WTAP-induced N6-methyladenosine of PD-L1 blocked T-cell-mediated antitumor activity under hypoxia in colorectal cancer.

N6-Methyladenosine (m6A) is a important process regulating gene expression post-transcriptionally. Programmed death ligand 1 (PD-L1) is a major immune inhibitive checkpoint that facilitates immune evasion and is expressed in tumor cells. In this research we discovered that Wilms' tumor 1-associated protein (WTAP) degradation caused by ubiquitin-mediated cleavage in cancer cells (colorectal cancer, CRC) under hypoxia was inhibited by Pumilio homolog 1 (PUM1) directly bound to WTAP. WTAP enhanced PD-L1 expression in a way that was m6A-dependent. m6A "reader," Insulin-like growth factor 2 mRNA-binding protein 2 (IGF2BP2) identified methylated PD-L1 transcripts and subsequently fixed its mRNA. Additionally, we found that T-cell proliferation and its cancer cell-killing effects were prevented by overexpression of WTAP in vitro and in vivo. Overexpression prevented T cells from proliferating and killing CRC by maintaining the expression of PD-L1. Further evidence supporting the WTAP-PD-L1 regulatory axis was found in human CRC and organoid tissues. Tumors with high WTAP levels appeared more responsive to anti-PD1 immunotherapy, when analyzing samples from patients undergoing treatment. Overall, our findings demonstrated a novel PD-L1 regulatory mechanism by WTAP-induced mRNA epigenetic regulation and the possible application of targeting WTAP as immunotherapy for tumor hypoxia.

Tanshinone IIA modulates cancer cell morphology and movement via Rho GTPases-mediated actin cytoskeleton remodeling.

Actin filaments form unique structures with robust actin bundles and cytoskeletal networks affixed to the extracellular matrix and interact with neighboring cells, which are crucial structures for cancer cells to acquire a motile phenotype. This study aims to investigate a novel antitumor mechanism by which Tanshinone IIA (Tan IIA) modulates the morphology and migration of liver cancer cells via actin cytoskeleton regulation. 97H and Huh7 exhibited numerous tentacle-like protrusions that interacted with neighboring cells. Following treatment with Tan IIA, 97H and Huh7 showed a complete absence of cytoplasmic protrusion and adherens junctions, thereby effectively impeding their migration capability. The fluorescence staining of F-actin and microtubules indicated that these tentacle-like protrusions and cell-cell networks were actin-based structures that led to morphological changes after Tan IIA treatment by retracting and reorganizing beneath the membrane. Tan IIA can reverse the actin depolymerization and cell morphology alterations induced by latrunculin A. Tan IIA down-regulated actin and Rho GTPases expression significantly, as opposed to inducing Rho signaling activation. Preventing the activity of proteasomes and lysosomes had no discernible impact on the modifications in cellular structure and protein expression induced by Tan IIA. However, as demonstrated by the puromycin labeling technique, the newly synthesized proteins were significantly inhibited by Tan IIA. In conclusion, Tan IIA can induce dramatic actin cytoskeleton remodeling by inhibiting the protein synthesis of actin and Rho GTPases, resulting in the suppression of tumor growth and migration. Targeting the actin cytoskeleton of Tan IIA is a promising strategy for HCC treatment.

Population genomic analysis provides evidence of the past success and future potential of South China tiger captive conservation.

BACKGROUND: Among six extant tiger subspecies, the South China tiger (Panthera tigris amoyensis) once was widely distributed but is now the rarest one and extinct in the wild. All living South China tigers are descendants of only two male and four female wild-caught tigers and they survive solely in zoos after 60 years of effective conservation efforts. Inbreeding depression and hybridization with other tiger subspecies were believed to have occurred within the small, captive South China tiger population. It is therefore urgently needed to examine the genomic landscape of existing genetic variation among the South China tigers. RESULTS: In this study, we assembled a high-quality chromosome-level genome using long-read sequences and re-sequenced 29 high-depth genomes of the South China tigers. By combining and comparing our data with the other 40 genomes of six tiger subspecies, we identified two significantly differentiated genomic lineages among the South China tigers, which harbored some rare genetic variants introgressed from other tiger subspecies and thus maintained a moderate genetic diversity. We noticed that the South China tiger had higher FROH values for longer runs of homozygosity (ROH > 1 Mb), an indication of recent inbreeding/founder events. We also observed that the South China tiger had the least frequent homozygous genotypes of both high- and moderate-impact deleterious mutations, and lower mutation loads than both Amur and Sumatran tigers. Altogether, our analyses indicated an effective genetic purging of deleterious mutations in homozygous states from the South China tiger, following its population contraction with a controlled increase in inbreeding based on its pedigree records. CONCLUSIONS: The identification of two unique founder/genomic lineages coupled with active genetic purging of deleterious mutations in homozygous states and the genomic resources generated in our study pave the way for a genomics-informed conservation, following the real-time monitoring and rational exchange of reproductive South China tigers among zoos.

Histopathological evaluation of the infection elicited by three species of Myxobolus (Cnidaria: Myxosporea), with identification of Myxobolusxiaoganensis n. sp. isolated from the silver carp Hypophthalmichthys molitrix.

Several parasites infecting the Asian carp species have been broadly spread along with the global fish trade. However, the diversity of specific parasite groups and their pattern of parasitism remain insufficiently elucidated even in their native regions. Here, we conducted a holistic identification and histological analysis of three Myxobolus species. The oblate myxospores of the first isolates were found infecting the spleen of silver carp Hypophthalmichthys molitrix, measuring 6.4 ± 0.4 (5.4-7.1) µm in length, 8.2 ± 0.4 (7.5-9.0) in width, and 5.9 ± 0.3 (5.2-6.2) in thickness. They were morphologically distinct from other congeners and regarded as a novel species, Myxobolus xiaoganensis n. sp. For the second isolates, we associated the formation of round plasmodia on the gill raker of silver carp with Myxobolus allotypica Chen, 1998. The third isolates, encapsulated in the intestinal serosa membrane of the grass carp Ctenopharyngodon idella, was proved to be conspecific to Myxobolus huasaensis Chen, 1998. While M. xiaoganensis n. sp. and M. huasaensis exhibited distinct origins, with genetic differences exceeding 4% from other congeners, M. allotypica displayed complete genetic congruence with an item available in Genbank. Histologically, myxospores of M. xiaoganensis n. sp. were scattered in the spleen, and the branchial and intestinal infections of M. allotypica and M. huasaensis were determined, respectively. Phylogenetic analysis demonstrated a non-monophyletic origin of both the Thelohanellus and Myxobolus genera, with a remarkable association of host affinity with myxosporean clustering.

Morphological, histological and phylogenetic identification of three species of Myxobolus (Myxosporea: Myxobolidae) infecting different carp lineages in China.

The global cultivation of common carp Cyprinus carpio is developed primarily for either food or recreational purposes and myxosporean infections causing significant economic losses are regularly troublesome for carp farmers. However, most myxosporean species are poorly characterized, making it difficult to correctly elaborate on their parasitism mode and determine pathogenicity. Here, based on an integrative approach, we supplemented fundamental information on three Myxobolus species infecting different carp lineages in China. Myxobolus elliptoides Wu et Chen, 1987; Nine new species of Myxosporida from freshwater fish of Wuhu lake, Hubei, China. Acta Hydrobiologica Sinica, 11, 161 formed yellowish pseudocysts in the anal fin of common carp and were observed with ellipsoidal myxospores and two polar capsules of distinct dimensions. The dermal tissue within the fin ray was regarded as the type site for growing plasmodia of M. elliptoides. Myxobolus basilamellaris Lom et Molnar, 1983; Myxobolus basilamellaris sp. n. (myxozoa, Myxosporea), a parasite of the gills of common carp (Cyprinus carpio L.). Folia Parasitologica, 30, 1 aggregated ellipsoidal myxospores at the base of the gill filaments of koi carp juvenile, causing significant tissue damage. Myxobolus artus Achmerov, 1960, enwrapped in diffuse pseudocysts, was observed throughout the trunk muscle of mirror carp. Its oblate myxospores were asymmetrical and contained two unequal-sized polar capsules. According to the localization of large plasmodia, inter- and intramuscular preferences were displayed by M. artus. For precise species identification, we provided the SSU rDNA sequences for each species of Myxobolus. Among them, M. elliptoides was molecularly characterized for the first time, showing the highest 94.21% identity to Thelohanellus sinensis (KY469292). Phylogenetically, the affinity of both M. artus and M. basilamellaris to their conspecific species derived from different carp lineages was highly supported. Moreover, the intermixed cluster of Myxobolus species, including M. elliptoides, with those of Thelohanellus species provided evidence for querying the monophyletic evolution history of these taxa. This work updates the appreciation of the concerned species and enhances our understanding of the parasite fauna of common carp.

Identification of Myxobolus distalisensis n. sp. (Cnidaria: Myxozoa) infecting yellow catfish Tachysurus fulvidraco (Richardson), with a supplement description of M. voremkhai (Akhmerov, 1960) Landsberg and Lom, 1991.

With growing scale of intensive fish cultivation, the risk of parasite infection in commercial fish is increased. Precisely identifying and characterizing the parasites that infect the farmed fish is critical to understanding the dynamics of their communities. Here, two species of Myxobolus were identified in farmed yellow catfish Tachysurus fulvidraco (Richardson) in China. Myxobolus distalisensis n. sp. developed plasmodia in the gill filaments, with oval to elliptical myxospores measuring 11.3 ± 0.6 (10.4-12.6) × 8.1 ± 0.3 (7.5-8.6) × 5.5 ± 0.2 (5.2-5.8) µm. Two pyriform polar capsules of equal size were measured 5.3 ± 0.4 (4.5-6.3) × 2.7 ± 0.1 (2.3-3) µm. Myxobolus voremkhai (Akhmerov, 1960) Landsberg and Lom, 1991 developed plasmodia in the gill arch and had a myxospore morphology similar to the conspecific isolates described in previous studies. The consensus sequences of M. distalisensis was remarkably distinct from those deposited in the GenBank, with exception of whereas M. voremkhai showing 99.84% identity. The genetic data on both isolates differed considerably from each other, revealing only 86.96% molecular identity. Histologically, M. distalisensis resided in the filament cartilage, and the aggressive proliferation of the sporogenic stages led to lytic cartilage corrosion. In contrast, plasmodia of M. voremkhai grossly observed at the base of the gill filament were embedded by the connective tissue in the gills arch. Phylogenetically, both isolates were separately placed in different subclades, indicating difference in their evolutionary history. Besides, the taxon under the family Myxobolidae was demonstrated non-monophyletic origins, and parasite radiation largely followed their host affinity.

LEGACY: Phase 2a Trial to Evaluate the Safety, Pharmacokinetics, and Pharmacodynamic Effects of the Anti-EL (Endothelial Lipase) Antibody MEDI5884 in Patients With Stable Coronary Artery Disease.

OBJECTIVE: Functional HDL (high-density lipoprotein) particles that facilitate cholesterol efflux may be cardioprotective. EL (endothelial lipase) hydrolyzes phospholipids promoting catabolism of HDL and subsequent renal excretion. MEDI5884 is a selective, humanized, monoclonal, EL-neutralizing antibody. We sought to determine the safety, pharmacokinetics, and pharmacodynamic effects of multiple doses of MEDI5884 in patients with stable coronary artery disease. Approach and Results: LEGACY was a phase 2a, double-blind, placebo-controlled, parallel-design trial that randomized 132 patients with stable coronary artery disease receiving high-intensity statin therapy to 3 monthly doses of 1 of 5 dose levels of MEDI5884 (50, 100, 200, 350, or 500 mg SC) or matching placebo. The primary end point was the safety and tolerability of MEDI5884 through the end of the study (day 151). Additional end points included change in HDL cholesterol and cholesterol efflux from baseline to day 91, hepatic uptake of cholesterol at day 91, changes in various other lipid parameters. The incidence of adverse events was similar between the placebo and MEDI5884 groups. In a dose-dependent manner, MEDI5884 increased HDL cholesterol up to 51.4% (P<0.0001) and global cholesterol efflux up to 26.2% ([95% CI, 14.3-38.0] P<0.0001). MEDI5884 increased HDL particle number up to 14.4%. At the highest dose tested, an increase in LDL (low-density lipoprotein) cholesterol up to 28.7% (P<0.0001) and apoB (apolipoprotein B) up to 13.1% (P=0.04) was observed with MEDI5884. However, at the potential target doses for future studies, there was no meaningful increase in LDL cholesterol or apoB. CONCLUSIONS: Inhibition of EL by MEDI5884 increases the quantity and quality of functional HDL in patients with stable coronary artery disease on high-intensity statin therapy without an adverse safety signal at the likely dose to be used. These data support further clinical investigation. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT03351738.

IgM-bearing B cell affinity subpopulations possess differential antigen sensitivity in rainbow trout.

The need for accurate assessments of in vitro generated antibody prompted examination of the effect of antigen on secreted antibody concentrations and affinities. It was found that the antigen concentrations commonly employed for in vitro stimulation were able to significantly compromise IgM titer and affinity estimates in rainbow trout. Specifically, IgM titers were underestimated with the high affinity antibodies being specifically blocked. To remedy this, pulsed antigen cultures were employed, and it was found to reveal more accurate IgM antibody titers and affinity estimates. Additionally, pulsed dose responses provided evidence that high antigen concentrations specifically suppressed high affinity B cell induction. Optimal expression of high affinity antibodies required exposure to lower concentrations of antigen. Each affinity subpopulation appeared to possess a graded sensitivity to each dose of antigen, revealing the complex dynamic for differential IgM-bearing B cell induction that is possible during a response. These results reveal not only the need for antigen removal prior to in vitro antibody secretion, but also the possible role of high zone immunological tolerance on IgM affinity maturation in rainbow trout.

Identification and functional characterization of CD154 in T cell-dependent immune response in Nile tilapia (Oreochromis niloticus).

CD154, a member of the TNF superfamily, is a multifunctional molecule highly expressed in activated T cells, and plays important roles in T cell-dependent humoral immune response. In this study, CD154 of Nile tilapia (Oreochromis niloticus) was identified, and its functions in the T cell-dependent immune response were demonstrated. The open reading frame (ORF) of OnCD154 is 699 bp, encoding a protein of 232 amino acids with a 23 amino acid transmembrane region. Amino acid sequence of OnCD154 is highly homologous to that of other teleost fish, especially rainbow trout. Quantitative real-time PCR (qRT-PCR) demonstrated that mRNA of OnCD154 is highly expressed in immune organs, especially in spleen, thymus, gills, head kidney, etc. In addition, the anti-OnCD154 polyclonal antibody (anti-(r)OnCD154) was successfully prepared, and it can react with natural protein in head kidney leukocytes. Following two immunizations with keyhole limpet hemocyanin (KLH) in vivo, the significantly up-regulated expression level of OnCD154 mRNA appeared earlier (fifth day) and higher (42.9 folds) in the second challenge than the first on in head kidney. Further, after stimulation with KLH in vitro, the expressions of T cell-dependent immune response-related molecules (activated T cell specific surface molecules CD3ε and CD154) and B cell differentiation-related molecules (Blimp1 and sIgM) and CD40 were significantly up-regulated in head kidney leukocytes. Moreover, the up-regulated expressions of these molecules were blocked with the treatment of anti-(r)OnCD154 antibody. Taken together, these results indicate that OnCD154 might get involved in T cell-dependent immune response, and provide a new insight into the humoral immune response of teleost fish.

Functional characterization of TNF-α in pufferfish (Takifugu obscurus) in immune response and apoptosis against Aeromonas hydrophila.

Tumour necrosis factor-α (TNF-α) is a multifunctional cytokine involved in immune system homeostasis, antimicrobial defence, regulation of apoptosis, cell proliferation and differentiation. Although the pro-inflammatory property of TNF-α has been made new progress, detailed research on host defence against bacterial infection and inducing apoptosis remains to be revealed in early vertebrates. Here, we reported the TNF-α homologue (ToTNF-α) from pufferfish (Takifugu obscurus). The open reading frame (ORF) of ToTNF-α was 753 bp, encoding a protein of 250 aa contained the TNF family signature and conserved cysteine residues. The mRNA expression of ToTNF-α had a wide range of tested tissues, with the highest expression in the skin. After Aeromonas hydrophila infection, the mRNA expression of ToTNF-α was significantly up-regulated both in vivo and in vitro experiments. After stimulation by recombinant protein of ToTNF-α ((r)ToTNF-α), the relative expressions of endogenous TNF-α, caspase 8, caspase 3, p53, and Bax inhibitor-1 in head kidney leucocytes were all notably up-regulated. These results showed that ToTNF-α might induce apoptosis depend on pro- and anti-apoptotic proteins at mRNA level. Moreover, flow cytometry analysis indicated that the (r)ToTNF-α can induce apoptosis of head kidney leucocytes. Taken together, these characteristics suggest that ToTNF-α can participate in immune response against A. hydrophila and induce apoptosis at mRNA and cellular level, which will help to understand the mechanism of apoptosis and immune response in teleost fish.

Characterisation of a copper/zinc superoxide dismutase from Pieris rapae and its role in protecting against oxidative stress induced by chlorantraniliprole.

Insecticide exposure typically leads to abnormally high levels of reactive oxygen species (ROS) and oxidative damage in insects. Superoxide dismutases (SODs) are potent antioxidant enzymes for ROS scavenging that are essential to protect insects against insecticide-induced oxidative injury. The small white butterfly, Pieris rapae, is an economically important lepidopteran pest of cruciferous crops, and the anthranilic diamide insecticide chlorantraniliprole is widely used to control this organism. However, whether chlorantraniliprole causes oxidative stress, and whether SODs are involved in ROS scavenging, remains unclear in P. rapae. In this study, an intracellular copper/zinc SOD (designated PrSOD1) gene was identified and characterised in P. rapae. The gene consists of four exons and three introns, and the PrSOD1 protein encoded by the gene has typical highly conserved features of CuZnSODs, including two signature motifs and seven Cu/Zn-interacting residues. Transcription of PrSOD1 was highest in the larval fat body and at the fifth-instar larval stage. Recombinant PrSOD1 protein expressed in Escherichia coli displayed antioxidant activity and high thermal and pH stability, confirming that PrSOD1 encodes a functional enzyme. Exposure to three sublethal doses of chlorantraniliprole for 6, 12 or 24 h resulted in significantly increased malondialdehyde concentration in P. rapae larvae, indicating insecticide-induced oxidative stress. Furthermore, both PrSOD1 transcription levels and CuZnSOD activity were quickly (6 and 12 h, respectively) upregulated in larvae subjected to chlorantraniliprole, strongly suggesting that PrSOD1 plays an important role in protecting against oxidative damage and possibly chlorantraniliprole tolerance in P. rapae.

Efficacy and safety of COVID-19 vaccines: a systematic review.

OBJECTIVE: To evaluate systematically the efficacy and safety of COVID-19 vaccines. METHODS: PubMed, Embase, Cochrane Library, Clinicaltrial.gov, CNKI, Wanfang Data, China Biomedical Literature Service System, and China Clinical Trial Registry were searched for randomized controlled trials of COVID-19 vaccines published up to December 31, 2020. The Cochrane bias risk assessment tool was used to assess the quality of studies. A qualitative analysis was performed on the results of clinical trials. RESULTS: Thirteen randomized, blinded, controlled trials, which involved the safety and efficacy of 11 COVID-19 vaccines, were included. In 10 studies, the 28-day seroconversion rate of subjects exceeded 80%. In two 10 000-scale clinical trials, the vaccines were effective in 95% and 70.4% of the subjects, respectively. The seroconversion rate was lower than 60% in only one study. In six studies, the proportion of subjects who had an adverse reaction within 28 days after vaccination was lower than 30%. This proportion was 30%-50% in two studies and > 50% in the other two studies. Most of the adverse reactions were mild to moderate and resolved within 24 hours after vaccination. The most common local adverse reaction was pain or tenderness at the injection site, and the most common systemic adverse reaction was fatigue, fever, or bodily pain. The immune response and incidence of adverse reactions to the vaccines were positively correlated with the dose given to the subjects. The immune response to the vaccines was worse in the elderly than in the younger population. In 6 studies that compared single-dose and double-dose vaccination, 4 studies showed that double-dose vaccination produced a stronger immune response than single-dose vaccination. CONCLUSIONS: Most of the COVID-19 vaccines appear to be effective and safe. Double-dose vaccination is recommended. However, more research is needed to investigate the long-term efficacy and safety of the vaccines and the influence of dose, age, and production process on the protective efficacy.

Identification and functional characterization of three caspases in Takifugu obscurus in response to bacterial infection.

Caspases are evolutionarily conserved proteases, which are inextricably linked with the apoptosis and immune system in mammals. However, the expression pattern and function of some caspases remain largely unknown in pufferfish. In this study, three different pufferfish caspases (caspase-2 (Pfcasp-2), caspase-3 (Pfcasp-3), and caspase-8 (Pfcasp-8)) were characterized, and their expression patterns and functions were determined following Aeromonas hydrophila infection. The open reading frames of Pfcasp-2, -3, and -8 are 1,320, 846, and 1455 bp, respectively. Analyses of sequence alignment and phylogenetic tree showed that casp-2, -3, and -8 share 52%-65%, 33%-40%, 63%-78% overall sequence identities with those of other vertebrates, respectively. 3D structures of Pfcasp-2, -3, and -8 enjoy conservation in core area together, while each owns a distinctive profile. Comparisons of deduced amino acid sequences indicated that Pfcaspases possessed the caspase domain and conserved active sites like 'HG' and 'QACXG' (X for R or G). qRT-PCR results revealed that Pfcasp-2, -3, and -8 were expressed constitutively in a wide range of organs, especially in immune-related organs including whole blood and kidney. In vitro, the expressions of the three caspases (Pfcasp-2, 3, and -8) and immune-related genes (IgM and IL-8) were significantly up-regulated in kidney leukocytes after A. Hydrophila challenge and inhibitors treatment. The expressions of Pfcasp-2 and Pfcasp-3 were successfully inhibited in the kidney leukocytes by Ac-DEVD-CHO (an inhibitor to caspase-3), but the expression of Pfcasp-8 was not affected. Cellular localization analysis showed that the distribution of Pfcasp-2, -3, and -8 was in cytoplasm. Further, overexpression of Pfcasp-2, -3, or -8 was found to cause DNA damage and apoptosis, suggesting that three caspases may be related to apoptosis and mediate different apoptosis pathways in pufferfish. Moreover, the expressions of these caspases were also up-regulated in whole blood and kidney after A. hydrophila challenge, indicating their possible involvement in the immune response against A. hydrophia stimulation. Taken together, the results of this study suggest that the caspase-2,-3, and -8 may play an important role in the apoptosis and immune response in pufferfish.

Proteome interrogation using gold nanoprobes to identify targets of arctigenin in fish parasites.

Gold nanoparticles (GNPs) are one of the most widely used nanomaterials in various fields. Especially, the unique chemical and physical properties make them as the promising candidates in drug target identification, unfortunately, little is known about their application in parasites. In this paper, GNPs were employed as new solid support to identify drug targets of natural bioactive compound arctigenin (ARG) against fish monogenean parasite Gyrodactylus kobayashi. Before target identification, GNPs with ARG on the surface showed the ability to enter the live parasites even the nucleus or mitochondria, which made the bound compounds capable of contacting directly with target proteins located anywhere of the parasites. At the same time, chemically modified compound remained the anthelminthic efficacy against G. kobayashii. The above results both provide assurance on the reliability of using GNPs for drug target-binding specificity. Subsequently, by interrogating the cellular proteome in parasite lysate, myosin-2 and UNC-89 were identified as the potential direct target proteins of ARG in G. kobayashii. Moreover, results of RNA-seq transcriptomics and iTRAQ proteomics indicated that myosin-2 expressions were down-regulated after ARG bath treatment both in transcript and protein levels, but for UNC-89, only in mRNA level. Myosin-2 is an important structural muscle protein expressed in helminth tegument and its identification as our target will enable further inhibitor optimization towards future drug discovery. Furthermore, our findings demonstrate the power of GNPs to be readily applied to other parasite drugs of unknown targets, facilitating more broadly therapeutic drug design in any pathogen or disease model.

Neovascularization by Sustained Delivery of G-CSF, EPO and VEGF Using Dextran/PLGA Microspheres.

BACKGROUND: Therapeutic neovascularization has some obstacles, such as it requires more than one proangiogenic factor, and these factors have short half-lives. To overcome these obstacles, combined delivery of granulocyte-colony stimulating factor (G-CSF), erythropoietin (EPO) and vascular endothelial growth factor (VEGF) using protein/dextran/poly (lactic-co-glycolic acid) (PLGA) sustained-release microspheres was proposed to promote neovascularization. METHODS: Dextran microparticles loaded with G-CSF, EPO or VEGF were prepared and encapsulated in PLGA microspheres to obtain protein-dextran-PLGA microspheres. The release behavior of microspheres was studied in vitro. The protein/dextran/PLGA microspheres were injected into the ischemic hindlimbs of rats. Neovascularization in ischemic muscle was measured. RESULTS: Microspheres released G-CSF, EPO and VEGF in vitro for more than 4 weeks. Combined therapy with VEGF, EPO and G-CSF promoted the expression of B-cell lymphoma-2 and stromal cell-derived factor 1, cellular proliferation and the incorporation of C-X-C chemokine receptor 4 positive cells. Capillary density and smooth muscle α-actin+ vessel density were higher in the combined treatment of VEGF, EPO and G-CSF than in the single factor treatment. CONCLUSIONS: The combined and sustained delivery of VEGF, EPO and G-CSF using dextran-PLGA microspheres had a more significant neovascularization effect than monotherapy with each factor alone. This combined therapy might be a promising treatment for ischemic vascular diseases.

Isolation of anti-Saprolegnia lignans from Magnolia officinalis and SAR evaluation of honokiol/magnolol analogs.

To control the fish fungal pathogen Saprolegnia, the effects of the petroleum ether extracts of Magnolia officinalis were evaluated by a rapeseed (Brassicanapus) microplate method in vitro. By loading on an open silica gel column and eluting with petroleum ether-ethyl acetate-methanol, honokiol (C18H18O2) and magnolol (C18H18O2) were isolated from Magnolia officinalis. Saprolegnia parasitica growth was inhibited significantly when honokiol concentration was >8.0 mg/L, and magnolol concentration was >9.0 mg/L, with EC50 values of 4.38 and 4.92 mg/L, respectively. Six honokiol and magnolol derivatives were designed, synthesized and evaluated for their anti-Saprolegnia activity. According to the results, double bond and hydroxyl played an important role in inhibiting Saprolegnia. Mechanistically, through the scanning electron microscope observation, honokiol and magnolol could cause the Saprolegnia parasitica mycelium tegumental damage including intensive wrinkles and nodular structures. Moreover, compared to traditional drugs kresoxim-methyl (LC50 = 0.66 mg/L) and azoxystrobin (LC50 = 2.71 mg/L), honokiol and magnolol showed a lower detrimental effect on zebrafish, with the LC50 values of 6.00 and 7.28 mg/L at 48 h, respectively. Overall, honokiol and magnolol were promising lead compounds for the development of commercial drugs anti-Saprolegnia.

Cytokine gene expression profiles in goldfish (Carassius auratus) during Gyrodactylus kobayashii infection.

Monogeneans of the genus Gyrodactylus are well-known pathogens causing huge mortalities in wild and cultured fish. Cytokine expression is one of most important host defense mechanisms against parasite infections. In this study, the expression pattern of the key pro-inflammatory (IL-1ß, IL-8, IFN-γ, TNF-α, IL-12 and iNOS) and anti-inflammatory cytokine genes (IL-10, TGFß and IL-4) of Gyrodactylus kobayashii infected goldfish (Carassius auratus) were determined by real-time quantitative PCR analysis. Our results showed that G. kobayashii infection caused increased expression of the pro-inflammatory cytokines including IFN-γ, TNF-α and iNOS in all detected tissues throughout the infection period. Among these genes, iNOS has the highest transcript level accompanied with increased nitric oxide (NO) concentration in the serum of all infected goldfish. The mRNA level of IL-1ß in the liver, spleen and head kidney was significantly up-regulated during the early stage of infection (days 2-8). While high expression level of IL-8 and IL-12 was observed during the elimination phase of infection (days 10-14). As for anti-inflammatory cytokines, the expression profiles of IL-10 were distinct from those of TGF-ß and IL-4. Specifically, the mRNA level of IL-10 did not increase in the spleen and head kidney during the early stage of infection, while increased expression of TGF-ß and IL-4 were likewise seen. Besides, all infected fish had significantly higher complement C3 but lower IgM levels than the non-infected fish. The results provide insights into the interaction between gyrodactylids and the fish host, and indicate that systemic cytokine responses are critical for controlling parasite infection in fish.

Immunosuppression-induced alterations in fish gut microbiota may increase the susceptibility to pathogens.

Intestinal bacteria play an important role in the health and provide a variety of beneficial effects to host. Immunosuppressant can reduce the immunity of host and increase the susceptibility to pathogens. But it is not clear whether the increased susceptibility caused by immunosuppressant is related to changes of gut microbiota. In this study, we used crucian carp administrated with dexamethasone to explore the effects of immunosuppressants on gut microbial communities and further evaluate the potential association between changes in gut microbiota and susceptibility to pathogens. The results of MANOVA based on the top 10 PCoA axis scores from unweighed/weighted UniFrac distances showed that administration of dexamethasone (P = 0.021) and the administration time (P = 0.027) had a significant impact on the gut microbial composition, regardless of pathogens infection status (P = 0.35). After administration with dexamethasone, the fish had higher abundance of Cetobacterium and lower abundance of Bacillus and Lactococcus, and the abundance of genus Bacillus, Pseudomonas and Lactococcus decreased along with prolong administration time of dexamethasone. The results may help us understand the correlation between the host susceptibility to pathogenic bacteria and gut microbial community shift, and extend our knowledge regarding the role of gut microbiota in keeping the balance between pathogenic and symbiotic bacteria.

Fabrication of Thermosensitive Cyclic Brush Copolymer with Enhanced Therapeutic Efficacy for Anticancer Drug Delivery.

Adaptation of cyclic brush polymer for drug delivery applications remains largely unexplored. Herein, cyclic brush copolymer of poly(2-hydroxyethyl methacrylate-g-poly(N-isopropylacrylamide-st-N-hydroxyethylacrylamide)) (cb-P(HEMA-g-P(NIPAAm-st-HEAAm))), comprising a cyclic core of PHEMA and thermosensitive brushes of statistical copolymer of P(NIPAAm-st-HEAAm), is designed and synthesized successfully via a graft-from approach using atom transfer free radical polymerization from a cyclic multimacroinitiator. The composition of the brush is optimized to endow the resulting cyclic brush copolymer with a lower critical solution temperature (LCST) slightly above the physiological temperature, but lower than the localized temperature of tumor tissue, which is suitable for the hyperthermia-triggered anticancer drug delivery. Critical aggregation concentration determination reveals better stability for the unimolecular nanoparticle formed by the cyclic brush copolymer than that formed by the bottlebrush analogue. The dramatically increased size with elevated temperatures from below to above the LCST confirms hyperthermia-induced aggregation for both formulations. Such structural destabilization promotes significantly the drug release at 40 °C. Most importantly, the drug-loaded cyclic brush copolymer shows enhanced in vitro cytotoxicity against HeLa cells than the bottlebrush counterpart. The better stability and higher therapeutic efficacy demonstrates that the thermosensitive cyclic brush copolymer is a better formulation than bottle brush copolymer for controlled drug release applications.

Molecular characterization of a typical 2-Cys thioredoxin peroxidase from the Asiatic rice borer Chilo suppressalis and its role in oxidative stress.

In insects, thioredoxin peroxidase (TPX) plays an important role in protecting against oxidative damage. However, studies on the molecular characteristics of TPXs in the Asiatic rice borer, Chilo suppressalis, are limited. In this work, a cDNA sequence (CsTpx3) encoding a TPX was identified from C. suppressalis. The deduced CsTPX3 protein shares high sequence identity and two positionally conserved cysteines with orthologs from other insect species, and was classified as a typical 2-Cys TPX. CsTpx3 was expressed most highly during the fifth-instar larval stage, and transcripts were most abundant in the midgut. Recombinant CsTPX3 protein expressed in Escherichia coli displayed the expected peroxidase activity by removing H2 O2 . Furthermore, CsTPX3 protected DNA from oxidative damage, and E. coli cells overexpressing CsTPX3 exhibited long-term resistance to oxidative stress. Exposure to various oxidative stressors, such as cold (8°C), heat (35°C), bacteria (E. coli), and two insecticides (chlorpyrifos and lambda-cyhalothrin), significantly upregulated transcription of CsTpx3. However, exposure to abamectin had no such effect. Our results provide valuable information for future studies on the antioxidant mechanism in this insect species.