RNA-seq and microRNA-seq analysis of Japanese flounder (Paralichthys olivaceus) larvae treated by thyroid hormones.

Flatfish pigmentation is a complex process, affected by environmental factors including light, nutrients, and hormones. Of those, the thyroid hormone has been reported to increase the albinism rate of Japanese flounder (Paralichthys olivaceus). However, the underlying mechanism remains unclear. In the present study, triiodothyronine (T3), thyroxine, and thiourea were introduced into P. olivaceus larvae from 16 to 57 days after hatching (DAH). By comparison of albinism rate, T3 treatment and control larvae of 42 DAH were chosen for mRNA and miRNA high-throughput sequencing analyses. A total of 337 miRNAs were identified via miRNA-seq, and 12 miRNAs exhibited significantly differential expression patterns in D42_T3 versus D42_Con (TPM > 10, fold change ≥ 1.5 or ≤ 0.67 and q ≤ 0.05). These differentially expressed miRNAs targeted 3658 putative genes, which further enriched to 10 GO terms (q < 0.05). RNA-seq identified 146 differentially expressed genes (DEGs) in D42_T3 versus D42_Con (|log2 fold change| > 1 and q < 0.005), including pigmentation-related genes such as the receptor tyrosine-protein kinase erbB-3, pro-opiomelanocortin A, and melanotransferrin, and the growth-related gene somatotropin. These DEGs were significantly enriched to 15 GO terms and 8 KEGG pathways (q < 0.05), which included several sugar metabolic pathways (glycolysis/gluconeogenesis and the pentose phosphate pathway). Integrated analysis revealed that 26 overlapping genes between DEGs and mRNAs were targeted by miRNAs. Furthermore, seven mRNA-miRNA pairs exhibited reversed regulation patterns. This provides important clues to understand the role of thyroid hormones in flatfish pigmentation.

Transcriptomics analysis revealing candidate networks and genes for the body size sexual dimorphism of Chinese tongue sole (Cynoglossus semilaevis).

The Chinese tongue sole (Cynoglossus semilaevis) is a typical female heterogamete species that exhibits female-biased sexual size dimorphism, which has severely hindered the sustainable development of the species in aquaculture. In the present study, four important somatotropic and reproductive tissues including brain, pituitary, liver, and gonad from 15 females and 15 males were used for transcriptome analysis via RNA-seq. A mean of 37,533,991 high-quality clean reads was obtained from each library and 806, 1482, 818, and 14,695 differentially expressed genes in female and male were identified from the brain, pituitary, liver, and gonad, respectively (fold change ≥ 2 and q < 0.05). Enrichment analyses of GO terms and KEGG pathways showed that nucleic acid-binding transcription factor activity, G-protein-coupled receptor activity, MAPK signaling pathway, steroid biosynthesis, and neuroactive ligand-receptor interaction may be involved in the sexual growth differences. Furthermore, via weighted gene co-expression network analyses, two modules (yellowgreen and salmon4) were identified to be significantly positive-correlated with female-biased sexual size dimorphism. An illustrated network map drawn by these two modules enabled the identification of a series of hub genes, including nipped-B-like protein A (nipbla), transcriptional activator protein Pur-beta-like (purb), and BDNF/NT-3 growth factors receptor (ntrk2). Detailed functional investigation of these networks and hub genes will further improve our understanding of the underlying molecular mechanism of sexual size dimorphism in fish.

Two skin cell lines from wild-type and albino Japanese flounder (Paralichthys olivaceus): establishment, characterization, virus susceptibility, efficient transfection, and application to albinism study.

In order to provide an applicable cell platform to study fish pathology and skin pigmentation, two cell lines derived from skin tissues of wild-type and albino Japanese flounder were established and named JFSK_wt and JFSK_alb, respectively. These two cell lines were cultured for 45 passages within approximately 300 days. JFSK_wt and JFSK_alb cells were maintained in Dulbecco's Modified Eagle's Medium and Ham's F-12 Nutrient Mixture (DMEM/F12) supplemented with antibiotics, fetal bovine serum (FBS), 2-mercaptoethanol (2-Me), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and basic fibroblast growth factor (bFGF). The optimal growth temperature for JFSK_wt and JFSK_alb cells was 24 °C, and microscopically, the two cell lines were composed of fibroblast-like cells. Chromosomal analysis revealed that JFSK_wt and JFSK_alb cells had an identical diploid karyotype with 2n = 48t. Results of viral inoculation assays revealed that both cell lines shared similar patterns of viral susceptibility to nervous necrosis virus (NNV). High transfection efficiency was observed in JFSK_wt and JFSK_alb cells transfected with a pEGFP-N3 reporter plasmid and Cy3-siRNA. The detection of dermal marker Dermo-1 showed that these two cells were both derived from the dermis. Finally, three genes involved in the melanogenesis pathway, including adenylate cyclase type 5 (adcy5), microphthalmia-associated transcription factor (mitf), and endothelin B receptor (ednrb), were downregulated in JFSK_alb versus JFSK_wt cells. Thus, the two cell lines, sampled from skin tissue of wild-type and albino Japanese flounder will be not only helpful for fish pathogen research but also beneficial for albinism-related gene function studies.

Characterization of a low-density lipoprotein receptor, Lrp13, in Chinese tongue sole (Cynoglossus semilaevis) and medaka (Oryzias latipes).

As an important economic marine species cultured in China, Chinese tongue sole (Cynoglossus semilaevis) has interested us due to its sexual dimorphism and ZW/ZZ sex determination system. In a previous study, dmrt1 was identified as a dosage-dependent male-determining gene. In the present study, a female-specific expressed gene, cse0440, initially annotated as lrp1b-like, was identified from chromosome W of C. semilaevis. In view of the differences between cse0440 and lrp1b in terms of expression pattern, a phylogenetic analysis containing 85 LRP proteins was constructed and provided an evidence to re-annotate cse0440 as cseLRP13. In addition, two orthologues of cseLRP13 were separately identified from W and Z chromosomes: cseLRP13-W and cseLRP13-Z. The subsequent multiple sequence alignment and syntenic arrangements of LRP13 in C. semilaevis, Japanese medaka (Oryzias latipes), large yellow croaker (Larimichthys crocea), striped bass (Morone saxatilis), white perch (Morone americana) and Fugu rubripes (Takifugu rubripes) further supported this re-annotation. RT-PCR and in situ hybridization revealed that cselrp13 was exclusively expressed in the oocytes and follicles of ovaries. These results suggested that lrp13 may play important roles in female reproduction. In future, with the advancement of micromanipulation in flatfish, the detailed function of two lrp13 orthologues in C. semilaevis will be elucidated.

Combined behavioral and EEG evidence for the 70 Hz frequency selection of short-term spinal cord stimulation in disorders of consciousness.

OBJECTIVES: This study investigated the prognostic effect of electroencephalography (EEG) instant effects of single spinal cord stimulation (SCS) on clinical outcome in disorders of consciousness (DOC) and the time-dependent brain response during the recovery of consciousness prompted by SCS. METHODS: Twenty three patients with DOC underwent short-term SCS (stSCS) implantation operation. Then, all patients received the postoperative EEG test including EEG record before (T1) and after (T2) single SCS session. Subsequently, 2 weeks stSCS treatment was performed and revised coma recovery scale (CRS-R) and EEG data were collected. Finally, they were classified into effective and ineffective groups at 3-month follow-up (T6). RESULTS: The parietal-occipital (PO) connectivity and clustering coefficients (CC) in the beta band of the effective group at the 1 week after the treatment (T5) were found to be higher than preoperative assessment (T0). Correlation analysis showed that the change in beta CC at T1/T2 was correlated with the change in CRS-R at T0/T6. In addition, the change in PO connectivity and CC in the beta at T0/T5 were also correlated with the change in CRS-R at T0/T5. CONCLUSION: SCS may facilitate the recovery of consciousness by enhancing local information interaction in posterior brain regions. And the recovery can be predicted by beta CC in the EEG test.

An NIR-II-emitting type-I photosensitizer for efficient hypoxic tumor phototheranostics.

A small molecule-based NIR-II type-I photosensitizer (IT-IC) with a strong push-pull effect and good planar π-conjugated structure was synthesized. The IT-IC NPs exhibited strong light absorption, outstanding NIR-II fluorescence emission, excellent photothermal conversion and efficient type-I/II ROS generation, showing encouraging therapeutic outcomes for hypoxic tumors.

The molecular mechanisms underlying optical isomer arbutin permeating the skin: The molecular interaction between arbutin and skin components.

Arbutin, a typical optical isomer, has garnered widespread acclaim in the whitening cosmetics for its favorable efficacy and safety. However, the molecular mechanisms underlying α-arbutin and ß-arbutin permeating across the skin have not elucidated clearly yet. Herein we aimed to unveil how α-arbutin and ß-arbutin interacted with keratin or SC lipids, further demonstrating their relationship with their drug permeability. We found that α-arbutin displayed significantly higher drug accumulation into the porcine skin than ß-arbutin within 24 h through in vitro permeation test. Moreover, α-arbutin predominantly induced the alternations of secondary structure of amide II during the drug permeation, which was favorable for α-arbutin permeation. On the contrary, ß-arbutin exhibited an observable effect on the stretching vibration of SC lipids, possessing a significantly stronger mixing energy, binding energy and compatibility with ceramide (Cer) than that of α-arbutin, which ultimately restricted its permeation. Interestingly, free fatty acids and ceramides of the SC lipids specifically utilized its oxygen atom of carboxyl group to dock the arbutin molecules, enhancing their affinity with ß-arbutin, as confirmed by molecular simulation and 13Carbon Nuclear Magnetic Resonance. Nevertheless, a favorable compatibility between α-arbutin and keratin was observed. It was emphasized that the distinct spatial configuration and opposite optical rotation of arbutin was the leading factor impacting the intermolecular force between arbutin and the SC, and resulted in a diverse drug permeation. In cellular and in vivo skin pharmacokinetic studies, α-arbutin also possessed a higher cellular uptake and topical bioavailability than ß-arbutin. This study revealed the transdermal permeation mechanisms of optical isomer arbutin at the molecular levels, providing methodological reference for the investigations of permeation behaviors of other isomers with similar spatial configuration.

Sterility of Aedes albopictus by X-ray Irradiation as an Alternative to γ-ray Irradiation for the Sterile Insect Technique.

The mosquito Aedes albopictus can transmit various arboviral diseases, posing a severe threat to human health. As an environmentally friendly method, sterile insect technology (SIT) is considered an alternative to traditional methods such as chemical pesticides to control Ae. albopictus. In SIT, the sterility of male mosquitoes can be achieved by γ-ray or X-ray radiation. Compared with γ-rays, X-rays are easier to obtain, cheaper, and less harmful. However, there is a lack of comparative assessment of these two types of radiation for SIT under the same controlled conditions. Here, we compared the effects of X-ray and γ-ray radiation on the sterility of Ae. albopictus males under laboratory-controlled conditions. Neither type of radiation affected the number of eggs but significantly reduced the survival time and hatch rate. The same dose of γ-rays caused a higher sterility effect on males than X-rays but had a more significant impact on survival. However, X-rays could achieve the same sterility effect as γ-rays by increasing the radiation dose. For example, X-rays of 60 Gy induced 99% sterility, similar to γ-rays of 40 Gy. In the test of male mating competitiveness, the induced sterility and the male mating competitiveness index were also identical at the same release ratio (sterile males/fertile males). At a release ratio of 7:1, nearly 80% of eggs failed to hatch. Sterile males produced by X-ray and γ-ray radiation had similar male competitiveness in competition with field males. In conclusion, a higher dose of X-rays is required to achieve the same sterility effect, compared to γ-rays. When γ-rays are not readily available, high-dose X-rays can be used instead. This study provides data supporting the selection of more suitable radiation for the field release of sterile male mosquitoes.

Copper-catalyzed asymmetric propargylic substitution with salicylaldehyde-derived imine esters.

Copper-catalyzed asymmetric propargylic substitution with salicylaldehyde-derived imine esters and propargylic carbonates has been successfully realized, generating a wide range of chiral amino acid derivatives containing propargylic groups with excellent results (up to 95% yield and 94% ee). The ortho-hydroxy group of the salicylaldehyde-derived imine esters is crucial to increase the reactivity and stabilize the azomethine ylide, which may be due to the formation of an intramolecular hydrogen bond between the hydroxyl group and the imine group. A series of synthetic transformations were carried out to access other important chiral compounds, which displayed the synthetic versatility.

Sequencing introduced false positive rare taxa lead to biased microbial community diversity, assembly, and interaction interpretation in amplicon studies.

BACKGROUND: Increasing studies have demonstrated potential disproportionate functional and ecological contributions of rare taxa in a microbial community. However, the study of the microbial rare biosphere is hampered by their inherent scarcity and the deficiency of currently available techniques. Sample-wise cross contaminations might be introduced by sample index misassignment in the most widely used metabarcoding amplicon sequencing approach. Although downstream bioinformatic quality control and clustering or denoising algorithms could remove sequencing errors and non-biological artifact reads, no algorithm could eliminate high quality reads from sample-wise cross contaminations introduced by index misassignment, making it difficult to distinguish between bona fide rare taxa and potential false positives in metabarcoding studies. RESULTS: We thoroughly evaluated the rate of index misassignment of the widely used NovaSeq 6000 and DNBSEQ-G400 sequencing platforms using both commercial and customized mock communities, and observed significant lower (0.08% vs. 5.68%) fraction of potential false positive reads for DNBSEQ-G400 as compared to NovaSeq 6000. Significant batch effects could be caused by stochastically introduced false positive or false negative rare taxa. These false detections could also lead to inflated alpha diversity of relatively simple microbial communities and underestimated that of complex ones. Further test using a set of cow rumen samples reported differential rare taxa by different sequencing platforms. Correlation analysis of the rare taxa detected by each sequencing platform demonstrated that the rare taxa identified by DNBSEQ-G400 platform had a much higher possibility to be correlated with the physiochemical properties of rumen fluid as compared to NovaSeq 6000 platform. Community assembly mechanism and microbial network correlation analysis indicated that false positive or negative rare taxa detection could lead to biased community assembly mechanism and identification of fake keystone species of the community. CONCLUSIONS: We highly suggest proper positive/negative/blank controls, technical replicate settings, and proper sequencing platform selection in future amplicon studies, especially when the microbial rare biosphere would be focused.

Stereodivergent synthesis via iridium-catalyzed asymmetric double allylic alkylation of cyanoacetate.

Methods that enable the rapid construction of multiple C-C bonds using a single catalyst with high diastereo- and enantio-control are particularly valuable in organic synthesis. Here, we report an Ir-catalyzed double allylic alkylation reaction in which bisnucleophilic cyanoacetate reacted successionally with electrophilic π-allyl-Ir species, producing various pseudo-C 2-symmetrical cyanoacetate derivatives in high yield with excellent stereocontrol. More challenging sequential allylic alkylation/allylic alkylation with two distinct allylic carbonates that can deliver the corresponding products bearing three contiguous tertiary-quaternary-tertiary stereocenters was also developed by using a modified catalytic system, which is revealed to be associated with the quasi-dynamic kinetic resolution of the initially formed diastereomeric monoallylation intermediates. Notably, stereodivergence for this sequential process depending on a single iridium catalyst was successfully realized, and up to six stereoisomers could be predictably prepared by combining the appropriate enantiomer of the chiral ligand for the iridium catalyst and adjusting the adding sequence of two distinct allylic precursors.

SENP1 in the retrosplenial agranular cortex regulates core autistic-like symptoms in mice.

Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder, causing defects of social interaction and repetitive behaviors. Here, we identify a de novo heterozygous gene-truncating mutation of the Sentrin-specific peptidase1 (SENP1) gene in people with ASD without neurodevelopmental delay. We find that Senp1+/- mice exhibit core autistic-like symptoms such as social deficits and repetitive behaviors but normal learning and memory ability. Moreover, we find that inhibitory and excitatory synaptic functions are severely affected in the retrosplenial agranular (RSA) cortex of Senp1+/- mice. Lack of Senp1 leads to increased SUMOylation and degradation of fragile X mental retardation protein (FMRP), also implicated in syndromic ASD. Importantly, re-introducing SENP1 or FMRP specifically in RSA fully rescues the defects of synaptic function and autistic-like symptoms of Senp1+/- mice. Together, these results demonstrate that disruption of the SENP1-FMRP regulatory axis in the RSA causes autistic symptoms, providing a candidate region for ASD pathophysiology.

Catalytic Asymmetric Umpolung Allylation/2-Aza-Cope Rearrangement for the Construction of α-Tetrasubstituted α-Trifluoromethyl Homoallylic Amines.

A general protocol for the preparation of enantioenriched α-tetrasubstituted α-trifluoromethyl homoallylic amines is disclosed. Despite the significant challenge in stereoselectivity control, Ir-catalyzed asymmetric cascade umpolung allylation/2-aza-Cope rearrangement of trifluoromethylated fluorenone imines with allylic carbonates was realized with excellent efficiency and remarkable stereoselectivity. These were enabled by the suitable protective imino moiety and an unexpectedly exclusive E-geometrical imine of the allylation intermediate. This methodology is also applicable to facile access to chiral α-trisubstituted α-trifluoromethyl homoallylic amines in similarly high yield and stereoselectivity.

The benefits of Tai Chi and brisk walking for cognitive function and fitness in older adults.

The purpose of this study was to investigate the benefits of exercises with different cognitive demands for cognitive functions (Executive and non-Executive) in healthy older adults. A cross-sectional design was adopted. In total, 84 healthy older adults were enrolled in the study. They were categorized into the Tai Chi group (TG), the brisk walking group (BG) or the control group (CG). Each participant performed the Stroop task and a digit comparison task. The Stroop task included the following three conditions: a naming condition, an inhibition condition and an executive condition. There were two experimental conditions in the digit comparison task: the non-delay condition and the delay condition. The results indicated that participants of the TG and BG revealed significant better performance than the CG in the executive condition of cognitive tasks and fitness. There was no significant difference of reaction time (RT) and accuracy rate in the inhibition and delay conditions of cognitive tasks and fitness between the TG and BG. The TG showed shorter reaction time in the naming and the executive conditions, and more accurate in the inhibition conditions than the BG. These findings demonstrated that regular participation in brisk walking and Tai Chi have significant beneficial effects on executive function and fitness. However, due to the high cognitive demands of the exercise, Tai Chi benefit cognitive functions (Executive and non-Executive) in older adults more than brisk walking does. Further studies should research the underlying mechanisms at the behavioural and neuroelectric levels, providing more evidence to explain the effect of high-cognitive demands exercise on different processing levels of cognition.

The integrated analysis of RNA-seq and microRNA-seq depicts miRNA-mRNA networks involved in Japanese flounder (Paralichthys olivaceus) albinism.

Albinism, a phenomenon characterized by pigmentation deficiency on the ocular side of Japanese flounder (Paralichthys olivaceus), has caused significant damage. Limited mRNA and microRNA (miRNA) information is available on fish pigmentation deficiency. In this study, a high-throughput sequencing strategy was employed to identify the mRNA and miRNAs involved in P. olivaceus albinism. Based on P. olivaceus genome, RNA-seq identified 21,787 know genes and 711 new genes by transcripts assembly. Of those, 235 genes exhibited significantly different expression pattern (fold change ≥2 or ≤0.5 and q-value≤0.05), including 194 down-regulated genes and 41 up-regulated genes in albino versus normally pigmented individuals. These genes were enriched to 81 GO terms and 9 KEGG pathways (p≤0.05). Among those, the pigmentation related pathways-Melanogenesis and tyrosine metabolism were contained. High-throughput miRNA sequencing identified a total of 475 miRNAs, including 64 novel miRNAs. Furthermore, 33 differentially expressed miRNAs containing 13 up-regulated and 20 down-regulated miRNAs were identified in albino versus normally pigmented individuals (fold change ≥1.5 or ≤0.67 and p≤0.05). The next target prediction discovered a variety of putative target genes, of which, 134 genes including Tyrosinase (TYR), Tyrosinase-related protein 1 (TYRP1), Microphthalmia-associated transcription factor (MITF) were overlapped with differentially expressed genes derived from RNA-seq. These target genes were significantly enriched to 254 GO terms and 103 KEGG pathways (p<0.001). Of those, tyrosine metabolism, lysosomes, phototransduction pathways, etc., attracted considerable attention due to their involvement in regulating skin pigmentation. Expression patterns of differentially expressed mRNA and miRNAs were validated in 10 mRNA and 10 miRNAs by qRT-PCR. With high-throughput mRNA and miRNA sequencing and analysis, a series of interested mRNA and miRNAs involved in fish pigmentation are identified. And the miRNA-mRNA regulatory network also provides a solid starting point for further elucidation of fish pigmentation deficiency.