Reduction of Z alpha-1 antitrypsin polymers in human iPSC-hepatocytes and mice by LRRK2 inhibitors.

Alpha-1 antitrypsin deficiency (A1ATD) is a life-threatening condition caused by inheritance of the SERPINA1 'Z' genetic variant (PiZ) driving AAT protein misfolding in hepatocytes. There remain no approved medicines for this disease. Here, we report the results of a small molecule screen performed in patient derived iPSC-hepatocytes that identified Leucine-rich repeat kinase-2 (LRRK2) as a potentially new therapeutic target. Of the commercially available LRRK2 inhibitors tested, we identified CZC-25146, a candidate with favorable pharmacokinetic properties, as being capable of reducing polymer load, increasing normal AAT secretion, and reducing inflammatory cytokines in both cells and PiZ mice. Mechanistically, this effect was achieved through induction of autophagy. Our findings support the use of CZC-25146 and LRRK2 inhibitors in hepatic proteinopathy research and their further investigation as novel therapeutic candidates for A1ATD.

Dual colorimetric and near-infrared fluorescence probe for Hg2+ detection and cell imaging.

Hg2+ in the environment endangers human health, and a convenient monitoring method is needed for the detection of Hg2+. In this study, we constructed a dual colorimetric near-infrared fluorescent probe (E)-2-(3-(3-(1,3-dithian-2-yl)-4-hydroxystyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile (YF-Hg), based on the malononitrile isophorone. YF-Hg can detect Hg2+ rapidly and sensitively, with fluorescence emission in the near-infrared region (659 nm) with an obvious color change from violet to red in the visible light range. In addition, the low toxicity and large Stokes shift (191 nm) of YF-Hg also suggest that it is a potential tool for live-cell fluorescence imaging.

Human iPSC-derived hepatocyte system models cholestasis with tight junction protein 2 deficiency.

Background & Aims: The truncating mutations in tight junction protein 2 (TJP2) cause progressive cholestasis, liver failure, and hepatocyte carcinogenesis. Due to the lack of effective model systems, there are no targeted medications for the liver pathology with TJP2 deficiency. We leveraged the technologies of patient-specific induced pluripotent stem cells (iPSC) and CRISPR genome-editing, and we aim to establish a disease model which recapitulates phenotypes of patients with TJP2 deficiency. Methods: We differentiated iPSC to hepatocyte-like cells (iHep) on the Transwell membrane in a polarized monolayer. Immunofluorescent staining of polarity markers was detected by a confocal microscope. The epithelial barrier function and bile acid transport of bile canaliculi were quantified between the two chambers of Transwell. The morphology of bile canaliculi was measured in iHep cultured in the Matrigel sandwich system using a fluorescent probe and live-confocal imaging. Results: The iHep differentiated from iPSC with TJP2 mutations exhibited intracellular inclusions of disrupted apical membrane structures, distorted canalicular networks, altered distribution of apical and basolateral markers/transporters. The directional bile acid transport of bile canaliculi was compromised in the mutant hepatocytes, resembling the disease phenotypes observed in the liver of patients. Conclusions: Our iPSC-derived in vitro hepatocyte system revealed canalicular membrane disruption in TJP2 deficient hepatocytes and demonstrated the ability to model cholestatic disease with TJP2 deficiency to serve as a platform for further pathophysiologic study and drug discovery. Lay summary: We investigated a genetic liver disease, progressive familial intrahepatic cholestasis (PFIC), which causes severe liver disease in newborns and infants due to a lack of gene called TJP2. By using cutting-edge stem cell technology and genome editing methods, we established a novel disease modeling system in cell culture experiments. Our experiments demonstrated that the lack of TJP2 induced abnormal cell polarity and disrupted bile acid transport. These findings will lead to the subsequent investigation to further understand disease mechanisms and develop an effective treatment.

[Preliminary observation on imbalance degree of impedance in extracellular fluid of cells in the twelve meridians in healthy subjects].

OBJECTIVE: To calculate the imbalance degree (IBD) of left-right meridian (IBD-LRM), IBD of exterior-interior meridian (IBD-EIM) and IBD of hand-foot meridians (IBD-HFM) of impedance in extracellular fluid of cells in twelve meridians of healthy subjects, so as to provide foundation for meridian diagnosis. METHODS: A total of 31 healthy volunteers were enrolled and bioelectrical impedance spectroscopy (BIS) was applied. The constant current (from 1 to 100 kHz, 200 µA) was connected into the bilateral twelve meridians through two excitation electrodes with a distance of 10 cm. Two measuring electrodes, with an interval of 5 cm, were set in between the two excitation electrodes to collect the voltage amplitude and phase. The Cole-Cole curve fitting was used to calculate the impedance of extracellular fluid of cells in the twelve meridians; the IBD-LRM, IBD-EIM and IBD-HFM as well as their absolute values were calculated. RESULTS: The impedance of extracellular fluid in the left side was higher than that in right side in the large intestine meridian, the small intestine meridian and the bladder meridian (P<0.05, P<0.01). The mean value of IBD-LRM of extracellular fluid was (4.0±1.4) %; the mean value of absolute value of IBD-LRM was (15.0±1.1) %; the maximum absolute value of IBD-LRM was the bladder meridian. The mean value of IBD-EIM was (3.3±1.0) %; the mean value of absolute value of IBD-EIM was (17.9±1.6) %; the maximum absolute value of IBD-EIM was the bladder meridian and the kidney meridian. The impedance of extracellular fluid of hand jueyin meridian, hand taiyin meridian and hand shaoyin meridian were lower than those of foot meridians. The mean value of IBD-HFM was (-2.6±1.1) %; the mean value of absolute value of IBD-HFM was (19.7±1.7) %; the maximum absolute value of IBD-HFM was shaoyang meridian; the imbalance of yin meridians was greater than yang meridians. There were significant differences in impedance of extracellular fluid between left and right and between hands and feet (P<0.05, P<0.01). CONCLUSION: The extracellular fluid of left-right meridians of healthy subjects is different, but the absolute value of IBD is low; the mean value of exterior meridian and interior meridian is very close, and the absolute value of IBD is medium; the impedance of the foot meridians are greater than the hand meridians, and the absolute value of IBD is relatively high.

[Comparison of Extracellular and Intracellular Fluid Distribution Between Stomach Meridian or Gallbladder Meridian and Their Neighboring Tissues of Lower Leg in Healthy Volunteers].

OBJECTIVE: To compare differences of extracellular fluid impedance (Re) and intracellular fluid impedance (Ri) between the Stomach(ST) Meridian or Gallbladder(GB) Meridian and their neighboring non-meridian sites of the left lower leg at the same level, so as to explore the distribution characteristics of body fluid in the meridian. METHODS: Sixteen healthy volunteers were enrolled in the present study. The Re and Ri were detected by using Ag/AgCl electrodes and a digital lock-in amplifier. The measuring electrodes (at an interval of about 3 cm) were separately fixed to the skin sites covering the running courses of the ST Meridian (in the lateral interspace of the anterior tibial muscle)and the GB Meridian (in the interspace of the anterior edge of the fibula), and the excitation electrodes (at an interval of about 9 cm) respectively fixed to the skin sites covering the anterior tibial muscle and the interspace between the anterior tibial muscle and the tibia (about 2 cm and 5 cm lateral to the ST and GB meridians, and about 3－4 cm and 6－8 cm lateral to the ST and GB meridians, respectively). A 100 µA constant current with frequencies from 1 kHz to 100 kHz delivered via an excitation electrode was applied to the site (control spots of the ST Meridian), and signals of the voltage amplitude and phase difference of the tissues fed to the lock-in amplifier via the measuring electrode were collected, followed by measuring those of the GB Meridian and control sites. The circumference of the lower leg around the two excitation and measuring electrodes was measured. Then the cole-cole curve fitting was performed to calculate the Ri and Re, as well as the intracellular fluid resistivity (ρi) and extracellular fluid resistivity (ρe) of the ST and GB meridians, the related muscles and interspace lateral to ST or GB (ST/GB) meridians at the same level. RESULTS: The Ri and Re (Ω) values of the ST, GB, the muscle lateral to ST/GB and the interspace lateral to ST/GB were 19.1±1.3 and 28.3±1.4, 15.8±1.9 and 25.7±2.0, 19.6±1.3 and 31.3±1.6, and 19.4±1.2 and 32.4±1.6, respectively. The Re values were significantly lower at the ST and GB meridians than at the muscle lateral to and the interspace lateral to both meridians (P<0.05). The ρi and ρe values (Ω•cm) of the ST, GB, the muscle lateral to and the interspace lateral to ST/GB were 658.9±78.5 and 953.8±75.3, 528.0±90.1 and 833.9±101.7, 669.9±71.8 and 1 059.8±86.0, 655.9±64.8 and 1 099.3±93.3, respectively. The ρi and ρe values were significantly lower at the GB Meridian Than at the other three locntions, and the ρe value of ST Meridian was significantly lower than those of the muscle lateral to and the interspace lateral to ST/GB meridians (P<0.01)．. CONCLUSION: The Ri, Re, ρi and ρe values of the ST and GB meridians are significantly lower than those of their neighboring tissues at the same levels of the lower leg, suggesting a more extracellular fluid in the meridian running course and providing evidence for our speculation that the meridian is a hydraulic resistance channel.

[Preliminary Observation on Imbalance Degree of Blood Perfusion on the Twelve Yuan-source Points in Healthy Subjects].

OBJECTIVE: To lay a foundation for meridian diagnosis by measuring the blood perfusion (BP) on yuan-source points of twelve meridians and calculating the normal range of imbalance degree (IBD) of left and right meridian (IBD-LRM), IBD of exterior-interior meridian (IBD-EIM) and IBD of hand-foot of the same name meridians (IBD-HFM) in healthy subjects. METHODS: BP at yuan-source points of twelve meridians was measured on 31 healthy volunteers by a Laser Doppler Line Scanner (LDLS). BP distribution and IBD-LRM, IBD-EIM, IBD-HFM were calculated. RESULTS: (1) Of the twelve meridians, BP was almost equal between the left and right of the same meridian. The mean value of IBD-LRM was (0.8±7.0)%. The absolute value of IBD-LRM ranged from (13.2±12.0)% to (22.9±15.6)%, with the mean value of (16.2±4.1)%, the IBD of gallbladder meridian of foot-shaoyang was the highest. (2) Of the six pairs of exterior-interior meridians, five pairs manifested as the interior (yin) meridians being larger than the exterior (yang) meridians in BP. The mean value of IBD-EIM was (-11.4±10.4)%. The absolute value of IBD-EIM ranged from (16.6±12.1)% to (36.6±15.6)%,with the mean value of (25.2±8.0)%, the IBD between pericardium meridian of hand-jueyin and triple energizer meridian of hand-shaoyang was the highest. (3)All of the hand-foot of the same name meridians were found hand meridians being larger than foot in BP. The mean value of IBD-HFM was (38.8±18.2)%.The absolute value of IBD-HFM ranged from (34.4±20.9)% to (59.6±12.0)%, with the mean value of (43.8±13.3)%, and IBD between heart meridian of hand-shaoyin and kidney meridian of foot-shaoyin was the highest. (4) The order of IBD absolute value in BP was the same as transcutaneous CO2 emission (TCE) measured before, and their IBD-LRM absolute values were close to each other. However, the absolute values of IBD-EIM and IBD-HFM existed some differences. CONCLUSIONS: The IBD-LRM of the twelve yuan-source points in BP of healthy subjects is small and in a balance state, but IBD-EIM and IBD-HFM are relatively high.

Molecular cloning, characterization and expression analysis of Wnt4, Wnt5, Wnt6, Wnt7, Wnt10 and Wnt16 from Litopenaeus vannamei.

The Wnt (Wg-type MMTV integration site) signaling represents as the negative regulator of virus-induced innate immune responses. Wnt genes act as ligands to activate the Wnt signaling. To know more about the information of Wnt genes in invertebrates, Litopenaeus vannamei Wnt genes (LvWnts) were identified and characterized. In this study, Six Wnt genes (LvWnt4, LvWnt5, LvWnt6, LvWnt7, LvWnt10 and LvWnt16) were obtained in L. vannamei. The complete cDNAs open reading frames (ORF) of LvWnt4, LvWnt5, LvWnt6, LvWnt7, LvWnt10 and LvWnt16 were 1077 bp, 1107 bp, 1350 bp, 1047 bp, 1509 bp and 1158 bp (GenBank accession no. KU169896, KU169897, KU169898, KU169899, KU169900 and KU169901), encoding 358, 368, 449, 348, 502 and 385 amino acid (aa) residues respectively. All the six members of LvWnts contain a Wnt1 domain, which is considered as an important feature of Wnt gene family. ClustalW analysis with amino acid sequences revealed that the proportion of identity with other species was more than 48% for all the LvWnts except LvWnt10 (36-41%). The phylogenetic relationship analysis illustrated that different subtype of Wnts formed their own separate branches and were placed in branch of invertebrates respectively with strong bootstrap support. The constitutive expressions of LvWnts were confirmed by RT-PCR in all the examined five developmental stages and eleven tissues of L. vannamei with different express patterns. LvWnt4, LvWnt5 and LvWnt10 were expressed highest in nerve while LvWnt6, LvWnt7 and LvWnt16 were expressed highest in intestine, stomach and gill, respectively. In addition, all the LvWnts were regulated by white spot syndrome virus (WSSV) challenges at different levels in hepatopancreas, gill and hemocytes, suggesting that Wnt genes may play a role in the defense against pathogenic virus infection in innate immune of L. vannamei.

Flightless-I (FliI) is a potential negative regulator of the Toll pathway in Litopenaeus vannamei.

Flightless-I (FliI) is a protein negatively modulates the Toll-like receptor (TLR) pathway through interacting with Myeloid differentiation factor 88 (MyD88). To investigate the function of FliI in innate immune responses in invertebrates, Litopenaeus vannamei FliI (LvFliI) was identified and characterized. The full-length cDNA of LvFliI is 4, 304 bp long, with an open reading frame (ORF) encoding a putative protein of 1292 amino acids, including 12 leucine-rich repeat (LRR) domains at the N-terminus and 6 gelsolin homology (GEL) domains at the C-terminus. The LvFliI protein was located in the cytoplasm and LvFliI mRNA was constitutively expressed in healthy L. vannamei, with the highest expression level in the muscle. LvFliI could be up-regulated in hemocytes after lipopolysaccharide (LPS), poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV) challenges, suggesting a stimulation response of LvFliI to bacterial and immune stimulant challenges. Upon LPS stimulation, overexpression of LvFliI in Drosophila Schneider 2 cells led to downregulation of Drosophila and shrimp antimicrobial peptide (AMP) genes. Knockdown of LvFliI by RNA interference (RNAi) resulted in an increase of the expression of three shrimp AMP genes (PEN2, crustin, and Lyz1). However, the mortality rates of LvFliI-knockdown shrimp in response to V. parahaemolyticus, S. aureus or WSSV infections were not significantly different from those of the control group. Taken together, all the results suggested that LvFliI may play a negative role in TLR signaling response in L. vannamei.

Litopenaeus vannamei NF-κB is required for WSSV replication.

Many viruses can hijack the host cell NF-κB as part of their life cycle, diverting NF-κB immune regulatory functions to favor their replications. There were several reports on the functions of Litopenaeus vannamei NF-κB (LvNF-κB) in White spot syndrome virus (WSSV) replication in vitro. Here, we studied the relationship between LvNF-κB family protein Dorsal (LvDorsal) and Relish (LvRelish) with WSSV replication in vivo. The expressions of LvDorsal and LvRelish were significantly upregulated by WSSV challenge. Virus loads and expression of viral envelope protein VP28 in LvDorsal or LvRelish silencing shrimps were significantly lower than the control shrimps injected with EGFP-dsRNA or PBS after challenge with 1×10(5) copies WSSV/shrimp. In addition to the LvDorsal activation of WSV069 (ie1) and WSV303 promoter that we have reported, LvRelish can also activate WSV069 (ie1) and WSV303 promoter by dual luciferase reporter assays through screening 40 WSSV gene promoters that have putative multiple NF-κB binding sites. The promoter activity of the WSV069 (ie1) by LvDorsal activation was significantly higher than that by LvRelish activation. WSSV replication in LvDorsal, LvRelish or WSV303 silencing shrimps were significantly inhibited. These results indicate that the L. vannamei NF-κB family proteins LvDorsal and LvRelish expressions are significantly activated by WSSV challenge and WSSV replication partially relied on the activations of LvDorsal and LvRelish in vivo.

[Effects of exogenous Ca2+ on the seed germination of Koelreuteria paniculata in limestone area of Southwest China under drought stress].

In this paper, different arid environments in the limestone area of Southwest China were simulated by using different concentrations (0%, 5%, 15%, 25%, and 30%) of PEG6000 (polyethylene glycol), and a gradient of exogenous Ca2+ (0, 15, 20, 25, and 30 mmol x L(-1) of CaCl2) was installed to simulate the high calcium soil environments, aimed to study the effects of different concentration PEG6000, CaCl2, and their interactions on the seed germination characteristics of Koelreuteria bipinnata, one of the preferred species in the ecological restoration in limestone area. Under the action of CaCl2 alone, medium or low concentration exogenous Ca2+ had lesser effect on the seed germination parameters, but high concentration exogenous Ca2+ had inhibition effect on the seed germination. Medium or low concentration CaC12 could increase the seedling fresh biomass. When the PEG6000 concentration increased from 0% to 25%, the seed germination rate, germination potential, germination index, and vigor index showed a significant decreasing trend, and the seedling fresh biomass decreased remarkably. When certain concentration of exogenous Ca2+ was exerted to the drought treatment groups, medium or low concentration CaCl2 improved the seed germination characteristics, which compensated the loss from drought stress to a certain degree. When the CaCl2 concentration was up to 30 mmol x L(-1), the seeds were hard to germinate. Additionally, no seeds germinated under the condition of PEG with the concentration of 30%. It was suggested that applying definite concentration of exogenous Ca2+ could increase the seedling fresh biomass under PEG6000 stress, and make the seedlings more adapt to arid environment.

Identification and function of leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2) in Litopenaeus vannamei.

Leucine-rich repeat flightless-I-interacting protein 2 (LRRFIP2) is a myeloid differentiation factor 88-interacting protein with a positive regulatory function in toll-like receptor signaling. In this study, seven LRRFIP2 protein variants (LvLRRFIP2A-G) were identified in Litopenaeus vannamei. All the seven LvLRRFIP2 protein variants encode proteins with a DUF2051 domain. LvLRRFIP2s were upregulated in hemocytes after challenged with lipopolysaccharide, poly I:C, CpG-ODN2006, Vibrio parahaemolyticus, Staphylococcus aureus, and white spot syndrome virus (WSSV). Dual-luciferase reporter assays in Drosophila Schneider 2 cells revealed that LvLRRFIP2 activates the promoters of Drosophila and shrimp AMP genes. The knockdown of LvLRRFIP2 by RNA interference resulted in higher cumulative mortality of L. vannamei upon V. parahaemolyticus but not S. aureus and WSSV infections. The expression of L. vannamei AMP genes were reduced by dsLvLRRFIP2 interference. These results indicate that LvLRRFIP2 has an important function in antibacterials via the regulation of AMP gene expression.

Molecular characterization and function of a p38 MAPK gene from Litopenaeus vannamei.

p38 mitogen-activated protein kinases (MAPKs) are broadly expressed from yeasts to mammals, and are involved in the regulation of cells responsible to various extracellular stimuli. In this study, a p38 MAPK gene (designated as Lvp38) from Litopenaeus vannamei, was cloned and characterized. It contained the conserved structures of a Thr-Gly-Tyr (TGY) motif and a substrate-binding site, Ala-Thr-Arg-Trp (ATRW). The tissue distribution patterns showed that Lvp38 was widely expressed in all examined tissues, with the highest expression in hemocytes, nerves, and intestines. Quantitative real-time PCR revealed that Lvp38 was upregulated in gills and hemocytes after infection with the Gram-negative Vibrio alginolyticus and the Gram-positive Staphylococcus aureus. Reporter gene assays indicated that Lvp38 activated the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp. Knockdown of Lvp38 by RNA interference (RNAi) resulted in a higher mortality of L. vannamei under V. alginolyticus and S. aureus infection, as well as a reduction in the expression of three shrimp AMP genes, namely, PEN4, crustin, and ALF2. Taken together, our data indicated that Lvp38 played a role in defending against bacterial infections.

Identification and function of myeloid differentiation factor 88 (MyD88) in Litopenaeus vannamei.

Myeloid differentiation factor 88 (MyD88) is a universal and essential signaling protein in Toll-like receptor/interleukin-1 receptor-induced activation of nuclear factor-kappa B. In this study, two MyD88 protein variants (LvMyD88 and LvMyD88-1) were identified in Litopenaeus vannamei. The LvMyD88 cDNA is 1,848 bp in length and contains an open reading frame (ORF) of 1,428 bp, whereas the LvMyD88-1 cDNA is 1,719 bp in length and has an ORF of 1,299 bp. Both variants encode proteins with death and Toll/interleukin-1 receptor domains and share 91% sequence identity. In healthy L. vannamei, the LvMyD88 genes were highly expressed in hemocytes but at a low level in the hepatopancreas. The LvMyD88s expression was induced in hemocytes after challenge with lipopolysaccharide, CpG-ODN2006, Vibrio parahaemolyticus, Staphyloccocus aureus, and white spot syndrome virus, but not by poly I∶C. Overexpression of LvMyD88 and LvMyD88-1 in Drosophila Schneider 2 cells led to activation of antimicrobial peptide genes and wsv069 (ie1), wsv303, and wsv371. These results suggested that LvMyD88 may play a role in antibacterial and antiviral response in L. vannamei. To our knowledge, this is the first report on MyD88 in shrimp and a variant of MyD88 gene in invertebrates.

Isolation and characterization of cDNAs encoding Ars2 and Pasha homologues, two components of the RNA interference pathway in Litopenaeus vannamei.

The RNA interference (RNAi) is an evolutionarily conserved protective mechanism in eukaryotes against parasitic foreign nucleic acids. Previous studies demonstrated that the RNAi mechanism is important for shrimp antiviral immunity. Here, we report the identification and functional analysis of two key components of the shrimp RNAi activity: Litopenaeus vannamei arsenite resistance gene 2 (LvArs2) and partner of drosha (LvPasha). The full-length cDNA of LvArs2 was 3470 bp, including a 5' untranslated region (UTR) of 167 bp, a 3' UTR of 639 bp, and an open reading frame (ORF) of 2664 bp that encoded 887 amino acid residues with an estimated molecular mass of 102.5 kDa. The full-length cDNA of LvPasha was 2654 bp, including a 5' UTR of 99 bp, a 3' UTR of 560 bp, and an ORF of 1995 bp that encoded 664 amino acid residues with an estimated molecular mass of 74.2 kDa. Co-immunoprecipitation demonstrated that LvArs2 interacted with L. vannamei Dicer2 (LvDcr2) and LvPasha in Drosophila Schneider 2 (S2) cells, suggesting that LvArs2 may be involved in regulation of the miRNA/siRNA pathways in L.vannamei. Subcellular localization assays demonstrated both LvArs2 and LvPasha proteins mainly presented in the nucleus. After Poly(C-G) stimulation, the expression of LvArs2 was suppressed and expression of LvPasha was enhanced in shrimp gills. These results suggest that LvArs2 and LvPasha may participate in the defense against RNA viruses in crustacea.

Identification and functional characterization of Dicer2 and five single VWC domain proteins of Litopenaeus vannamei.

Dicer (Dcr) is the key protein of the RNA interference (RNAi) pathway. To investigate the role of the RNAi pathway in shrimp anti-viral immunity, Litopenaeus vannamei Dcr2 (designated as LvDcr2) was identified and characterized. The full-length cDNA of LvDcr2 was 5513bp long, with an open reading frame encoding a putative protein of 1502 amino acids. In addition, five proteins homologous to the single von Willebrand factor type C (VWC) domain protein (SVC) were also identified in L. vannamei and named LvSVC1-5. These LvSVCs were between 102 and 190 amino acids in length and all contained a motif similar to Drosophila melanogaster SVC proteins (DmSVCs). By co-immunoprecipitation assays and pull-down assays, we demonstrated that LvDcr2, L. vannamei Argonaute 2 (LvAgo2), and L. vannamei transactivating response RNA-binding protein isoform 1 (LvTRBP1) interacted with each other. A luciferase reporter assay indicated that the promoters of LvSVC1, LvSVC4, LvSVC5, and DmSVC Vago (DmVago) were activated by LvDcr2 as well as by Drosophila Dcr2 (DmDcr2). Real-time RT-PCR showed that LvDcr2 and LvSVCs were up-regulated in immune responses against Poly(C-G) or WSSV challenge. These results suggested that LvDcr2 formed complexes with LvAgo2 and LvTRBP1 to act as the cores of shrimp small interfering RNA (siRNA)-induced silencing complex (siRISC)/siRISC-loading complex (siRLC), role in shrimp siRNA pathway. Furthermore, these results also suggested that LvDcr2 may engage in non-specific activation of anti-viral immunity.