A Blueprint of Microstructures and Stage-Specific Transcriptome Dynamics of Cuticle Formation in Bombyx mori.

Insect cuticle is critical for the environmental adaptability and insecticide resistance of insects. However, there is no clear understanding of the structure and protein components of the cuticle during each developmental stage of holometabolous insects, and knowledge about the protein components within each layer is vague. We conducted serial sectioning, cuticular structure analysis, and transcriptome sequencing of the larval, pupal, and adult cuticles of Bombyx mori. The deposition processes of epicuticle, exocuticle, and endocuticle during larval, pupal, and adult cuticle formation were similar. Transcriptome analysis showed that these cuticle formations share 74% of the expressed cuticular protein (CP) genes and 20 other structural protein genes, such as larval serum protein and prisilkin. There are seven, six, and eleven stage-specific expressed CP genes in larval, pupal, and adult cuticles, respectively. The types and levels of CP genes may be the key determinants of the properties of each cuticular layer. For example, the CPs of the RR-2 protein family with high contents of histidine (His) are more essential for the exocuticle. Functional analysis suggested that BmorCPAP1-H is involved in cuticle formation. This study not only offers an in-depth understanding of cuticle morphology and protein components but also facilitates the elucidation of molecular mechanisms underlying cuticle formation in future studies.

Subcellular stoichiogenomics reveal cell evolution and electrostatic interaction mechanisms in cytoskeleton.

BACKGROUND: Eukaryotic cells contain a huge variety of internally specialized subcellular compartments. Stoichiogenomics aims to reveal patterns of elements usage in biological macromolecules. However, the stoichiogenomic characteristics and how they adapt to various subcellular microenvironments are still unknown. RESULTS: Here we first updated the definition of stoichiogenomics. Then we applied it to subcellular research, and detected distinctive nitrogen content of nuclear and hydrogen, sulfur content of extracellular proteomes. Specially, we found that acidic amino acids (AAs) content of cytoskeletal proteins is the highest. The increased charged AAs are mainly caused by the eukaryotic originated cytoskeletal proteins. Functional subdivision of the cytoskeleton showed that activation, binding/association, and complexes are the three largest functional categories. Electrostatic interaction analysis showed an increased electrostatic interaction between both primary sequences and PPI interfaces of 3D structures, in the cytoskeleton. CONCLUSIONS: This study creates a blueprint of subcellular stoichiogenomic characteristics, and explains that charged AAs of the cytoskeleton increased greatly in evolution, which offer material basis for the eukaryotic cytoskeletal proteins to act in two ways of electrostatic interactions, and further perform their activation, binding/association and complex formation.

Cuticular protein defective Bamboo mutant of Bombyx mori is sensitive to environmental stresses.

Insect cuticle acts as a primary protective barrier against environment stresses that may directly impact the insect body. Here, we report the mechanical defense function of a structural cuticular protein, BmorCPH24, to environmental stresses using a silkworm Bamboo (Bo) mutant with this gene mutation. Ultraviolet (UV) irradiation and topical application of an acetone insecticide were used as environmental stresses to determine the differences in susceptibility between Bo and wild-type larvae. UV irradiation resulted in a sunburn phenotype in the Bo strains earlier than the wild-type indicating the sensitivity of Bo. Higher malondialdehyde (MDA) content and a lower survival ratio were also observed in the Bo strains. Treatment with deltamethrin revealed that Bo larvae were more sensitive to insecticides than the wild-type. Furthermore, cuticle analysis by microsection revealed thinner cuticle and a significant decrease in the endocuticle layer (∼64.0%) in Bo. These results suggest that BmorCPH24 mutation can lead to deficiency in resources required to construct the cuticle in Bo resulting in thin cuticle and reduced resistance to UV and insecticides. These results provide us new insight into the role of structural cuticular proteins in insect cuticle against environment stresses.

Mutation of a lepidopteran-specific PMP-like protein, BmLSPMP-like, induces a stick body shape in silkworm, Bombyx mori.

BACKGROUND: Lepidoptera is one of the largest orders of insects, some of which are major pests of crops and forests. The cuticles of lepidopteran pests play important roles in defense against insecticides and pathogens, and are indispensable for constructing and maintaining extracellular structures and locomotion during their life cycle. Lepidopteran-specific cuticular proteins could be potential targets for lepidopteran pest control. But information on this is limited. Our research aimed to screen the lepidopteran-specific cuticular proteins using the lepidopteran model, the silkworm, to explore the molecular mechanism underlying the involvement of cuticular proteins in body shape construction. RESULTS: Positional cloning showed that BmLSPMP-like, a gene encoding a lepidopteran-specific peritrophic matrix protein (PMP) like protein which includes a peritrophin A-type chitin-binding domain (CBM_14), is responsible for the stick (sk) mutation. BmLSPMP-like is an evolutionarily conserved gene that exhibits synteny in Lepidoptera and underwent purifying selection during evolution. Expression profiles demonstrated that BmLSPMP-like is expressed in chitin-forming tissues, testis and ovary, and accumulates in the cuticle. BmLSPMP-like knockout, generated with CRISPR/Cas9, resulted in a stick-like larval body shape phenotype. Over-expression of BmLSPMP-like in the sk mutant rescued its abnormal body shape. The results showed that BmLSPMP-like may be involved in assemblage in the larval cuticle. CONCLUSION: Our results suggested that the dysfunction of BmLSPMP-like may result in a stick body shape phenotype in silkworm, through the regulation of the arrangement of the chitinous laminae and cuticle thickness. Our study provides new evidence of the effects of LSPMP-likes on lepidopteran body shape formation, metamorphosis and mortality, which could be an eco-friendly target for lepidopteran pest management. © 2022 Society of Chemical Industry.

Excess melanin precursors rescue defective cuticular traits in stony mutant silkworms probably by upregulating four genes encoding RR1-type larval cuticular proteins.

Melanin and cuticular proteins are vital cuticle components in insects. Cuticular defects caused by mutations in cuticular protein-encoding genes can obstruct melanin deposition. The effects of changes in melanin on the expression of cuticular protein-encoding genes, the cuticular and morphological traits, and the origins of these effects are unknown. We found that the cuticular physical characteristics and the expression patterns of larval cuticular protein-encoding genes markedly differed between the melanic and non-melanic integument regions. By using four p multiple-allele color pattern mutants with increasing degrees of melanism (+p, pM, pS, and pB), we found that the degree of melanism and the expression of four RR1-type larval cuticular protein-encoding genes (BmCPR2, BmLcp18, BmLcp22, and BmLcp30) were positively correlated. By modulating the content of melanin precursors and the expression of cuticular protein-encoding genes in cells in tissues and in vivo, we showed that this positive correlation was due to the induction of melanin precursors. More importantly, the melanism trait introduced into the BmCPR2 deletion strain Dazao-stony induced up-regulation of three other similar chitin-binding characteristic larval cuticular protein-encoding genes, thus rescuing the cuticular, morphological and adaptability defects of the Dazao-stony strain. This rescue ability increased with increasing melanism levels. This is the first study reporting the induction of cuticular protein-encoding genes by melanin and the biological importance of this induction in affecting the physiological characteristics of the cuticle.

Stoichiogenomics reveal oxygen usage bias, key proteins and pathways associated with stomach cancer.

Stomach cancer involves hypoxia-specific microenvironments. Stoichiogenomics explores environmental resource limitation on biological macromolecules in terms of element usages. However, the patterns of oxygen usage by proteins and the ways that proteins adapt to a cancer hypoxia microenvironment are still unknown. Here we compared the oxygen and carbon contents ([C]) between proteomes of stomach cancer (hypoxia) and two stomach glandular cells (normal). Key proteins, genome locations, pathways, and functional dissection associated with stomach cancer were also studied. An association of oxygen content ([O]) and protein expression level was revealed in stomach cancer and stomach glandular cells. For differentially expressed proteins (DEPs), oxygen contents in the up regulated proteins were3.2%higherthan that in the down regulated proteins in stomach cancer. A total of 1,062 DEPs were identified; interestingly none of these proteins were coded on Y chromosome. The up regulated proteins were significantly enriched in pathways including regulation of actin cytoskeleton, cardiac muscle contraction, pathway of progesterone-mediated oocyte maturation, etc. Functional dissection of the up regulated proteins with high oxygen contents showed that most of them were cytoskeleton, cytoskeleton associated proteins, cyclins and signaling proteins in cell cycle progression. Element signature of resource limitation could not be detected in stomach cancer for oxygen, just as what happened in plants and microbes. Unsaved use of oxygen by the highly expressed proteins was adapted to the rapid growth and fast division of the stomach cancer cells. In addition, oxygen usage bias, key proteins and pathways identified in this paper laid a foundation for application of stoichiogenomics in precision medicine.

Genome-wide and expression-profiling analyses suggest the main cytochrome P450 genes related to pyrethroid resistance in the malaria vector, Anopheles sinensis (Diptera Culicidae).

BACKGROUND: Anopheles sinensis is one of the major malaria vectors. However, pyrethroid resistance in An. sinensis is threatening malaria control. Cytochrome P450-mediated detoxification is an important pyrethroid resistance mechanism that has been unexplored in An. sinensis. In this study, we performed a comprehensive analysis of the An. sinensis P450 gene superfamily with special attention to their role in pyrethroid resistance using bioinformatics and molecular approaches. RESULTS: Our data revealed the presence of 112 individual P450 genes in An. sinensis, which were classified into four major clans (mitochondrial, CYP2, CYP3 and CYP4), 18 families and 50 subfamilies. Sixty-seven genes formed nine gene clusters, and genes within the same cluster and the same gene family had a similar gene structure. Phylogenetic analysis showed that most of An. sinensis P450s (82/112) had very close 1: 1 orthology with Anopheles gambiae P450s. Five genes (AsCYP6Z2, AsCYP6P3v1, AsCYP6P3v2, AsCYP9J5 and AsCYP306A1) were significantly upregulated in three pyrethroid-resistant populations in both RNA-seq and RT-qPCR analyses, suggesting that they could be the most important P450 genes involved in pyrethroid resistance in An. sinensis. CONCLUSION: Our study provides insight on the diversity of An. sinensis P450 superfamily and basis for further elucidating pyrethroid resistance mechanism in this mosquito species. © 2018 Society of Chemical Industry.

Identification of carboxylesterase genes associated with pyrethroid resistance in the malaria vector Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: Carboxylesterases (CCEs) are one of three large detoxification enzyme families. Some CCEs are active on synthetic insecticides with ester structures. Anopheles sinensis is an important malaria vector in eastern Asia. This study identified and characterized the CCE genes in the A. sinensis genome and determined CCE genes associated with pyrethroid resistance using RNA sequencing (RNA-seq) and quantitative reverse transcription - polymerase chain reaction (qRT-PCR), in A. sinensis from Anhui, Chongqing, and Yunnan in China. RESULTS: Fifty-seven putative CCEs were identified and placed into three classes, 12 subfamilies and 14 clades through phylogenetic and homology analyses. Exon sizes ranged from 31 to 4317 bp, with 49 CCEs having two to five exons and eight having six to 11 exons. A total of 183 introns were recognized with sizes ranging from 31 to 4317 bp. The 57 CCEs were located on 14 scaffolds, with 70% located on four scaffolds. The alpha-esterase subfamily was significantly expanded compared with that of Anopheles gambiae. In a pyrethroid-resistant strain, RNA-seq detected five upregulated CCE genes and qRT-PCR detected 12 upregulated CCE genes. The α-esterase 10 (AsAe10) and acetylcholinesterase 1 (AsAce1) genes were the main CCE genes associated with pyrethroid resistance. CONCLUSION: This information will be useful for further study of the CCE gene family and pyrethroid resistance mechanisms mediated by CCEs. © 2017 Society of Chemical Industry.

Suppression of Laccase 2 severely impairs cuticle tanning and pathogen resistance during the pupal metamorphosis of Anopheles sinensis (Diptera: Culicidae).

BACKGROUND: Phenol oxidases (POs) catalyze the oxidation of dopa and dopamine to melanin, which is crucial for cuticle formation and innate immune maintenance in insects. Although, Laccase 2, a member of the PO family, has been reported to be a requirement for melanin-mediated cuticle tanning in the development stages of some insects, whether it participates in cuticle construction and other physiological processes during the metamorphosis of mosquito pupae is unclear. METHODS: The association between the phenotype and the expression profile of Anopheles sinensis Laccase 2 (AsLac2) was assessed from pupation to adult eclosion. Individuals showing an expression deficiency of AsLac2 that was produced by RNAi and their phenotypic defects and physiological characterizations were compared in detail with the controls. RESULTS: During the dominant expression period, knockdown of AsLac2 in pupae caused the cuticle to be unpigmented, and produced thin and very soft cuticles, which further impeded the eclosion rate of adults as well as their fitness. Moreover, melanization immune responses in the pupae were sharply decreased, leading to poor resistance to microorganism infection. Both the high conservation among Laccase 2 homologs and a very similar genomic synteny of the neighborhood in Anopheles genus implies a conservative function in the pupal stage. CONCLUSIONS: To our knowledge, this is the first study to report the serious phenotypic defects in mosquito pupae caused by the dysfunction of Laccase 2. Our findings strongly suggest that Laccase 2 is crucial for Anopheles cuticle construction and melanization immune responses to pathogen infections during pupal metamorphosis. This irreplaceability provides valuable information on the application of Lacccase 2 and/or other key genes in the melanin metabolism pathway for developing mosquito control strategies.

Knockdown of NogoA prevents MPP+­induced neurotoxicity in PC12 cells via the mTOR/STAT3 signaling pathway.

NogoA is a myelin­associated protein, which is important in the inhibition of axonal fiber growth and in regeneration following injury of the mammalian central nervous system. A previous study suggested that NogoA may be key in the process of Parkinson's disease (PD), which is the second most common chronic neurodegenerative disorder worldwide. The regulatory mechanism underlying the effect of NogoA on the process of PD remains to be fully elucidated. The present study aimed to investigate the effect and underlying mechanism of NogoA on cellular viability, apoptosis and autophagy induced by 1-methyl-4-phenylpyridinium (MPP+) in PC12 cells, a commonly used in vitro PD model. PC12 cells were treated with 1 mM MPP+ for 24 h and the cells were harvested for western blotting. The results demonstrated that the protien expression levels of NogoA were increased in the PC12 cells treated with MPP+. Subsequently, NogoA small interfering RNA was synthesized and transfected into PC12 cells to silence the expression of NogoA. NogoA knockdown significantly reduced the MPP+­induced decrease in cell viability and apoptosis, detected using a cell counting kit­8 and flow cytometric analysis, respectively. Interference in the expression of NogoA increased the MPP+­induced decrease in mitochondrial membrane potential, determined quantitatively by flow cytometry using JC-1 dye, and the protein levels of Beclin­1. In addition, MPP+ treatment activated the mammalian target of rapamycin (mTOR)/signal transducer and activator of transcription 3 (STAT3) signaling pathway. Knockdown of NogoA significantly inhibited the expression levels of mTOR and STAT3. Furthermore, overexpression of NogoA had similar neurotoxic effects on the PC12 cells as MPP+ treatment. Treatment with rapamycin, an inhibitor of the mTOR/STAT3 signaling pathway had a similar effect to that of NogoA knockdown in the MPP+­treated PC12 cells. Taken together, the results from the present study demonstrated that NogoA may regulate MPP+­induced neurotoxicity in PC12 cells via the mTOR/STAT3 signaling pathway and provided an explanation regarding the regulatory mechanism of NogoA on the process of PD.

A novel PINK1- and PARK2-dependent protective neuroimmune pathway in lethal sepsis.

Although the PINK1-PARK2 pathway contributes to the pathogenesis of Parkinson disease, its roles in sepsis (a major challenge for critical care) were previously unknown. Here, we show that pink1-/- and park2-/- mice are more sensitive to polymicrobial sepsis-induced multiple organ failure and death. The decrease in the circulating level of the neurotransmitter dopamine in pink1-/- and park2-/- mice accelerates the release of a late sepsis mediator, HMGB1, via HIF1A-dependent anaerobic glycolysis and subsequent NLRP3-dependent inflammasome activation. Genetic depletion of Nlrp3 or Hif1a in pink1-/- and park2-/- mice confers protection against lethal polymicrobial sepsis. Moreover, pharmacological administration of dopamine agonist (e.g., pramipexole), HMGB1-inhibitor (e.g., neutralizing antibody or glycyrrhizin), or NLRP3-inhibitor (e.g., MCC950) reduces septic death in pink1-/- and park2-/- mice. The mRNA expression of HIF1A and NLRP3 is upregulated, whereas the mRNA expression of PINK1 and PARK2 is downregulated in peripheral blood mononuclear cells of patients with sepsis. Thus, an impaired PINK1-PARK2-mediated neuroimmunology pathway contributes to septic death and may represent a novel therapeutic target in critical care medicine.

HMGB1 in health and disease.

Complex genetic and physiological variations as well as environmental factors that drive emergence of chromosomal instability, development of unscheduled cell death, skewed differentiation, and altered metabolism are central to the pathogenesis of human diseases and disorders. Understanding the molecular bases for these processes is important for the development of new diagnostic biomarkers, and for identifying new therapeutic targets. In 1973, a group of non-histone nuclear proteins with high electrophoretic mobility was discovered and termed high-mobility group (HMG) proteins. The HMG proteins include three superfamilies termed HMGB, HMGN, and HMGA. High-mobility group box 1 (HMGB1), the most abundant and well-studied HMG protein, senses and coordinates the cellular stress response and plays a critical role not only inside of the cell as a DNA chaperone, chromosome guardian, autophagy sustainer, and protector from apoptotic cell death, but also outside the cell as the prototypic damage associated molecular pattern molecule (DAMP). This DAMP, in conjunction with other factors, thus has cytokine, chemokine, and growth factor activity, orchestrating the inflammatory and immune response. All of these characteristics make HMGB1 a critical molecular target in multiple human diseases including infectious diseases, ischemia, immune disorders, neurodegenerative diseases, metabolic disorders, and cancer. Indeed, a number of emergent strategies have been used to inhibit HMGB1 expression, release, and activity in vitro and in vivo. These include antibodies, peptide inhibitors, RNAi, anti-coagulants, endogenous hormones, various chemical compounds, HMGB1-receptor and signaling pathway inhibition, artificial DNAs, physical strategies including vagus nerve stimulation and other surgical approaches. Future work further investigating the details of HMGB1 localization, structure, post-translational modification, and identification of additional partners will undoubtedly uncover additional secrets regarding HMGB1's multiple functions.