA-Ser(UCN) gene, was found to co-exist with 1555(A--> G) mutation in two maternal members. CONCLUSION: The cosegregation of 955-960(insC) and 1555(A--> G) implies that 955-960(insC) may synergistically cause hearing loss in the presence of an 1555(A--> G) mutation, serving as an aggravating factor to enhance the sensitivity to aminoglycosides, and may sometimes increase the penetrance of 1555(A--> G) mutation.

[Mutations analysis in a pedigree with maternally inherited sensorineural hearing loss].

OBJECTIVE: To analyze the mutations in a pedigree with maternally inherited sensorineural hearing loss, and to investigate whether 235delC heterozygote mutation in gap junction protein beta 2 (GJB2) gene modulates the severity of hearing loss associated with the A1555G mitochondrial mutation. METHODS: The PCR products were digested with the Alw26 I restriction enzyme, followed by direct sequencing to detect the mitochondrial mutations in 72 members of a core pedigree of an extensive family with matrilineal nonsyndromic deafness; 235delC mutation of the GJB2 gene was screened in this family by using the Apa I restriction enzyme and direct sequencing. RESULTS: The A1555G mutation of the mitochondrial DNA was present in all 27 members of maternal line, out of them, 21 members had phenotype of deafness (77.8%), with a high penetrance. Only three maternal line members of 72 members possessed 235delC heterozygote mutations, and the three had different phenotypes. CONCLUSION: The A1555G homozygous mutation of mitochondrial DNA is the susceptive etiological factor of nonsyndromic deafness in this family, but in the study of this pedigree, the 235delC heterozygous mutation in GJB2 gene may not aggravate the symptoms of hearing loss associated with the A1555G mitochondrial mutation.