The structure of a 12-segmented dsRNA reovirus: New insights into capsid stabilization and organization.

Infecting a wide range of hosts, members of Reovirales (formerly Reoviridae) consist of a genome with different numbers of segmented double stranded RNAs (dsRNA) encapsulated by a proteinaceous shell and carry out genome replication and transcription inside the virion. Several cryo-electron microscopy (cryo-EM) structures of reoviruses with 9, 10 or 11 segmented dsRNA genomes have revealed insights into genome arrangement and transcription. However, the structure and genome arrangement of 12-segmented Reovirales members remain poorly understood. Using cryo-EM, we determined the structure of mud crab reovirus (MCRV), a 12-segmented dsRNA virus that is a putative member of Reovirales in the non-turreted Sedoreoviridae family, to near-atomic resolutions with icosahedral symmetry (3.1 Å) and without imposing icosahedral symmetry (3.4 Å). These structures revealed the organization of the major capsid proteins in two layers: an outer T = 13 layer consisting of VP12 trimers and unique VP11 clamps, and an inner T = 1 layer consisting of VP3 dimers. Additionally, ten RNA dependent RNA polymerases (RdRp) were well resolved just below the VP3 layer but were offset from the 5-fold axes and arranged with D5 symmetry, which has not previously been seen in other members of Reovirales. The N-termini of VP3 were shown to adopt four unique conformations; two of which anchor the RdRps, while the other two conformations are likely involved in genome organization and capsid stability. Taken together, these structures provide a new level of understanding for capsid stabilization and genome organization of segmented dsRNA viruses.

"Hard" Emulsion-Induced Interface Super-Assembly: A General Strategy for Two-Dimensional Hierarchically Porous Metal-Organic Framework Nanoarchitectures.

Two-dimensional (2D) hierarchically porous metal-organic framework (MOF) nanoarchitectures with tailorable meso-/macropores hold great promise for enhancing mass transfer kinetics, augmenting accessible active sites, and thereby boosting performance in heterogeneous catalysis. However, achieving the general synthesis of 2D free-standing MOF nanosheets with controllable hierarchical porosity and thickness remains a challenging task. Herein, we present an ingenious "hard" emulsion-induced interface super-assembly strategy for preparing 2D hierarchically porous UiO-66-NH2 nanosheets with highly accessible pore channels, tunable meso-/macropore sizes, and adjustable thicknesses. The methodology relies on transforming the geometric shape of oil droplet templates within appropriate oil-in-water emulsions from conventional zero-dimensional (0D) "soft" liquid spheres to 2D "hard" solid sheets below the oil's melting/freezing point. Subsequent surfactant exchange on the surface of 2D "hard" emulsions facilitates the heterogeneous nucleation and interfacial super-assembly of in situ formed mesostructured MOF nanocomposites, serving as structural units, in a loosely packed manner to produce 2D MOF nanosheets with multimodal micro/meso-/macroporous systems. Importantly, this strategy can be extended to prepare other 2D hierarchically porous MOF nanosheets by altering metal-oxo clusters and organic ligands. Benefiting from fast mass transfer and highly accessible Lewis acidic sites, the resultant 2D hierarchically porous UiO-66-NH2 nanosheets deliver a fabulous catalytic yield of approximately 96% on the CO2 cycloaddition of glycidyl-2-methylphenyl ether, far exceeding the yield of approximately 29% achieved using conventional UiO-66-NH2 microporous crystals. This "hard" emulsion-induced interface super-assembly strategy paves a new path toward the rational construction of elaborate 2D nanoarchitecture of hierarchical MOFs with tailored physicochemical properties for diverse potential applications.

Wavelength-polarization-multiplexed multichannel perfect vortex array generator based on dielectric metasurface.

The multi-channel perfect vortex (PV) array based on metasurface has important applications in optical communication, particle manipulation, quantum optics, and other fields due to its ultra-thin structure and excellent wavefront control ability. However, it is very challenging to utilize a single metasurface to simultaneously achieve independent channel PV arrays at different wavelengths with low crosstalk and low structural complexity. Here, we propose and design a single rectangular structured metasurface based on TiO2, achieving a multi-channel PV beam array with dual-wavelength and dual-polarization multiplexing. Simulation and experimental results show that when two orthogonal linearly polarized beams with wavelengths of 532 nm and 633 nm are incident on the metasurface, clear PV arrays with corresponding topological charge arrangements can be obtained in different diffraction regions of the same observation plane. The metasurface proposed in this article can enhance the channel capacity of a PV beam array through wavelength-polarization-multiplexing, thus having important application potential in spatial information transmission, high-dimensional information storage, and secure information encryption.

Negative Cross-Talk between TLR2/4-Independent AMPKα1 and TLR2/4-Dependent JNK Regulates S. pneumoniae-Induced Mucosal Innate Immune Response.

Streptococcus pneumoniae is major cause of otitis media (OM) and life-threatening pneumonia. Overproduction of mucin, the major component of mucus, plays a critical role in the pathogenesis of both OM and pneumonia. However, the molecular mechanisms underlying the tight regulation of mucin upregulation in the mucosal epithelium by S. pneumoniae infection remain largely unknown. In this study, we show that S. pneumoniae pneumolysin (PLY) activates AMP-activated protein kinase α1 (AMPKα1), the master regulator of energy homeostasis, which is required for S. pneumoniae-induced mucin MUC5AC upregulation in vitro and in vivo. Moreover, we found that PLY activates AMPKα1 via cholesterol-dependent membrane binding of PLY and subsequent activation of the Ca2+- Ca2+/calmodulin-dependent kinase kinase ß (CaMKKß) and Cdc42-mixed-lineage protein kinase 3 (MLK3) signaling axis in a TLR2/4-independent manner. AMPKα1 positively regulates PLY-induced MUC5AC expression via negative cross-talk with TLR2/4-dependent activation of MAPK JNK, the negative regulator of MUC5AC expression. Moreover, pharmacological inhibition of AMPKα1 suppressed MUC5AC induction in the S. pneumoniae-induced OM mouse model, thereby demonstrating its therapeutic potential in suppressing mucus overproduction in OM. Taken together, our data unveil a novel mechanism by which negative cross-talk between TLR2/4-independent activation of AMPKα1 and TLR2/4-dependent activation of JNK tightly regulates the S. pneumoniae PLY-induced host mucosal innate immune response.

Tetrameric Acetyl-CoA Acetyltransferase 1 Is Important for Tumor Growth.

Mitochondrial acetyl-CoA acetyltransferase 1 (ACAT1) regulates pyruvate dehydrogenase complex (PDC) by acetylating pyruvate dehydrogenase (PDH) and PDH phosphatase. How ACAT1 is "hijacked" to contribute to the Warburg effect in human cancer remains unclear. We found that active, tetrameric ACAT1 is commonly upregulated in cells stimulated by EGF and in diverse human cancer cells, where ACAT1 tetramers, but not monomers, are phosphorylated and stabilized by enhanced Y407 phosphorylation. Moreover, we identified arecoline hydrobromide (AH) as a covalent ACAT1 inhibitor that binds to and disrupts only ACAT1 tetramers. The resultant AH-bound ACAT1 monomers cannot reform tetramers. Inhibition of tetrameric ACAT1 by abolishing Y407 phosphorylation or AH treatment results in decreased ACAT1 activity, leading to increased PDC flux and oxidative phosphorylation with attenuated cancer cell proliferation and tumor growth. These findings provide a mechanistic understanding of how oncogenic events signal through distinct acetyltransferases to regulate cancer metabolism and suggest ACAT1 as an anti-cancer target.

Caspase-3-Triggered Intracellular Gadolinium Nanoparticle Formation for T1-Weighted Magnetic Resonance Imaging of Apoptosis In Vivo.

Apoptosis, with a hallmark of upregulated protease Caspase-3, has been frequently imaged with various probes to reveal the therapeutic efficiencies of different drugs. However, activatable molecular probes with programmable self-assembling behaviors that enable enhanced T1-weighted magnetic resonance imaging (MRI) of apoptosis remain scarce. Herein, taking advantage of a CBT-Cys click reaction, we rationally designed a Caspase-3-activatable self-assembling probe Ac-Asp-Glu-Val-Asp-Cys(StBu)-Lys(DOTA(Gd))-CBT (DEVDCS-Gd-CBT) for apoptosis imaging in vivo. After Caspase-3 cleavage in apoptotic cells, DEVDCS-Gd-CBT underwent CBT-Cys click reaction to form a cyclic dimer, which self-assembled into Gd nanoparticles. With this probe, enhanced T1-weighted MR images of apoptosis were achieved at low magnetic fields in vitro, in cis-dichlorodiamineplatinum-induced apoptotic cells and in tail-amputation-simulated apoptotic zebrafish. We anticipate that the smart probe DEVDCS-Gd-CBT could be applied for T1-weighted MRI of apoptosis-related diseases in the clinic in the future.

Enhanced Photoacoustic Imaging of Urokinase-Type Plasminogen Activator Activity in Tumors.

Photoacoustic (PA) imaging of urokinase-type plasminogen activator (uPA) activity in vivo holds high promise for early diagnosis of breast cancer. Molecular probes with resisted fluorescence (FL) emission for enhanced PA signals of uPA activity have not been reported. Herein, we proposed a molecular probe Cbz-Gly-Gly-Arg-Phe-Phe-IR775 (Z-GGRFF-IR775) which, upon uPA cleavage, assembled into nanoparticles FF-IR775-NP with quenched fluorescence but enhanced PA signals. Experimental results validated that, upon uPA activation, Z-GGRFF-IR775 exhibited 4.7-fold, 4.1-fold, and 2.9-fold higher PA signals over those in uPA inhibitor-treated control groups in vitro, in MDA-MB-231 cells, and in a tumor-bearing mouse model, respectively. We anticipate that this probe could be applied for highly sensitive PA imaging of uPA activity in early stage malignant tumors in the near future.

Dual-wavelength hologram of high transmittance metasurface.

In this work, a simple dielectric metasurface hologram is proposed and designed by combining the electromagnetic vector analysis method and the immune algorithm, which can realize the holographic display of dual wavelength orthogonal-linear polarization light in visible light band, solve the problem of low efficiency of the traditional design method of metasurface hologram, and effectively improve the diffraction efficiency of metasurface hologram. The titanium dioxide metasurface nanorod based on rectangular structure is optimized and designed. When the x-linear polarized light with wavelength of 532 nm and y-linear polarized light with wavelength of 633 nm are incident on the metasurface respectively, different display output images with low cross-talk can be obtained on the same observation plane, and the transmission efficiencies of x-linear and y-linear polarized light are as high as 68.2% and 74.6% respectively in simulation. Then the metasurface is fabricated by Atomic Layer Deposition method. The experimental results are consistent with the design results, which proves that the metasurface hologram designed by this method can completely realize the feasibility of wavelength and polarization multiplexing holographic display, and has potential application value in holographic display, optical encryption, anti-counterfeiting, data storage and other fields.

Long-infrared dual-wavelength linear-polarization-multiplexed confocal metalens based on an all-silicon dielectric.

The metalens has vast applications in biomedicine and industrial manufacturing due to their ultrathin structure and vital ability to manipulate the properties of light waves for long-infrared systems. However, it is difficult for metalens to achieve the confocal function with high focusing efficiency, wide wavelength bandwidth, and low structural complexity. Here, we propose and experimentally demonstrate an all-silicon dielectric metalens composed of arrays of minimalist meta-atoms with a single rectangular nanopillar arranged on a periodic square lattice substrate, which realizes the confocal function of the orthogonal-linear-polarized light with wavelengths of 10.6 µm and 9.3 µm, with focusing efficiencies of 64.94% and 60.03%, respectively. Also, it reveals nearly the diffraction-limited focusing performance. In addition, the metalens can realize precise long-infrared thermal imaging. Moreover, the proposed metalens is compatible with the standard complementary metal oxide semiconductor processes, which can effectively reduce the manufacturing cost and provide a feasible solution for developing planar integrated multifunctional micro-nanophotonic devices in the long-infrared field.

A coupled awareness-epidemic model on a multi-layer time-varying network.

Social interactions have become more complicated and changeable under the influence of information technology revolution. We, thereby, propose a multi-layer activity-driven network with attractiveness considering the heterogeneity of activated individual edge numbers, which aims to explore the role of heterogeneous behaviors in the time-varying network. Specifically, three types of individual behaviors are introduced: (i) self-quarantine of infected individuals, (ii) safe social distancing between infected and susceptible individuals, and (iii) information spreading of aware individuals. Epidemic threshold is theoretically derived in terms of the microscopic Markov chain approach and the mean-field approach. The results demonstrate that performing self-quarantine and maintaining safe social distance can effectively raise the epidemic threshold and suppress the spread of diseases. Interestingly, individuals' activity and individuals' attractiveness have an equivalent effect on epidemic threshold under the same condition. In addition, a similar result can be obtained regardless of the activated individual edge numbers. The epidemic outbreak earlier in a situation of the stronger heterogeneity of activated individual edge numbers.

Characterization of the genomic and transcriptional structure of chicken NRG4 gene.

Neuregulin 4 (NRG4) is an important adipocytokine, which plays crucial roles in maintaining energy balance, regulating glucose and lipid metabolism, and preventing non-alcoholic fatty liver disease in mammals. At present, the genomic organization, transcript and protein isoforms of human NRG4 gene have been fully explored. Previous studies in our laboratory have shown that the NRG4 gene is expressed in chicken adipose tissue, but the chicken NRG4 (cNRG4) genomic structure, transcript and protein isoforms are still unknown. To this end, in this study, the genomic and transcriptional structure of the cNRG4 gene were systematically investigated using rapid amplification of cDNA ends (RACE) and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that the coding region (CDS) of the cNRG4 gene was small, but it had a very complex transcriptional structure characterized by multiple transcription start sites, alternative splicing, intron retention, cryptic exons, and alternative polyadenylation, thus leading to production of four 5?UTR isoforms (cNRG4 A, cNRG4 B, cNRG4 C, and cNRG4 D) and six 3?UTR isoforms (cNRG4 a, cNRG4 b, cNRG4 c, cNRG4 d, cNRG4 e, and cNRG4 f) of the cNRG4 gene. The cNRG4 gene spanned 21,969 bp of genomic DNA (Chr.10:3,490,314~3,512,282) and consisted of 11 exons and 10 introns. Compared with the cNRG4 gene mRNA sequence (NM_001030544.4), two novel exons and one cryptic exon of the cNRG4 gene were identified in this study. Bioinformatics analysis, RT-PCR, cloning and sequencing analysis showed that the cNRG4 gene could encode three protein isoforms (cNRG4-1, cNRG4-2 and cNRG4-3). This study lays a foundation for further research on the function and regulation of the cNRG4 gene.

[A cross-sectional study of enterovirus nucleic acid test with throat swabs for term late neonates during coronavirus disease 2019]. / æ–°åž‹å† çŠ¶ç— æ¯’æ„ŸæŸ“æœŸé—´è¶³æœˆçš„æ™šæœŸæ–°ç”Ÿå„¿å’½æ‹­å­è‚ é“ç— æ¯’æ ¸é ¸æ£€æµ‹æ¨ªæ–­é¢ç ”ç©¶.

OBJECTIVES: To investigate the positive rate of enterovirus (EV) nucleic acid in throat swabs of term late neonates hospitalized during the coronavirus disease 2019 (COVID-19) epidemic and the clinical characteristics of the neonates. METHODS: A single-center cross-sectional study was performed on 611 term late infants who were hospitalized in the neonatal center from October 2020 to September 2021. Throat swabs were collected on admission for coxsackie A16 virus/EV71/EV universal nucleic acid testing. According to the results of EV nucleic acid test, the infants were divided into a positive EV nucleic acid group (8 infants) and a negative EV nucleic acid group (603 infants). Clinical features were compared between the two groups. RESULTS: Among the 611 neonates, 8 tested positive for EV nucleic acid, with a positive rate of 13.1‰, among whom 7 were admitted from May to October. There was a significant difference in the proportion of infants contacting family members with respiratory infection symptoms before disease onset between the positive and negative EV nucleic acid groups (75.0% vs 10.9%, P<0.001). There were no significant differences between the two groups in demographic data, clinical symptoms, and laboratory test results (P>0.05). CONCLUSIONS: There is a certain proportion of term late infants testing positive for EV nucleic acid in throat swabs during the COVID-19 epidemic, but the proportion is low. The clinical manifestations and laboratory test results of these infants are non-specific. Transmission among family members might be an important cause of neonatal EV infection.

Hedgehogs as Amplifying Hosts of Severe Fever with Thrombocytopenia Syndrome Virus, China.

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tickborne bandavirus mainly transmitted by Haemaphysalis longicornis ticks in East Asia, mostly in rural areas. As of April 2022, the amplifying host involved in the natural transmission of SFTSV remained unidentified. Our epidemiologic field survey conducted in endemic areas in China showed that hedgehogs were widely distributed, had heavy tick infestations, and had high SFTSV seroprevalence and RNA prevalence. After experimental infection of Erinaceus amurensis and Atelerix albiventris hedgehogs with SFTSV, we detected robust but transitory viremias that lasted for 9-11 days. We completed the SFTSV transmission cycle between hedgehogs and nymph and adult H. longicornis ticks under laboratory conditions with 100% efficiency. Furthermore, naive H. longicornis ticks could be infected by SFTSV-positive ticks co-feeding on naive hedgehogs; we confirmed transstadial transmission of SFTSV. Our study suggests that the hedgehogs are a notable wildlife amplifying host of SFTSV in China.

The impact of the global and local awareness diffusion on epidemic transmission considering the heterogeneity of individual influences.

In this paper, we propose a coupled awareness-epidemic spreading model considering the heterogeneity of individual influences, which aims to explore the interaction between awareness diffusion and epidemic transmission. The considered heterogeneities of individual influences are threefold: the heterogeneity of individual influences in the information layer, the heterogeneity of individual influences in the epidemic layer and the heterogeneity of individual behavioral responses to epidemics. In addition, the individuals' receptive preference for information and the impacts of individuals' perceived local awareness ratio and individuals' perceived epidemic severity on self-protective behavior are included. The epidemic threshold is theoretically established by the microscopic Markov chain approach and the mean-field approach. Results indicate that the critical local and global awareness ratios have two-stage effects on the epidemic threshold. Besides, either the heterogeneity of individual influences in the information layer or the strength of individuals' responses to epidemics can influence the epidemic threshold with a nonlinear way. However, the heterogeneity of individual influences in the epidemic layer has few effect on the epidemic threshold, but can affects the magnitude of the final infected density.

Molecular characterization and functional analysis of TRAF6 in the spotted sea bass (Lateolabrax maculatus).

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a crucial adapter protein in the toll-like receptor signaling pathway that triggers downstream molecules involved in innate immunity. Although TRAF6 has been well studied in mammals, the molecular information and function of TRAF6 in fish is still limited. Here, we identified and analyzed a TRAF6 homolog (LmTRAF6) from the spotted sea bass (Lateolabrax maculatus). Similar to its counterparts in mammals and other fish species, LmTRAF6 shares the domain topology containing one N-terminal RING, two TRAF-type zinc fingers, a coiled-coil region and a C-terminal MATH domain. Despite a sequence similarity of 60% with mammalian TRAF6s, LmTRAF6 shares higher similarities with teleost homologs (~68%-93%). The coding region of LmTRAF6 gene contains seven exons and six introns, which is consistent to the genetic organization in grouper and rock bream, but not in zebrafish, common carp and tetrapods (the sixth intron was lost resulting in a combined exon). Quantitative real-time polymerase chain reaction analysis revealed that LmTRAF6 transcripts were ubiquitously expressed in all tested tissues and upregulated after Vibrio. harveyi and S. agalactiae infection. LmTRAF6 could assist HEK293T cells to survive by inhibiting apoptosis under both V. harveyi and S. agalactiae stimulation. Intracellular localization showed that LmTRAF6 was localized mainly in the cytoplasm. Overexpression of wild-type (WT) LmTRAF6 and the truncated form of △MATH increased the ability of NF-κB in HEK293T cells, whereas truncations, including the △RING and △coiled-coil domain, did not significantly activate NF-κB, indicating that the RING finger and coiled-coil domain play crucial roles in downstream signal transduction. In addition, overexpression of LmTRAF6-WT significantly increased the activation of NF-κB in HEK293T cells under V. harveyi and S. agalactiae stimulation. These results suggest that LmTRAF6 activates NF-κB and plays a potential role in the immune defense system against bacterial infection.

Horizontal gene transfer contributes to virulence and antibiotic resistance of Vibrio harveyi 345 based on complete genome sequence analysis.

BACKGROUND: Horizontal gene transfer (HGT), which is affected by environmental pollution and climate change, promotes genetic communication, changing bacterial pathogenicity and drug resistance. However, few studies have been conducted on the effect of HGT on the high pathogenicity and drug resistance of the opportunistic pathogen Vibrio harveyi. RESULTS: V. harveyi 345 that was multidrug resistant and infected Epinephelus oanceolutus was isolated from a diseased organism in Shenzhen, Southern China, an important and contaminated aquaculture area. Analysis of the entire genome sequence predicted 5678 genes including 487 virulence genes contributing to bacterial pathogenesis and 25 antibiotic-resistance genes (ARGs) contributing to antimicrobial resistance. Five ARGs (tetm, tetb, qnrs, dfra17, and sul2) and one virulence gene (CU052_28670) on the pAQU-type plasmid p345-185, provided direct evidence for HGT. Comparative genome analysis of 31 V. harveyi strains indicated that 217 genes and 7 gene families, including a class C beta-lactamase gene, a virulence-associated protein D gene, and an OmpA family protein gene were specific to strain V. harveyi 345. These genes could contribute to HGT or be horizontally transferred from other bacteria to enhance the virulence or antibiotic resistance of 345. Mobile genetic elements in 71 genomic islands encoding virulence factors for three type III secretion proteins and 13 type VI secretion system proteins, and two incomplete prophage sequences were detected that could be HGT transfer tools. Evaluation of the complete genome of V. harveyi 345 and comparative genomics indicated genomic exchange, especially exchange of pathogenic genes and drug-resistance genes by HGT contributing to pathogenicity and drug resistance. Climate change and continued environmental deterioration are expected to accelerate the HGT of V. harveyi, increasing its pathogenicity and drug resistance. CONCLUSION: This study provides timely information for further analysis of V. harveyi pathogenesis and antimicrobial resistance and developing pollution control measurements for coastal areas.

Chaperone-mediated autophagy: Advances from bench to bedside.

Protein homeostasis or proteostasis is critical for proper cellular function and survival. It relies on the balance between protein synthesis and degradation. Lysosomes play an important role in degrading and recycling intracellular components via autophagy. Among the three types of lysosome-based autophagy pathways, chaperone-mediated autophagy (CMA) selectively degrades cellular proteins with KFERQ-like motif by unique machinery. During the past several years, significant advances have been made in our understanding of how CMA itself is modulated and what physiological and pathological processes it may be involved in. One particularly exciting discovery is how other cellular stress organelles such as ER signal to CMA. As more proteins are identified as CMA substrates, CMA function has been associated with an increasing number of important cellular processes, organelles, and diseases, including neurodegenerative diseases. Here we will summarize the recent advances in CMA biology, highlight ER stress-induced CMA, and discuss the role of CMA in diseases.

Large mode area microstructured fiber supporting 56 super-OAM modes.

In this paper, a kind of super-mode orbital angular momentum microstructured fiber (SM-OAM-MSF) is proposed. By introducing 20 Ge-doped equiangular cylindrical inclusions in the ring-core region, mode coupling mechanism is employed in the formation of super-OAM (SOAM) modes. Specifically, the double degenerated out-of-phase SMs are first generated by the coupling of individual core mode, then the quadruple degenerated SOAM modes are formed by combining two components of the out-of-phase SMs with a phase difference of ±π/2. Theoretical analysis and numerical results reveal that the effective index difference (Δneff) between adjacent out-of-phase SM groups are strongly influenced by the parameters of the individual core except the ring-core's width. Therefore, large mode area and SOAM modes' index separation larger than 1.0×10-4 can be achieved simultaneously in our proposed SM-OAM-MSF. Through careful fiber design, HE1,1 and HE2,1 are used in the formation of SMs and SOAM modes. Simulations show that all the nine SOAM groups originating from HE1,1 mode and the first five SOAM groups stemming from normal coupling of HE2,1 mode can be supported above 1.0µm, that are 56 SOAM modes in total. The highest purity is 99.86% for SOAM±2,1±,5 mode. And the maximum mode area (Aeff) value reaches up to 638.88µm2 at 1.55µm, which is nearly eight times larger compared to that of conventional ring-core MSFs.

DRAM1 regulates autophagy and cell proliferation via inhibition of the phosphoinositide 3-kinase-Akt-mTOR-ribosomal protein S6 pathway.

BACKGROUND: Macroautophagy (hereafter autophagy) is a tightly regulated process that delivers cellular components to lysosomes for degradation. Damage-regulated autophagy modulator 1 (DRAM1) induces autophagy and is necessary for p53-mediated apoptosis. However, the signalling pathways regulated by DRAM1 are not fully understood. METHODS: HEK293T cells were transfected with FLAG-DRAM1 plasmid. Autophagic proteins (LC3 and p62), phosphorylated p53 and the phosphorylated proteins of the class I PI3K-Akt-mTOR-ribosomal protein S6 (rpS6) signalling pathway were detected with Western blot analysis. Cellular distribution of DRAM1 was determined with immunostaining. DRAM1 was knocked down in HEK293T cells using siRNA oligos which is confirmed by quantitative RT-PCR. Cells were serum starved for 18 h after overexpression or knockdown of DRAM1 to decrease the rpS6 activity to the basal level, and then the cells were stimulated with insulin growth factor, epidermal growth factor or serum. rpS6 phosphorylation and rpS6 were detected with Western blotting. Similarly, after overexpression or knockdown of DRAM1, phosphorylation of IGF-1Rß and IGF-1R were examined with Western blotting. Cell viability was determined with CCK-8 assay and colony formation assay. Finally, human cancer cells Hela, SW480, and HCT116 were transfected with the FLAG-DRAM1 plasmid and phosphorylated rpS6 and rpS6 were detected with Western blot analysis. RESULTS: DRAM1 induced autophagy and inhibited rpS6 phosphorylation in an mTORC1-dependent manner in HEK293T cells. DRAM1 didn't affect the phosphorylated and total levels of p53. Furthermore, DRAM1 inhibited the activation of the PI3K-Akt pathway stimulated with growth factors or serum. DRAM1 was localized at the plasma membrane and regulate the phosphorylation of IGF-1 receptor. DRAM1 decreased cell viability and colony numbers upon serum starvation. Additionally, DRAM1 inhibited rpS6 phosphorylation in several human cancer cells. CONCLUSIONS: Here we provided evidence that DRAM1 inhibited rpS6 phosphorylation in multiple cell types. DRAM1 inhibited the phosphorylation of Akt and the activation of Akt-rpS6 pathway stimulated with growth factors and serum. Furthermore, DRAM1 regulated the activation of IGF-1 receptor. Thus, our results identify that the class I PI3K-Akt-rpS6 pathway is regulated by DRAM1 and may provide new insight into the potential role of DRAM1 in human cancers.

Molecular characterization and functional analysis of Akirin from black tiger shrimp (Penaeus monodon).

Akirin, which are members of the NF-κB signaling pathway, play critical roles in regulating the expression of antimicrobial peptides. In the present study, the Akirin gene from Penaeus monodon was identified from a transcriptome database and designated as PmAkirin. The complete sequence of the PmAkirin cDNA was 1508 bp, encoding a protein of 213 amino acids, and it showed 99% amino acid identity to the Litopenaeus vannamei Akirin. Two predicted nuclear localization signals (NLSs) were found, and the amino acid sequence alignments showed that PmAkirin was highly conserved at the N-terminus and C-terminus. PmAkirin expression was found to be the highest in the hemolymph, followed by the heart, gill, stomach, hepatopancreas, intestine, and muscle. When challenged with Vibrio parahaemolyticus infection, the PmAkirin mRNA and three antimicrobial peptides (AMPs: PmALF2, PmALF3, and PmCrus4) were upregulated. However, another five AMPs (PmALF6, PmCrus1, PmPEN3a, PmPEN3b, and PmPEN5) were downregulated by V. parahaemolyticus infection. Silencing PmAkirin by dsRNA significantly decreased the expression of the eight AMPs, which lead to an increase in the blood concentration of V. parahaemolyticus and higher mortality in the shrimp. In contrast, the overexpression of PmAkirin significantly increased the expression of the eight AMPs, which led to a reduction in the blood concentration of V. parahaemolyticus and promoted the survival of the shrimp. Taken together, we concluded that PmAkirin plays an important role in regulating the expression of AMPs in black tiger shrimp to defend against V. parahaemolyticus infection.