Evaluating solubility, stability, and inclusion complexation of oxyresveratrol with various ß-cyclodextrin derivatives using advanced computational techniques and experimental validation.

Oxyresveratrol (OXY), a natural stilbenoid in mulberry fruits, is known for its diverse pharmacological properties. However, its clinical use is hindered by low water solubility and limited bioavailability. In the present study, the inclusion complexes of OXY with ß-cyclodextrin (ßCD) and its three analogs, dimethyl-ß-cyclodextrin (DMßCD), hydroxypropyl-ß-cyclodextrin (HPßCD) and sulfobutylether-ß-cyclodextrin (SBEßCD), were investigated using in silico and in vitro studies. Molecular docking revealed two binding orientations of OXY, namely, 4',6'-dihydroxyphenyl (A-form) and 5,7-benzenediol ring (B-form). Molecular Dynamics simulations suggested the formation of inclusion complexes with ßCDs through two distinct orientations, with OXY/SBEßCD exhibiting maximum atom contacts and the lowest solvent-exposed area in the hydrophobic cavity. These results corresponded well with the highest binding affinity observed in OXY/SBEßCD when assessed using the MM/GBSA method. Beyond traditional simulation methods, Ligand-binding Parallel Cascade Selection Molecular Dynamics method was employed to investigate how the drug enters and accommodates within the hydrophobic cavity. The in silico results aligned with stability constants: SBEßCD (2060 M-1), HPßCD (1860 M-1), DMßCD (1700 M-1), and ßCD (1420 M-1). All complexes exhibited a 1:1 binding mode (AL type), with SBEßCD enhancing OXY solubility (25-fold). SEM micrographs, DSC thermograms, FT-IR and 1H NMR spectra confirm the inclusion complex formation, revealing novel surface morphologies, distinctive thermal behaviors, and new peaks. Notably, the inhibitory impact on the proliferation of breast cancer cell lines, MCF-7, exhibited by inclusion complexes particularly OXY/DMßCD, OXY/HPßCD, and OXY/SBEßCD were markedly superior compared to that of OXY alone.

Enhancing solubility and stability of piperine using ß-cyclodextrin derivatives: computational and experimental investigations.

Piperine (PP), a natural alkaloid found in black pepper, possesses significant bioactivities. However, its use in pharmaceutical applications is hindered by low water solubility and susceptibility to UV light degradation. To overcome these challenges, we investigated the potential of ß-cyclodextrin (ßCD) and its derivatives with dimethyl (DMßCD), hydroxy-propyl (HPßCD) and sulfobutyl-ether (SBEßCD) substitutions to enhance the solubility and stability of PP. This study employed computational and experimental approaches to examine the complexation between PP and ßCDs. The results revealed the formation of two types of inclusion complexes: the P-form and M-form involving the insertion of piperidine moiety and the methylene-di-oxy-phenyl moiety, respectively. These complexes primarily rely on van der Waals interactions. Among the three derivatives, the PP/SBEßCD complex exhibited the highest stability followed by HPßCD, as attributed to maximum atom contacts and minimal solvent accessibility. Solubility studies confirmed the formation of inclusion complexes in a 1:1 ratio. Notably, the stability constant of the inclusion complex was approximately two-fold higher with SBEßCD and HPßCD compared to ßCD. The DSC thermograms provided confirmation of the formation of the inclusion complex between the host and guest. These findings highlight the potential of ßCD derivatives to effectively encapsulate PP, improving its solubility and presenting new opportunities for its pharmaceutical applications.Communicated by Ramaswamy H. Sarma.

Enhancing solubility and stability of sorafenib through cyclodextrin-based inclusion complexation: in silico and in vitro studies.

Sorafenib (SOR) is an oral multikinase inhibitor that effectively hampers the growth and spread of cancer cells by targeting angiogenesis and proliferation. However, SOR tablets (Nexavar) have limited oral bioavailability, ranging from 38% to 49%, due to their low water solubility. To address this issue, cyclodextrins (CDs), widely used to enhance the solubility and stability of lipophilic drugs by encapsulating them within their molecular structure, were considered in this study. We focused on ß-cyclodextrin (ßCD) and its derivatives, including hydroxypropyl-ß-cyclodextrin (HPßCD), dimethyl-ß-cyclodextrin (DMßCD), sulfobutylether-ß-cyclodextrin (SBEßCD), and compared them with γ-cyclodextrin (γCD) for generating inclusion complexes with SOR. The 200 ns molecular dynamics simulations revealed that SOR could form inclusion complexes with all CDs in two possible orientations: pyridine group insertion (P-form) and chlorobenzotrifluoride group insertion (C-form), primarily driven by van der Waals interactions. Among the four ßCD derivatives studied, SOR exhibited the highest number of atom contacts with SBEßCD and demonstrated the lowest solvent accessibility within the hydrophobic cavity of SBEßCD. These findings correlated with the highest binding affinity of SOR/SBEßCD complex determined by SIE, MM/GBSA, and MM/PBSA methods. Experimental results further supported our computational predictions, in which SBEßCD exhibited a stability constant of 940 M-1 at 25 °C, surpassing ßCD's stability constant of 210 M-1. Taken together, our results suggest that the modified CDs, particularly SBEßCD, hold promising potential as an efficient molecular encapsulating agent for SOR, offering improved solubility and stability for this lipophilic drug.

Effects of PmDOME and PmSTAT knockdown on white spot syndrome virus infection in Penaeus monodon.

Janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling pathway plays an important role in antiviral immunity. This research reports the full-length DOME receptor gene in Penaeus monodon (PmDOME) and examines the effects of PmDOME and PmSTAT silencing on immune-related gene expressions in shrimp hemocytes during white spot syndrome virus (WSSV) infection. PmDOME and PmSTAT were up-regulated in shrimp hemocytes upon WSSV infection. Suppression of PmDOME and PmSTAT showed significant impacts on the expression levels of ProPO2 (melanization), Vago5 (interferon-like protein) and several antimicrobial peptides, including ALFPm3, Penaeidin3, CrustinPm1 and CrustinPm7. Silencing of PmDOME and PmSTAT reduced WSSV copy numbers and delayed the cumulative mortality caused by WSSV. We postulated that suppression of the JAK/STAT signaling pathway may activate the proPO, IFN-like antiviral cytokine and AMP production, resulting in a delay of WSSV-related mortality.

Insight into the Molecular Weight of Hydrophobic Starch Laurate-Based Adhesives for Paper.

Instead of using finite petroleum-based resources and harmful additives, starch can be used as a biodegradable, low-cost, and non-toxic ingredient for green adhesives. This work employs K3PO4 catalyzed transesterifications of cassava starch and methyl laurate at varying reaction times (1-10 h), resulting in the enhanced hydrophobicity of starch laurates. At longer reaction times, starch laurates having higher degrees of substitution (DS) were obtained. While starch laurates are the major products of transesterification, relatively low-molecular-weight byproducts (1%) were detected and could be hydrolyzed starches based on gel permeation chromatography results. Contact angle measurements confirmed the relatively high hydrophobicity of the modified starches compared with that of native starch. The modified starches were then employed to prepare water-based adhesives on paper (without any additional additives). Notably, the shear strength of the esterified starch adhesives appears to be independent of the DS of esterified samples, hence the transesterification reaction times. Additionally, the shear strength of water-based adhesives (0.67-0.73 MPa) for bonding to paper substrates is superior to that of two other commercially available glues by a factor of 10 to 80 percent.

In Vitro and In Silico Study on the Molecular Encapsulation of α-Tocopherol in a Large-Ring Cyclodextrin.

α-tocopherol is the physiologically most active form of vitamin E, with numerous biological activities, such as significant antioxidant activity, anticancer capabilities, and anti-aging properties. However, its low water solubility has limited its potential use in the food, cosmetic, and pharmaceutical industries. One possible strategy for addressing this issue is the use of a supramolecular complex with large-ring cyclodextrins (LR-CDs). In this study, the phase solubility of the CD26/α-tocopherol complex was investigated to assess the possible ratios between host and guest in the solution phase. Next, the host-guest association of the CD26/α-tocopherol complex at different ratios of 1:2, 1:4, 1:6, 2:1, 4:1, and 6:1 was studied by all-atom molecular dynamics (MD) simulations. At 1:2 ratio, two α-tocopherol units interact spontaneously with CD26, forming an inclusion complex, as supported by the experimental data. In the 2:1 ratio, a single α-tocopherol unit was encapsulated by two CD26 molecules. In comparison, increasing the number of α-tocopherol or CD26 molecules above two led to self-aggregation and consequently limited the solubility of α-tocopherol. The computational and experimental results indicate that a 1:2 ratio could be the most suitable stoichiometry to use in the CD26/α-tocopherol complex to improve α-tocopherol solubility and stability in inclusion complex formation.

Starch Chemical Composition and Molecular Structure in Relation to Physicochemical Characteristics and Resistant Starch Content of Four Thai Commercial Rice Cultivars Differing in Pasting Properties.

Variations in starch pasting properties, considered an alternative potential quality classification parameter for rice starches, are directly controlled by the diverse starch molecular composition and structural features. Here, the starch characteristics of four rice cultivars (i.e., RD57, RD29, KDML105, and RD6) differing in pasting properties were assessed, and their relationship was determined. The results revealed that protein and moisture contents and their crystalline type were similar among the four rice starches. However, their molecular compositions and structures (i.e., reducing sugar and amylose contents, amylopectin branch chain-length distributions, granule size and size distribution, and degree of crystallinity) significantly varied among different genotypes, which resulted in distinct swelling, solubility, gelatinization, retrogradation, and hydrolytic resistance properties. The swelling power and gelatinization enthalpy (∆H) were positively correlated with C-type granule and relative crystallinity, but were negatively correlated with amylose content, B-type granule and median particle size (d(0.5)). Conversely, the water solubility and resistant starch content negatively correlated with C-type granule, but positively correlated with amylose content, B-type granule, and d(0.5). The gelatinization onset temperature (To(g)), and retrogradation concluding temperatures (Tc(r)), enthalpy (∆H(r)), and percentage (R%) were positively impacted by the amount of protein, amylose, and B1 chains (DP 13-24), while they were negatively correlated with short A chains (DP 6-12). Collectively, the starch physicochemical and functional properties of these Thai rice starches are attributed to an interplay between compositional and structural features. These results provide decisive and crucial information on rice cultivars' suitability for consumption as cooked rice and for specific industrial applications.

Identification of crucial amino acid residues involved in large ring cyclodextrin synthesis by amylomaltase from Corynebacterium glutamicum.

Amylomaltase can be used to synthesize large ring cyclodextrins (LR-CDs), applied as drug solubilizer, gene delivery vehicle and protein aggregation suppressor. This study aims to determine the functional amino acid positions of Corynebacterium glutamicum amylomaltase (CgAM) involved in LR-CD synthesis by site-directed mutagenesis approach and molecular dynamic simulation. Mutants named Δ167, Y23A, P228Y, E231Y, A413F and G417F were constructed, purified, and characterized. The truncated CgAM, Δ167 exhibited no starch transglycosylation activity, indicating that the N-terminal domain of CgAM is necessary for enzyme activity. The P228Y, A413F and G417F produced larger LR-CDs from CD36-CD40 as compared to CD29 by WT. A413F and G417F mutants produced significantly low LR-CD yield compared to the WT. The A413F mutation affected all tested enzyme activities (starch tranglycosylation, disproportionation and cyclization), while the G417F mutation hindered the cyclization activity. P228Y mutation significantly lowered the k cat of disproportionation activity, while E231Y mutant exhibited much higher k cat and K m values for starch transglycosylation, compared to that of the WT. In addition, Y23A mutation affected the kinetic parameters of starch transglycosylation and cyclization. Molecular dynamic simulation further confirmed these mutations' impacts on the CgAM and LR-CD interactions. Identified functional amino acids for LR-CD synthesis may serve as a model for future modification to improve the properties and yield of LR-CDs.

White spot syndrome virus impact on the expression of immune genes and gut microbiome of black tiger shrimp Penaeus monodon.

The gut microbiome plays an essential role in the immune system of invertebrates and vertebrates. Pre and pro-biotics could enhance the shrimp immune system by increasing the phenoloxidase (PO), prophenoloxidase (ProPO), and superoxide dismutase activities. During viral infection, the host immune system alteration could influence the gut microbiome composition and probably lead to other pathogenic infections. Since the JAK/STAT pathway is involved in white spot syndrome virus (WSSV) infection, we investigated the intestine immune genes of STAT-silenced shrimp. During WSSV infection, expression levels of PmVago1, PmDoral, and PmSpätzle in PmSTAT-silenced shrimp were higher than normal. In addition, the transcription levels of antimicrobial peptides, including crustinPm1, crustinPm7, and PmPEN3, were higher in WSSV-challenged PmSTAT-silenced shrimp than the WSSV-infected normal shrimp. Meanwhile, PmSTAT silencing suppressed PmProPO1, PmProPO2, and PmPPAE1 expressions during WSSV infection. The microbiota from four shrimp tested groups (control group, WSSV-infected, PmSTAT-silenced, and PmSTAT-silenced infected by WSSV) was significantly different, with decreasing richness and diversity due to WSSV infection. The relative abundance of Bacteroidetes, Actinobacteria, and Planctomycetes was reduced in WSSV-challenged shrimp. However, at the species level, P. damselae, a pathogen to human and marine animals, significantly increased in WSSV-challenged shrimp. In constrast, Shewanella algae, a shrimp probiotic, was decreased in WSSV groups. In addition, the microbiota structure between control and PmSTAT-silenced shrimp was significantly different, suggesting the importance of STAT to maintain the homeostasis interaction with the microbiota.

Computational design of Lactobacillus Acidophilus α-L-rhamnosidase to increase its structural stability.

α-L-rhamnosidase catalyzes hydrolysis of the terminal α-L-rhamnose from various natural rhamnoglycosides, including naringin and hesperidin, and has various applications such as debittering of citrus juices in the food industry and flavonoid derhamnosylation in the pharmaceutical industry. However, its activity is lost at high temperatures, limiting its usage. To improve Lactobacillus acidophilus α-L-rhamnosidase stability, we employed molecular dynamics (MD) to identify a highly flexible region, as evaluated by its root mean square fluctuation (RMSF) value, and computational protein design (Rosetta) to increase rigidity and favorable interactions of residues in highly flexible regions. MD results show that five regions have the highest flexibilities and were selected for design by Rosetta. Twenty-one designed mutants with the best ΔΔG at each position and ΔΔG < 0 REU were simulated at high temperature. Eight designed mutants with ΔRMSF of highly flexible regions lower than -10.0% were further simulated at the optimum temperature of the wild type. N88Q, N202V, G207D, Q209M, N211T and Y213K mutants were predicted to be more stable and could maintain their native structures better than the wild type due to increased hydrogen bond interactions of designed residues and their neighboring residues. These designed mutants are promising enzymes with high potential for stability improvement.

Production of Large-Ring Cyclodextrins by Amylomaltases.

Amylomaltase is a well-known glucan transferase that can produce large ring cyclodextrins (LR-CDs) or so-called cycloamyloses via cyclization reaction. Amylomaltases have been found in several microorganisms and their optimum temperatures are generally around 60-70 °C for thermostable amylomaltases and 30-45 °C for the enzymes from mesophilic bacteria and plants. The optimum pHs for mesophilic amylomaltases are around pH 6.0-7.0, while the thermostable amylomaltases are generally active at more acidic conditions. Size of LR-CDs depends on the source of amylomaltases and the reaction conditions including pH, temperature, incubation time, and substrate. For example, in the case of amylomaltase from Corynebacterium glutamicum, LR-CD productions at alkaline pH or at a long incubation time favored products with a low degree of polymerization. In this review, we explore the synthesis of LR-CDs by amylomaltases, structural information of amylomaltases, as well as current applications of LR-CDs and amylomaltases.

Molecular structure and properties of cassava-based resistant maltodextrins.

In-depth molecular structure and properties of cassava-derived resistant maltodextrins (RMDs) were determined. Cassava starch was dextrinized with 0.04% or 0.06% HCl at 120 °C for 60-180 min to obtain resistant dextrins (RDs), which were further hydrolyzed by α-amylase to produce RMDs. Prolonging dextrinization duration decreased proportion of α-1,4 linkages and α-/ß-reducing ends but increased fraction of indigestible α-/ß-1,6, ß-1,4, ß-1,2 linkages, degree of branching (DB), degree of polymerization, relative molecular weight, and total dietary fiber (TDF) content of the RMDs. Moreover, RMDs had greater proportion of ß-glycosidic linkages, α-/ß-reducing end, DB, TDF, and low molecular weight dietary fiber (LMWDF) content than their RD counterparts. Potential prebiotic activity score was higher in RMDs with abundant LMWDF fraction but low DB. Slight difference in the glass transition temperature of maximally freeze-concentrated unfrozen phase (Tg') and unfrozen water content was found among RMDs. However, RMDs had lower Tg' than their RD counterparts.

Salt tolerance at vegetative stage is partially associated with changes in grain quality and starch physicochemical properties of rice exposed to salinity stress at reproductive stage.

BACKGROUND: Rice yield and grain quality are highly sensitive to soil salinity. Distinct rice genotypes respond to salinity stress differently. To explore the variation in grain yield and grain trait adaptation to moderate, reproductive-stage salinity stress (4 dS/m electrical conductivity), four rice cultivars differing in degrees of vegetative salt tolerance, including Pokkali (salt-tolerant), RD15 (moderately salt-tolerant), KDML105 (moderately salt-susceptible) and IR29 (salt-susceptible), were examined. RESULTS: Grain fertility and 100-grain weight of RD15, KDML105 and IR29, as well as grain morphology of KDML105 and IR29, were significantly disturbed. Interestingly, grain starch accumulation in RD15 and KDML105 was enhanced under stress. However, only RD15 showed changes in starch physicochemical properties, including increased granule diameter, decreased gelatinization peak temperature (Tp ) and decreased retrogradation onset temperature (To ). Notably, Pokkali maintained productivity, grain quality, and starch properties, while the grain quality of IR29 remained unchanged under salinity stress. Multivariate analysis displayed clear separation of productivity, grain morphology, and starch variables of RD15 in the salt-treated group relative to the control group, suggesting that it was the cultivar most impacted by salt stress despite its moderate salt-tolerance at vegetative stage. CONCLUSION: Our results demonstrate specific salinity responses among the rice genotypes, and suggest discrepancies between degrees of salt tolerance at vegetative stage versus the ability to maintain both grain quality and starch properties in response to salinity stress imposed at reproductive stage. © 2021 Society of Chemical Industry.

Leukocyte telomere length is not shortened in methamphetamine dependence or methamphetamine-induced psychosis but is increased following traumatic events.

OBJECTIVE: This study aims to examine the effects of methamphetamine (MA) use and dependence and MA withdrawal symptoms on the telomere length and whether shortening of the latter is associated with MA-induced psychosis (MIP). METHODS: This study included 185 MA-abuse, 118 MA-dependent, and 67 MIP patients, diagnosed using DSM-IV criteria. The Semi-structured Assessment for Drug Dependence and Alcoholism (SSADDA) questionnaire was employed to collect MA-related data. MIP was confirmed using the Methamphetamine Experience Questionnaire (MEQ). The leukocyte telomere length was measured using real-time polymerase chain reaction measuring the Telomere/Single gene ratio (T/S ratio). Data were analysed using multivariate statistical analyses. RESULTS: There were no significant associations between the T/S ratio and severity of MA-use, MIP, and MA withdrawal symptoms. MIP was significantly predicted by alcohol dependence, antisocial personality disorder, and MA-use severity. There were significantly positive associations between the T/S ratio and previous traumatic and life-threatening events. The T/S ratio was not affected by alcohol and nicotine dependence. Alcohol and nicotine dependence, antisocial personality disorder, and severity of MA use increased risk of MA withdrawal symptoms. CONCLUSION: MIP and MA-use severity are not associated with leukocyte telomere length, but previous traumatic and life-threatening events are associated with increased telomere length.

Yield, Grain Quality, and Starch Physicochemical Properties of 2 Elite Thai Rice Cultivars Grown under Varying Production Systems and Soil Characteristics.

Rice production systems and soil characteristics play a crucial role in determining its yield and grain quality. Two elite Thai rice cultivars, namely, KDML105 and RD6, were cultivated in two production systems with distinct soil characteristics, including net-house pot production and open-field production. Under open-field system, KDML105 and RD6 had greater panicle number, total grain weight, 100-grain weight, grain size, and dimension than those grown in the net-house. The amounts of reducing sugar and long amylopectin branch chains (DP 25-36) of the RD6 grains along with the amounts of long branch chains (DP 25-36 and DP ≥ 37), C-type starch granules, and average chain length of the KDML105 were substantially enhanced by the open-field cultivation. Contrastingly, the relative crystallinity of RD6 starch and the amounts of short branch chains (DP 6-12 and DP 13-24), B- and A-type granules, and median granule size of KDML105 starch were significantly suppressed. Consequently, the open-field-grown RD6 starch displayed significant changes in its gelatinization and retrogradation properties, whereas, certain retrogradation parameters and peak viscosity (PV) of KDML105 starches were differentially affected by the distinct cultivating conditions. This study demonstrated the influences of production systems and soil characteristics on the physicochemical properties of rice starches.

Enhancement of large ring cyclodextrin production using pretreated starch by glycogen debranching enzyme from Corynebacterium glutamicum.

Synthesis of large-ring cyclodextrins (LR-CDs) in any significant amount has been challenging. This study enhanced the LR-CDs production by Thermus filiformis amylomaltase (TfAM) enzyme by starch pretreatment using glycogen debranching enzyme from Corynebacterium glutamicum (CgGDE). CgGDE pretreated tapioca starch gave LR-CD conversion of 31.2 ± 2.2%, compared with LR-CDs produced from non-treated tapioca starch (16.0 ± 2.4%). CgGDE pretreatment enhanced amylose content by approximately 30%. Notably, a shorter incubation time of 1 h is sufficient for CgGDE starch pretreatment to produce high LR-CD yield, compared with 6 h required for the commercial isoamylase. High-Performance Anion Exchange Chromatography coupled with Pulsed Amperometric Detection (HPAEC-PAD) and Gel Permeable Chromatography (GPC) revealed that CgGDE is more efficient than the commercial isoamylase in debranching tapioca starch and gave lower molecular weight products. In addition, lower amount of by-products (linear oligosaccharides) were detected in cyclization reaction when using CgGDE-pretreated starch. In conclusion, CgGDE is a highly effective enzyme to promote LR-CD synthesis from starch with a shorter incubation time than the commercial isoamylase.

A GH13 α-glucosidase from Weissella cibaria uncommonly acts on short-chain maltooligosaccharides.

α-Glucosidase (EC 3.2.1.20) is a carbohydrate-hydrolyzing enzyme which generally cleaves α-1,4-glycosidic bonds of oligosaccharides and starch from the nonreducing ends. In this study, the novel α-glucosidase from Weissella cibaria BBK-1 (WcAG) was biochemically and structurally characterized. WcAG belongs to glycoside hydrolase family 13 (GH13) and to the neopullanase subfamily. It exhibits distinct hydrolytic activity towards the α-1,4 linkages of short-chain oligosaccharides from the reducing end. The enzyme prefers to hydrolyse maltotriose and acarbose, while it cannot hydrolyse cyclic oligosaccharides and polysaccharides. In addition, WcAG can cleave pullulan hydrolysates and strongly exhibits transglycosylation activity in the presence of maltose. Size-exclusion chromatography and X-ray crystal structures revealed that WcAG forms a homodimer in which the N-terminal domain of one monomer is orientated in proximity to the catalytic domain of another, creating the substrate-binding groove. Crystal structures of WcAG in complexes with maltose, maltotriose and acarbose revealed a remarkable enzyme active site with accessible +2, +1 and -1 subsites, along with an Arg-Glu gate (Arg176-Glu296) in front of the active site. The -2 and -3 subsites were blocked by Met119 and Asn120 from the N-terminal domain of a different subunit, resulting in an extremely restricted substrate preference.

Solubility enhancement of poorly water soluble domperidone by complexation with the large ring cyclodextrin.

The water solubility of domperidone (DMP) could be improved by complexation with large ring cyclodextrins (LR-CDs). LR-CDs contain a relatively hydrophobic cavity that is capable of entrapping the molecules to form inclusion complexes. The complex formation capability of mixture LR-CDs having a degree of polymerization (DP) of 22-48, with DMP was investigated. The phase solubility profile of mixture LR-CD/DMP was classified as AN-type, resulting in increased DMP solubility in water by 3-fold. Various physicochemical techniques confirmed the mixture LR-CD/DMP complex formation. Single LR-CD with DP of 26, 27, 28, 29, 30, 33 and 34 (CD26 ~ CD34) were isolated from LR-CD mixtures using ODS column for HPLC separation. The CD33/DMP complex has demonstrated the most significant improvement compared to other single LR-CD complexes with a 2.7-fold increase in DMP solubility. The molecular dynamic result revealed that DMP formed stable complexes with CD33 by positioned fully encapsulated inside the cavity and covered by 13-14 subunits of CD33.

PmAP2-ß depletion enhanced activation of the Toll signaling pathway during yellow head virus infection in the black tiger shrimp Penaeus monodon.

Yellow head virus (YHV) is a pathogen which causes high mortality in penaeid shrimp. Previous studies suggested that YHV enters shrimp cells via clathrin-mediated endocytosis. This research investigated the roles of clathrin adaptor protein 2 subunit ß (AP-2ß) from Penaeus monodon during YHV infection. PmAP2-ß was continuously up-regulated more than twofold during 6-36 hpi. Suppression of PmAP2-ß significantly reduced YHV copy numbers and delayed shrimp mortality. Quantitative RT-PCR revealed that knockdown of PmAP2-ß significantly enhanced the expression level of PmSpätzle, a signaling ligand in the Toll pathway, by 30-fold at 6 and 12 hpi. Moreover, the expression levels of gene components in the Imd and JAK/STAT signaling pathways under the suppression of PmAP2-ß during YHV infection were also investigated. Interestingly, anti-lipopolysaccharide factor isoform 3 (ALFPm3) was up-regulated by 40-fold in PmAP2-ß knockdown shrimp upon YHV infection. In addition, silencing of PmAP2-ß dramatically enhanced crustinPm1 expression in YHV-infected shrimp. Knockdown of ALFPm3 and crustinPm1 significantly reduced shrimp survival rate. Taken together, this work suggested that PmAP2-ß-deficiency promoted the Toll pathway signalings, resulting in elevated levels of ALFPm3 and crustinPm1, the crucial antimicrobial peptides in defence against YHV.

Role of Clathrin Assembly Protein-2 Beta Subunit during White Spot Syndrome Virus Infection in Black Tiger Shrimp Penaeus monodon.

White spot syndrome virus (WSSV) is one of the most lethal viruses severely affecting shrimp industry. This disease can cause 100% mortality of farmed shrimp within a week. This work aims to characterize clathrin assembly proteins in Penaeus monodon and investigate their roles in WSSV entry. In general, clathrin assembly proteins form complexes with specific receptors and clathrins, leading to clathrin-mediated endocytosis. Adaptor protein 2 (AP-2), which is responsible for endocytosis at plasma membrane, consists of four subunits including α, ß2, µ2 and σ2. Knockdown of clathrin coat AP17, or σ subunit of AP-2 dramatically reduced WSSV infectivity. Similar results were observed, when shrimp were pre-treated with chlorpromazine (CPZ), an inhibitor of clathrin-dependent endocytosis. The complete open reading frames of AP-2ß and µ subunits of P. monodon are reported. PmAP-2 ß was up-regulated about 4-fold at 6 and 36 h post-WSSV infection. Knockdown of PmAP-2ß delayed shrimp mortality during WSSV infection, of which WSSV intermediate early 1 gene expression was also down-regulated. Immunogold-labelling and transmission electron microscopy revealed that PmAP-2ß co-localized with WSSV particles at plasma membrane. In addition, PmAP-2ß-silencing significantly affected the expression levels of PmSTAT, PmDOME, PmDorsal and ALFPm3 during WSSV infection. It is possible that PmAP-2ß is associated with the JAK/STAT and the Toll pathway.