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Background: Previous prospective studies of associations between circulating inflammation-related molecules and pancreatic cancer risk have included limited numbers of markers. Methods: We conducted a case–cohort study nested within the Japan Public Health Center-based Prospective Study Cohort II. We selected a random subcohort (n = 774) from a total of 23,335 participants aged 40 to 69 years who returned a questionnaire and provided blood samples at baseline. During the follow-up period from 1993 to 2010, we identified 111 newly diagnosed pancreatic cancer cases, including one case within the subcohort. Plasma concentrations of 62 inflammatory markers of chemokines, cytokines, and growth factors were measured by a Luminex fluorescent bead-based assay. Cox regression models were applied to estimate HR and 95% confidence intervals (CI) for pancreatic cancer risk for quartiles of marker levels adjusted for potential confounders. Results: The HR (95% CI) for the highest versus the lowest category of C–C motif ligand chemokine 8/monocyte chemoattractant protein 2 (CCL8/MCP2) was 2.03 (1.05–3.93; Ptrend = 0.048). After we corrected for multiple comparisons, none of the examined biomarkers were associated with pancreatic cancer risk at P-value Conclusions: We found no significant associations between 62 inflammatory markers and pancreatic cancer risk. Impact: The suggestive association with circulating levels of leukocyte recruiting cytokine CCL8/MCP2 may warrant further investigation.

In this study, we obtained four alternative splicing transcripts of intracellular copper/zinc superoxide dismutase 1 (icCuZnSOD1) in Oxya chinensis. OcicCuZnSOD1a has all common characteristics of CuZnSOD family and is a canonical CuZnSOD. OcicCuZnSOD1b is missing a Zn binding site. OcicCuZnSOD1c lacks Zn ion and is a Cu-only SOD. OcicCuZnSOD1d is missing a CuZnSOD conserved sequence and lacks the E-loop, a conserved disulfide bond, and an active site arginine. OcicCuZnSOD1a was the most heat-resistant and OcicCuZnSOD1c was the most unstable at high temperatures above 55 °C. They were stable at a wide pH range, especially in alkaline conditions. The four variants expressed at the throughout developmental stages and had various tissue expression patterns. OcicCuZnSOD1a and OcicCuZnSOD1d were significantly induced by 8.79 mM CuCl2 and OcicCuZnSOD1b was significantly up-regulated by 14.67 mM CuCl2. OcicCuZnSOD1a was significantly inhibited by 19.13 mM ZnSO4 while OcicCuZnSOD1d were significantly induced by 22.61 mM ZnSO4. Disc diffusion assay showed that the four isoforms of OcicCuZnSOD1 made the killing zones smaller surrounding the CdCl2-soaked filter discs. However, the reduction ratios of OcicCuZnSOD1a were the highest. These results implied that the four transcripts played roles in defense against CdCl2-induced oxidative stress while OcicCuZnSOD1a had stronger antioxidant capacity.

CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.

A novel healthy diet index for dietary quality can be used to assess food intake. After the Great East Japan Earthquake in 2011, the Fukushima Health Management Survey collected dietary data using a short-form food frequency questionnaire (FFQ). The current study included eligible participants (n = 64,909) aged 16–84 years who answered the FFQ in 2011. The year- and sex-specific dietary patterns were determined via principal component analysis. Based on the typical Japanese, juice/dairy, and meat patterns, healthy diet index (HDI) scores were assigned for food items, resulting in Spearman’s correlation coefficients of 0.730, −0.227, and −0.257, respectively. The mean (standard deviation) of the HDI scores (range: 1–18) were 9.89 (2.68) in men and 9.96 (2.58) in women. Older individuals, women, nonsmokers, those in good health and with regular physical exercise, and those who did not transfer residences had a high HDI score. In the confirmatory analysis, the adjusted odds ratio (95% confidence interval) of the highest vs. the lowest quartiles of HDI scores was 0.87 (0.80, 0.94) for overweight, 0.89 (0.81, 0.97) for large waist circumference, and 0.73 (0.66, 0.80) for dyslipidemia. The HDI score obtained using the FFQ can be applied to evaluate dietary profiles.

Cytochrome P450 monooxygenases (CYPs) play essential physiological functions in insects. CYP303A1 is highly conserved in insect species studied to date, and shows an indispensable role for adult eclosion in both Locusta migratoria and Drosophila melanogaster. However, how CYP303A1 is regulated to control insect developmental process remains uninvestigated. In this study, we discovered functional binding sites for miR-184 in the coding sequence of LmCYP303A1. The luciferase reporter assay showed that miR-184 could target LmCYP303A1 and regulate its expression in vitro. Furthermore, overexpression of miR-184 through microinjection of agomir to locusts reduced the transcripts of LmCYP303A1 and led to the abnormal molting, which is similar to the phenotype of silencing LmCYP303A1 by direct injection of dsLmCYP303A1 to locusts. Meanwhile, down regulation of miR-184 by injection of antagomir increased the LmCYP303A1 transcript and caused molting defects. These findings suggested that miR-184 could target LmCYP303A1 to regulate the molting process in L. migratoria, which might be considered as a novel target for pest control. This article is protected by copyright. All rights reserved.

BACKGROUND Knickkopf (Knk) proteins play crucial roles in the formation of insect cuticle. Recent studies in the holometabolous insect red flour beetle (Tribolium castaneum) have shown that three Knk genes encoding TcKnk, TcKnk2 and TcKnk3 play different but essential roles at different developmental stages and in different tissues. However, the functions of Knk genes had not been fully explored in hemimetabolous insects such as the migratory locust Locusta migratoria. RESULTS We identified three transcripts of LmKnk-like genes LmKnk2 and LmKnk3 with the full-length cDNA sequences, which were named as LmKnk2, LmKnk3-FL and LmKnk3-5'. These three transcripts were highly expressed before molting and mainly expressed in the integument. Among these genes, silencing only LmKnk3-5' by RNA interference (RNAi) caused molting defects and high mortality of the locusts. Injection of dsLmKnk3-5' dramatically decreased chitin content, but did not affect cuticle laminar ultra-structures in the integument. After the knockdown of LmKnk3-5' transcript, lipid deposition on the cuticle surface was impeded, and locusts exhibited increased susceptibility to each of four insecticides in three different classes. However, no visible phenotypic changes were observed after LmKnk2 or LmKnk3-FL was silenced by RNAi. CONCLUSION We demonstrate that LmKnk3-5' is essential for cuticle formation in L. migratoria. This contrasts the findings that the cognate protein in T. castaneum TcKnk3-5' is dispensable for cuticle formation and survival. Hence, we provide some evidence that the function of Knk-type proteins may be species-specific. We therefore think that LmKnk3-5' may be a good target for the application of RNAi-based technologies for species-specific insect pest management. © 2020 Society of Chemical Industry.

Objective: Dietary patterns more closely resemble actual eating behaviours because multiple food groups, not a single food group or nutrient, are considered. The present study aimed to identify and assess changes of dietary patterns in Fukushima residents. Design: Dietary data were collected using a short-form FFQ in annual Fukushima Health Management Survey between 2011 and 2013 after the Great East Japan Earthquake. Year- and sex-specific dietary patterns were determined by the principal component analysis. Setting: Evacuation and nonevacuation zones in Fukushima, Japan. Participants: Eligible participants aged ≥16 years answered the FFQ (n 67 358 in 2011, n 48 377 in 2012 and n 40 742 in 2013). Results: Three identified dietary patterns were assessed similarly in men and women and among years: typical, juice and meat. In total participants, the Spearman’s correlation coefficients between two survey years were 0·70–0·74 for the typical, 0·58–0·66 for the juice and 0·50–0·54 for the meat pattern scores. Adjusted for sociodemographic factors, evacuees had lower typical pattern scores, higher juice pattern scores and the same meat pattern scores compared with non-evacuees. The means of typical pattern scores in evacuees and it of juice pattern scores in non-evacuees continued declining over years. Similar profiles of dietary patterns and trends of pattern scores were observed in participants (n 22 805) who had provided three dietary assessments. Conclusions: Changes of dietary patterns have been observed between 2011 and 2013. Careful investigation of those with low intake of typical pattern foods and promotion of them, particularly in evacuees, are needed.

Dicers belong to a class of large RNase III multidomain ribonucleases and are central components of the RNA interference (RNAi) pathways. In insects, Dicer-2 has been known to cleave long double-stranded RNA (dsRNA) in small interfering RNA (siRNA)-mediated-RNAi pathway. However, Dicer-1 is responsible for cleaving precursor microRNAs (pre28 miRNAs) in miRNA-mediated RNAi pathway. In this study, we identified one LmDicer-1 and two LmDicer-2 (LmDicer-2a and LmDicer-2b) genes in Locusta migratoria. The RNAi of RNAi assay showed that knockdown of each of the Dicer genes reduced RNAi efficiency against a target gene (Lmβ-Tubulin), suggesting that all these genes participated in the siRNA-mediated RNAi pathway. Sequence analyses of the siRNAs generated from dsLmβ-Tubulin after silencing each LmDicer gene showed no significant difference in the pattern of siRNAs mapped to dsLmβ-Tubulin. This result indicated that all the three LmDicers are capable of generating siRNAs from the dsRNA. We then generated recombinant proteins consisting of different domains using Escherichia coli expression system and incubated each recombinant protein with dsLmβ-Tubulin. We found that the recombinant Dicer proteins successfully cleaved dsLmβ-Tubulin. However, LmDicer-2a-R lacking dsRBD domain lost activity, suggesting that dsRBD domain is critical for Dicer function. Furthermore, overexpression of these proteins in Drosophila S2 cells improved RNAi efficiency. Our siRNA affinity chromatography and LC-MS/MS analysis identified LmDicer-2a, LmDicer-2b, LmR2D2, LmAgo2a, LmAgo1, LmStaufen and LmTARBP2 as constituents of RNA-induced silencing complex. Taken together, these data show that both LmDicer-1 and two LmDicer-2s all participate in siRNA-mediated RNAi pathway and likely contribute to high RNAi efficiency in L. migratoria.

Thitarodes (Lepidoptera, Hepialidae) is the only genus that hosts to the Ophiocordyceps sinensis, a traditional Chinese medicine considered as a powerful medicinal supplement. In this study, the complete mitochondrial genomes (mitogenomes) of two species, T. damxungensis and T. pui, have been sequenced, which are 15,928 bp and 15,362 bp in size respectively, and both contain 13 protein-coding genes (PCGs), 2 rRNAs, 22 tRNAs and an AT-rich region. Like other hepialoids, the gene arrangement of the mitogenomes of T. damxungensis and T. pui is identical to the ancestral arrangement but differs from those of other lepidopteran species on account of the different arrangements of trnM, trnI, and trnQ. The size of AT-rich region is 545 bp in T. damxungensis and 1030 bp in T. pui. Tandem repetition in the AT-rich region is responsible for the length difference of the A + T-rich region in both species. In Hepialidae, the phylogenetic study based on the dataset of the sequences that combined the protein-coding genes and RNA genes suggested that the species T. yunnanensis should still belong to the genus Thitarodes rather than Ahamns, which is different from the results based on the traditional phylogeny.

Background The aryl hydrocarbon receptor (AhR) belongs to the bHLH-PAS (basic Helix-Loop-Helix - Period/ARNT/Single minded) family of transcription factors. AhR is a ligand-activated transcription factor, which participates in the sensing and transmitting stimuli of endogenous and exogenous chemicals, and subsequently activates the transcription of genes related to various physiological and detoxification functions. Result In this study, a single full-length LmAhR sequence was cloned and characterized. RNA interference (RNAi) and insecticide bioassays showed that LmAhR plays a vital role in chlorpyrifos susceptibility. To better identify aryl hydrocarbon receptor from locusta migratoria (LmAhR)-regulated genes involved in chlorpyrifos susceptibility, a comparative transcriptome analysis was performed using double-stranded (ds)GFP- and dsLmAhR-injected Locusta migratoria. Differential gene expression analysis identified 145 down-regulated and 67 up-regulated genes (P ≤ 0.05 and fold change ≥2) in dsLmAhR-knockdown insects. We selected 27 down-regulated genes and verified their expression levels using reverse transcription quantitative PCR. Finally, one glutathione S-transferase (GST) gene (LmGSTd7) was selected as a candidate detoxification gene and was further validated via RNAi and chlorpyrifos bioassays. Conclusion Our data suggest that AhR is associated with chlorpyrifos susceptibility via the regulation of LmGSTd7 expression in L. migratoria. © 2019 Society of Chemical Industry.

The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.

Lead (Pb) is known to be toxic to many organisms. Oxidative stress is a major mechanism of its toxicity. This research aims to investigate the effects of Pb on hydrogen peroxide (H2O2) and malonedialdehyde (MDA) contents, activities and mRNA levels of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx)) after Oxya chinensis were acutely treated with lead acetate for 24 h. The results showed that the LD50-24 h value of lead acetate to O. chinensis was 1541.89 (1431.19–1655.77) μg g−1 body H2O2 and MDA contents were elevated after Pb administration, which suggested that Pb induced the overproduction of ROS and caused oxidative stress. SOD activities were significantly inhibited 40.42% of the control by 280 μg μL−1 Pb. CAT activities were increased while GPx activities had no significant changes. Different types of antioxidant-related genes had various responses to Pb stress. The transcriptions of icCuZnSOD2 and ecCuZnSOD2 were significantly inhibited by different concentrations of Pb. MnSOD mRNA levels showed the concentration-dependent rise with the Pb concentrations increase. The expressions of ecCuZnSOD1, CAT1, and GPx were significantly up-regulated while the transcriptions of icCuZnSOD1 and CAT2 had no significant changes. Alteration of activities and mRNA expressions of antioxidant enzymes implied that Pb-induced antioxidant defenses were related to modifications at enzymatic and transcriptional levels. The profiles of antioxidant enzymes and H2O2 and MDA contents and relationships among the parameters indicated that the cooperation of multiple antioxidants rather than a single factor might be responsible for the antioxidant defenses against Pb stress.

Cells produce an extracellular matrix (ECM) with a stereotypic organization that is important for tissue function. The insect cuticle is a layered ECM that mainly consists of the polysaccharide chitin and associated proteins adopting a quasi-crystalline structure. Our understanding of the molecular mechanisms deployed during construction of the highly ordered protein-chitin ECM so far is limited. In this study, we report on the role of the chitin deacetylase 1 (LmCDA1) in the organization of the protein-chitin ECM in the migratory locust Locusta migratoria, and LmCDA1 localizes predominantly to the apical tier of the protein-chitin ECM, but it is also found in lower regions. Reduction of LmCDA1 function correlates with lower amounts of chitin and impedes conversion of chitin to chitosan by deacetylation. Establishment of the quasi-crystalline architecture of the protein-chitin ECM is, however, independent of LmCDA1 activity, but it is dependent on another chitin deacetylase, LmCDA2, which has no detectable effects on chitin deacetylation and, as shown previously, no influence on chitin content. Our data reveal that LmCDA1 and LmCDA2 act in parallel and independently from each other in defining the dimensions of the cuticle. Both enzymes are non-uniformly distributed within the protein-chitin matrix, suggesting a site-autonomous function.

Halloween genes are involved in the biosynthesis of the molting hormone, which plays a key role in insect ecdysis, development, metamorphosis, and reproduction. Our previous work identified five Halloween genes from Locusta migratoria, but their functions are currently unknown. In this study, the sequences of these five Halloween genes were analyzed and characterized. LmCYP307a2, LmCYP306a1, LmCYP302a1, and LmCYP315a1 were primarily expressed in the prothoracic glands, while LmCYP314a1 was universally expressed in peripheral tissues, especially in the ovaries and Malpighian tubules. All five Halloween genes were mainly expressed from the 5th to the 7th d in 5th-instar nymphs. RNA interference (RNAi) silencing of LmCYP307a2 resulted in severe molting delays and molting failure, which could be rescued by supplementary 20-hydroxyecdysone. A hematoxylin and eosin staining analysis suggested that the RNAi of LmCYP307a2 inhibited the ecdysis process by inhibiting the apolysis and degradation of the old cuticle, and by promoting the synthesis of a new cuticle. Quantitative reverse transcription polymerase chain reaction results showed that the expressions of LmE74, LmCht5, and LmCht10 were dramatically down-regulated, while that of LmChsI was substantially up-regulated, after knockdown of LmCYP307a2. The results suggest that LmCYP307a2 is related to the molt process via regulation of chitin synthesis and degradation.

To investigate associations between dietary patterns and the risk of impaired kidney function, we analyzed data from 14,732 participants (40&ndash, 89 years) who completed the baseline diet questionnaire of The Fukushima Health Management Survey in 2011. The incidence of chronic kidney disease (CKD) (estimated glomerular filtration rate (eGFR) &lt, 60 mL/min/1.73 m2 or proteinuria (&ge, 1+ by dipstick test)) and annual changes in eGFR were assessed from 2012 to 2015. Three major dietary patterns were identified. The adjusted cumulative incidence ratio of the highest vs. lowest tertile of a vegetable diet scores was 0.90 (95% confidence interval (CI): 0.82, 1.00) for eGFR &lt, 60 mL/min/1.73 m2, 0.68 (95% CI: 0.52, 0.90) for proteinuria, and 0.88 (95% CI: 0.80, 0.97) for CKD (P for trend = 0.031, 0.007, and 0.005, respectively). The incident risk of CKD in the highest tertile of juice diet scores was 18% higher than the lowest tertile. The odds ratio of the highest vs. lowest tertile of vegetable diet scores was 0.85 (95% CI: 0.75, 0.98) in the rapidly decreasing eGFR group (P for trend = 0.009). We did not observe significant associations for the meat dietary pattern. A Japanese vegetable diet could reduce the risk of developing impaired kidney function and CKD.

Simple Summary Insect reproduction is an important and complicated process required for producing healthy individuals and maintaining their population abundance. Thus, it could become a valuable target for insect biological control. To date, many factors and pathways have been revealed to be involved in this reproductive process, but it is still far from a full understanding of the molecular network underlying this process. We herein investigated a RNA helicase, DEAD-box protein 6 (DDX6) in Locusta migratoria, a global, destructive pest, and found that knockdown LmDDX6 downregulated expression levels of juvenile hormone receptor gene methoprene-tolerant and its target genes 78-kDa glucose-regulated proteins, thus reducing vitellogenin expression and ultimately impairing the ovary development and oocyte maturation. These results demonstrate that LmDDX6 is a key player in female locust reproduction, providing, thus, a novel target for locust biological control. Abstract DEAD-box protein 6 (DDX6) is a member of the DDX RNA helicase family that exists in all eukaryotes. It has been extensively studied in yeast and mammals and has been shown to be involved in messenger ribonucleoprotein assembly, mRNA storage, and decay, as well as in miRNA-mediated gene silencing. DDX6 participates in many developmental processes but the biological function of DDX6 in insects has not yet been adequately addressed. Herein, we characterized the LmDDX6 gene that encodes the LmDDX6 protein in Locusta migratoria, a global, destructive pest. LmDDX6 possesses five motifs unique to the DDX6 subfamily. In the phylogenetic tree, LmDDX6 was closely related to its orthologs in Apis dorsata and Zootermopsis nevadensis. RT-qPCR data revealed high expression of LmDDX6 in the ovary, muscle, and fat body, with a declining trend in the ovary after adult ecdysis. LmDDX6 knockdown downregulated the expression levels of the juvenile hormone receptor Met, and genes encoding Met downstream targeted Grp78-1 and Grp78-2, reduced LmVg expression, and impaired ovary development and oocyte maturation. These results demonstrate that LmDDX6 plays an essential role in locust female reproduction and, thus, could be a novel target for locust biological control.

DDX3 represents a well-defined subfamily of DEAD-box RNA helicase and exerts multiple functions in RNA metabolism, cell cycle, tumorigenesis, signal pathway, and fertility. Our previous study has shown that LmDDX3, the ortholog of DDX3 in Locusta migratoria, is ubiquitously expressed, and with a high abundance in testis and ovary. Knockdown of LmDDX3 results in a lethal phenotype in nymph, but it still remains unclear for its role in reproductive process. In this study, we therefore characterized LmDDX3 expression in female adult locust and analyzed its function in oocyte development. LmDDX3 was expressed in all tissues examined with significant more transcripts in ovary and hindgut. In ovary, a strong expression level was detected at the day just after adult eclosion, and a dramatic reduction then occurred during the oocyte development. LmDDX3 RNAi led to a reduced vitellogenin (Vg) expression in fat body via partially at least, the JH signaling pathway, and caused an upregulation of vitellogenin receptor (VgR) in ovary, and thus blocked the ovarian development and oocyte maturation. Sequence and phylogenetic analysis indicated that LmDDX3 was closely related to termite DDX3. Taken together, these data reveal a critical role for LmDDX3 in regulating the transcription of Vg and VgR, two major factors in vitellogenesis that is a key process required for ovary development and oocyte maturation in locust, and contribute thereof a new putative target for locust biological control.

The efficiency of RNA interference (RNAi) varies substantially among different insect species. Rapid degradation of double-stranded RNA (dsRNA) by dsRNA-degrading nucleases (dsRNases) has been implicated to cause low RNAi efficiency in several insect species. In this study, we identified four dsRNase genes (OfdsRNase1, OfdsRNase2, OfdsRNase3 and OfdsRNase4) from the Asian corn borer (Ostrinia furnacalis) transcriptome database. Bioinformatic analyses showed that each deduced protein sequence contained endonuclease NS domains and signal peptides. Gene expression analysis revealed that OfdsRNase2 was exclusively expressed in the midgut of larvae. RNAi efficiency was investigated in 2-d-old fifth-instar larvae (high expression of dsRNase2) and 2-d-old pupae (low expression of dsRNase2) by feeding or injecting dsRNA targeting a marker gene that encodes the lethal giant larvae protein (OfLgl). Our results showed that OfLgl only partially silenced the expression of OfLgl in pupae, but not in larvae, suggesting that OfdsRNase2 could contribute to lower RNAi efficiency in larval stages. This hypothesis was supported by our RNAi-of-RNAi experiment using a tissue culture technique where the silencing efficiency against the reporter gene, OfHex1, was significantly improved after knockdown of OfdsRNase2. When double luciferase assays were performed to evaluate the role of the four dsRNases in vitro, only OfdsRNase2 expressed in S2 cells significantly affected RNAi efficiency by degrading dsRNA. Taken together, our results suggested that the degradation of dsRNA by OfdsRNase2 in the midgut contributed to low RNAi efficiency in O. furnacalis larvae.

CRISPR-Cas12c/d proteins share limited homology with Cas12a and Cas9 bacterial CRISPR RNA (crRNA)-guided nucleases used widely for genome editing and DNA detection. However, Cas12c (C2c3)- and Cas12d (CasY)-catalyzed DNA cleavage and genome editing activities have not been directly observed. We show here that a short-complementarity untranslated RNA (scoutRNA), together with crRNA, is required for Cas12d-catalyzed DNA cutting. The scoutRNA differs in secondary structure from previously described tracrRNAs used by CRISPR-Cas9 and some Cas12 enzymes, and in Cas12d-containing systems, scoutRNA includes a conserved five-nucleotide sequence that is essential for activity. In addition to supporting crRNA-directed DNA recognition, biochemical and cell-based experiments establish scoutRNA as an essential cofactor for Cas12c-catalyzed pre-crRNA maturation. These results define scoutRNA as a third type of transcript encoded by a subset of CRISPR-Cas genomic loci and explain how Cas12c/d systems avoid requirements for host factors including ribonuclease III for bacterial RNA-mediated adaptive immunity.

Background We hypothesized that early postoperative administration of celecoxib would reduce pain scores and improve sleep quality and active range of motion after total knee arthroplasty (TKA) under general anesthesia. Methods Patients in the celecoxib group received 400 mg of celecoxib 2 hours after TKA, followed 6 hours later by 200 mg of celecoxib. Patients in the control group received 400 mg of celecoxib the second day after surgery. Patients in both group had access to patient-controlled analgesia fentanyl. The primary outcome measure was the patient-reported visual analog scale (VAS) pain score the second day after TKA. The secondary outcome measure was sleep quality (days 1, 2, and 7 postoperatively). Active knee joint range of motion was assessed on days 2 and 7 postoperatively, and VAS pain scores were evaluated on postoperative days 1 to 7. Total fentanyl consumption was also assessed. Results Compared to the control group, the celecoxib group had significantly lower median VAS pain scores on postoperative days 1 and 2, significantly less nocturnal awakening (in minutes) and frequency of body motion, and better sleep efficacy on postoperative day 1. The celecoxib group also had a significantly better median flexion angle (°) on postoperative days 2 and 7, and lower cumulative fentanyl consumption. Conclusion Early administration of celecoxib after TKA was associated with significantly reduced early VAS pain scores and improved sleep quality and active knee flexion angles. Thus, the early administration of celecoxib after TKA under general anesthesia may reduce pain and improve sleep quality and functional recovery. Levels of Evidence Level II, therapeutic study. Trial Registration UMIN-CTR 000014624 (July 23, 2014).

Background Cuticular hydrocarbons (CHCs) have a critical role in preventing desiccation and penetration of xenobiotics in insects. Previous studies have shown that cytochrome P450 subfamily 4G (CYP4G) enzymes are oxidative decarbonylases, essential for CHC biosynthesis. However, it is unclear whether there are functional differences between the two CYP4G genes in most insects. In Locusta migratoria, we identified two CYP4G genes (LmCYP4G62 and LmCYP4G102). LmCYP4G102 plays a critical role in the synthesis of CHCs, but the function of LmCYP4G62 is unknown. Results We identified, characterized, and compared two LmCYP4G genes, based on L. migratoria transcriptomic and genomic databases. RT-qPCR showed that both were highly expressed in tissues with which oenocytes are associated, the integument and fat body. Immunostaining indicated that LmCYP4G62 and LmCYP4G102 were highly abundant in oenocytes in these tissues. However, the two enzymes had a different subcellular distribution, with LmCYP4G62 localized on the plasma membrane and LmCYP4G102 dispersed throughout the oenocyte cytoplasm, presumably on the endoplasmic reticulum. RNA interference-mediated gene silencing against each of the two genes resulted in reduced CHC contents, in all classes for LmCYP4G102, but mostly shorter chain CHCs for LmCYP4G62. Silencing of both genes resulted in increased insecticide penetration through the cuticle, and increased locust susceptibility to desiccation and insecticides. Conclusion Our studies suggest that both LmCYP4G62 and LmCYP4G102 contribute to hydrocarbon biosynthesis and play key roles in protecting locusts from water loss and insecticide penetration, but they are not fully redundant. Further, the two LmCYP4G genes might be used as new targets for insect pest management. © 2020 Society of Chemical Industry.

Chitinases, key enzymes involved in degradation of chitin, have been repeatedly shown to play an indispensable role during insect post-embryonic molting processes at stage transitions. However, how chitinases affect insect embryonic development remains to be analyzed. In this study, we investigated the role of chitinase 5-1 (LmCht5-1) during embryonic development of the hemimetabolous insect Locusta migratoria. LmCht5-1 transcript levels were high in pro-nymphs during late embryogenesis. The respective protein localized to both the pro-nymphal and, to a much lesser extent, the newly formed nymphal cuticle. After injection of double stranded RNA against LmCht5-1 into 8 days old embryos, LmCht5-1 transcripts were strongly reduced. Most of dsLmCht5-1-injected pro-nymphs failed to develop to first-instar nymphs and died at or before hatching. Histological analyzes showed that degradation of the pro-nymph cuticle was blocked in these animals. At the ultra-structural level, we found that LmCht5-1 was needed for the degradation of the lamellar procuticle, while the separation of the procuticle from the epicuticle and epidermis (apolysis) was independent of LmCht5-1 function. Taken together, our results indicate that LmCht5-1 and other yet unknown degrading enzymes act in parallel at distinct positions of the cuticle during molting of the pro-nymph to the first-instar nymph during locust embryogenesis.

Cytochrome P450 monooxygenases (P450s) are important enzymes for biotransformations of various endogenous and xenobiotic substances in various organisms. In this study, we examined microsomal P450 protein content and enzymatic activity in four major detoxification tissues dissected from fifth-instar nymphs of the migratory locust (Locusta migratoria). The highest microsomal P450 protein content was found in the gastric caeca (a part of the midgut), followed by the midgut, Malpighian tubules and fat bodies. Microsomal P450s showed the highest aromatic hydroxylation, O-dealkylation and O-dearylation activities towards six of the seven model substrates examined in the fat bodies. Although the gastric caeca showed the highest P450 protein content, the enzymatic activities towards six of the seven model substrates were the lowest in this tissue. Further, the midgut, gastric caeca and fat bodies showed significant metabolic activities towards two pyrethroid insecticides (deltamethrin and fluvalinate), but no significant activities towards the other four insecticides (malathion, chlorpyrifos, carbaryl and methoprene). These results support our conclusions: 1) total P450 protein content alone cannot be reliably used to predict its enzymatic activity, and 2) insect P450 enzymatic activity is both tissue and substrate dependent.

Purified Brazilin from Sappan wood extract has been reported with significant antitumor effect, especially on human T24 cells and bladder cancer mouse models. Brazilin can significantly induce expression of c-Fos and GADD45β and transfection expression of c-Fos and GADD45β in T24 cells can induce significant cell morphology changes, reduced viability and cell death, while transfection of siRNA-c-Fos and siRNA-GADD45β can reverse the induced cell death. Co-transfection of both c-Fos and GADD45β into T24 cells resulted in a significantly additive effect when compared to single transfection with only c-Fos or GADD45β. Meanwhile, transfection of interfering siRNA-c-Fos or siRNA-GADD45β can partially rescue the cell viability and siRNA co-transfection showed increased rescue rate. The transfection expression and interference with pcDNA3.1-c-Fos/siRNA-c-Fos or pcDNA3.1-GADD45β/siRNA-GADD45β did not affect each other's expression. Moreover, analysis of c-Fos and GADD45β regulated genes and signal pathways showed that no common regulated genes or pathways were present. All the results indicated that c-Fos and GADD45β mediate independent Brazilin-inducible genes and pathways. © 2018 IUBMB Life, 70(11):1101-1110, 2018.

Cytochrome P450 monooxygenases (CYPs) belong to a large superfamily of heme-containing enzymes catalyzing at least 60 different types of chemically distinct reactions. Insect CYPs play key roles in biotransformation of insecticides and plant chemicals, and are implicated in insecticide resistance and insect adaptation to their host plants. Insect CYPs are well studied in model insects, but little is known about the CYP superfamily in paurometabolous insects. We employed Illumina sequencing technology to identify 71 partial and 78 full-length open reading frames (ORFs) of LmCYP genes from the migratory locust (Locusta migratoria), one of the most destructive paurometabolous insect pests in the world. Seventy-eight LmCYPs with complete ORFs were formally named and classified into 19 families and 43 subfamilies. The majority of LmCYPs were mainly expressed in nymphal and adult stages, but LmCYP expression varied widely among thirteen different tissues examined. Regulatory elements were predicted in the promoter regions of LmCYP genes, and subsequent exposure of locusts to 12 different exogenous chemicals showed that 2-tridecanone and xanthotoxin were the most effective at increasing LmCYP expression. Our results represent the first transcriptome-wide analysis of the LmCYP superfamily from migratory locust, and provide a foundation for understanding the physiological functions, functional diversity, evolution, and regulatory mechanisms controlling the expression of the CYP gene superfamily in the locust.