Transcriptomic and antiviral analyses of PoIFN-Delta5 against porcine enteric viruses in porcine intestinal epithelial cells.

The interferon-delta family was first reported in domestic pigs and belongs to the type I interferon (IFN-I) family. The enteric viruses could cause diarrhea in newborn piglets with high morbidity and mortality. We researched the function of the porcine IFN-delta (PoIFN-δ) family in the porcine intestinal epithelial cells (IPEC-J2) cells infected with porcine epidemic diarrhea virus (PEDV). Our study found that all PoIFN-δs shared a typical IFN-I signature and could be divided into five branches in the phylogenic tree. Different strains of PEDV could induce typical IFN transitorily, and the virulent strain AH2012/12 had the strongest induction of porcine IFN-δ and IFN-alpha (PoIFN-α) in the early stage of infection. In addition, it was found that PoIFN-δ5/6/9/11 and PoIFN-δ1/2 were highly expressed in the intestine. PoIFN-δ5 had a better antiviral effect on PEDV compared to PoIFN-δ1 due to its higher induction of ISGs. PoIFN-δ1 and PoIFN-δ5 also activated JAK-STAT and IRS signaling. For other enteric viruses, transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus (PoRV), PoIFN-δ1 and PoIFN-δ5 both showed an excellent antiviral effect. Transcriptome analyses uncovered the differences in host responses to PoIFN-α and PoIFN-δ5 and revealed thousands of differentially expressed genes were mainly enriched in the inflammatory response, antigen processing and presentation, and other immune-related pathways. PoIFN-δ5 would be a potential antiviral drug, especially against porcine enteric viruses. These studies were the first to report the antiviral function against porcine enteric viruses and broaden the new acquaintances of this type of interferon though not novelly discovered.

Disruption of T-box transcription factor eomesa results in abnormal development of median fins in Oujiang color common carp Cyprinus carpio.

Median fins are thought to be ancestors of paired fins which in turn give rise to limbs in tetrapods. However, the developmental mechanisms of median fins remain largely unknown. Nonsense mutation of the T-box transcription factor eomesa in zebrafish results in a phenotype without dorsal fin. Compared to zebrafish, the common carp undergo an additional round of whole genome duplication, acquiring an extra copy of protein-coding genes. To verify the function of eomesa genes in common carp, we established a biallelic gene editing technology in this tetraploidy fish through simultaneous disruption of two homologous genes, eomesa1 and eomesa2. We targeted four sites located upstream or within the sequences encoding the T-box domain. Sanger sequencing data indicated the average knockout efficiency was around 40% at T1-T3 sites and 10% at T4 site in embryos at 24 hours post fertilization. The individual editing efficiency was high to about 80% at T1-T3 sites and low to 13.3% at T4 site in larvae at 7 days post fertilization. Among 145 mosaic F0 examined at four months old, three individuals (Mutant 1-3) showed varying degrees of maldevelopment in the dorsal fin and loss of anal fin. Genotyping showed the genomes of all three mutants were disrupted at T3 sites. The null mutation rates on the eomesa1 and eomesa2 loci were 0% and 60% in Mutant 1, 66.7% and 100% in Mutant 2, and 90% and 77.8% in Mutant 3, respectively. In conclusion, we demonstrated a role of eomesa in the formation and development of median fins in Oujiang color common carp and established an method that simultaneously disrupt two homologous genes with one gRNA, which would be useful in genome editing in other polyploidy fishes.

The synergy of thanatin and cathelicidin-BF-15a3 combats Escherichia coli O157:H7.

Escherichia coli O157:H7 is a pathogen that commonly causes foodborne illness and represents a health hazard to consumers. The combined use of synergistic antimicrobial peptides (AMPs) is a promising way to improve the microbiological safety of foods. In this study, we detected the synergistic interactions between thanatin and BF-15a3 to reduce their usage and obtain more efficient antibacterial activity. The minimal inhibitory concentrations (MICs) of thanatin and BF-15a3 against 49 E. coli O157:H7 strains were ranged from 2 to 8 µg/mL and 4-32 µg/mL, showed a general inhibitory effect on E. coli O157:H7 strains, respectively, even multidrug-resistant strains. Their fractional inhibitory concentration index (FICI) was 0.375, which suggested that their combination presented synergistic antibacterial effect against E. coli O157:H7. The killing kinetic curves indicated that the 0.25 × MIC combination had equivalent bactericidal effects to 1 × MIC thanatin or BF-15a3. When AMP combinations were used to treat eukaryotic cells to evaluate the hemolytic characteristics against rabbit erythrocytes and cytotoxicity against human embryonic kidney 293T (HEK-293T) cells and intestinal porcine enterocyte J2 (IPEC-J2) cells, no magnified adverse effects were observed, exhibiting higher specificity to bacteria and lower toxicity to eukaryotic cells. Compared with bacteriostasis of thanatin or BF-15a3 alone, the proportion of membrane-damaged bacteria treated with the synergetic combination did not appear a significant rise, interestingly the Zeta potential of them greatly decreased and their cell membrane permeability significantly increased. Besides, more release of ions and cytoplasm were detected, confirming a more severe loss of membrane integrity. These results suggested that the synergistic action mode of thanatin and BF-15a3 is likely attributed to damage aggravation to E. coli membrane. When applying in fresh-cut lettuce and cucumber, their combination allowed for 2.5 log CFU/piece reductions of E. coli O157:H7 in 24 h. In conclusion, the combination of thanatin and BF-15a3 showed excellent synthetic efficacy to kill E. coli O157:H7 in vitro under lower MICs than single use of them.

Achog1 is required for the asexual sporulation, stress responses and pigmentation of Aspergillus cristatus.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea; hypotonic conditions only induce its sexual development to produce ascospores, while hypertonic conditions only induce its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the role of Achog1, which is homologous to hog1 from Saccharomyces cerevisiae, in sporulation, different kinds of stress responses and pigment production was investigated. Deletion mutants of Achog1 were obtained by homologous recombination. Phenotypic observations showed that the time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in the deletion mutants of Achog1 in hypertonic media, indicating that Achog1 plays a positive role in asexual development. Stress sensitivity tests showed that ΔAchog1 strains were sensitive to hyperosmolarity, and the order of the sensitivity of ΔAchog1 to different osmotic regulators was 3 M sucrose >3 M NaCl >3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. pH sensitivity tests indicated that Achog1 inhibited the growth of A. cristatus under alkaline stress. Additionally, pigmentation was decreased in the Achog1 deletion mutants compared with the WT. All the above developmental defects were reversed by the reintroduction of the Achog1 gene in ΔAchog1. Pull-down and LC-MS/MS analysis showed that the expression levels of proteins interacting with Achog1 were significantly different under low and high osmotic stress, and proteins related to conidial development were present only in the cultures treated with hyperosmotic stress. Transcription profiling data showed that Achog1 suppressed the expression of several genes related to asexual development, osmotic and oxidative stress resistance. On the basis of gene knockout, pull-down mass spectrometry and RNA-seq analyses, a regulatory pathway for Achog1 was roughly identified in A. cristatus.

The role of Acpbs2 in the asexual sporulation, stress response and carbon metabolism of Aspergillus cristatus.

Aspergillus cristatus is the dominant fungus during the fermentation of Fuzhuan brick tea, hypotonic conditions only induced its sexual development to produce ascospores, while hypertonic conditions only induced its asexual development to produce conidia, indicating that osmotic stress can regulate spore production in A. cristatus. However, the underlying regulatory mechanism is unclear. In this study, the roles of Acpbs2, which is homologous to pbs2 from Saccharomyces cerevisiae, in sporulation, stress responses, the color of colonies, and carbon metabolism were explored in A. cristatus. Deletion mutants of Acpbs2 were obtained by homologous recombination. The time required to produce conidia was delayed, and the number of conidia produced was significantly reduced in hypertonic media in ΔAcpbs2 by phenotypic observations, indicating that Acpbs2 plays a positive role in asexual development. Stress sensitivity tests showed that the order of the sensitivity of ΔAcpbs2 to different osmotic regulators was 3 M NaCl > 3 M sucrose > 3 M sorbitol. Moreover, the deletion mutants were sensitive to high oxidative stress. The growth of the Acpbs2 deletion mutant was inhibited under alkaline-pH stress, indicating that Acpbs2 is involved in high pH stress tolerance. Additionally, compared with the wild type, the colony color of the Acpbs2 deletion mutant became lighter. All the above developmental defects were reversed by the reintroduction of the Acpbs2 gene in ΔAcpbs2. Transcriptome data showed that Acpbs2 regulated the expression of several genes related to conidial development, osmotic stress, oxidative stress, and carbon metabolism. More importantly, the interaction between Acpbs2 and its downstream gene Achog1 was verified by yeast two-hybrid assays. We speculated that this interaction might regulate the osmotic stress response, the oxidative stress response, and asexual sporulation in A. cristatus, which will be one of the focuses of our future research.

Nonstructural Protein 1 of Variant PEDV Plays a Key Role in Escaping Replication Restriction by Complement C3.

Zoonotic coronaviruses represent an ongoing threat to public health. The classical porcine epidemic diarrhea virus (PEDV) first appeared in the early 1970s. Since 2010, outbreaks of highly virulent PEDV variants have caused great economic losses to the swine industry worldwide. However, the strategies by which PEDV variants escape host immune responses are not fully understood. Complement component 3 (C3) is considered a central component of the three complement activation pathways and plays a crucial role in preventing viral infection. In this study, we found that C3 significantly inhibited PEDV replication in vitro, and both variant and classical PEDV strains induced high levels of interleukin-1ß (IL-1ß) in Huh7 cells. However, the PEDV variant strain reduces C3 transcript and protein levels induced by IL-1ß compared with the PEDV classical strain. Examination of key molecules of the C3 transcriptional signaling pathway revealed that variant PEDV reduced C3 by inhibiting CCAAT/enhancer-binding protein ß (C/EBP-ß) phosphorylation. Mechanistically, PEDV nonstructural protein 1 (NSP1) inhibited C/EBP-ß phosphorylation via amino acid residue 50. Finally, we constructed recombinant PEDVs to verify the critical role of amino acid 50 of NSP1 in the regulation of C3 expression. In summary, we identified a novel antiviral role of C3 in inhibiting PEDV replication and the viral immune evasion strategies of PEDV variants. Our study reveals new information on PEDV-host interactions and furthers our understanding of the pathogenic mechanism of this virus. IMPORTANCE The complement system acts as a vital link between the innate and the adaptive immunity and has the ability to recognize and neutralize various pathogens. Activation of the complement system acts as a double-edged sword, as appropriate levels of activation protect against pathogenic infections, but excessive responses can provoke a dramatic inflammatory response and cause tissue damage, leading to pathological processes, which often appear in COVID-19 patients. However, how PEDV, as the most severe coronavirus causing diarrhea in piglets, regulates the complement system has not been previously reported. In this study, for the first time, we identified a novel mechanism of a PEDV variant in the suppression of C3 expression, showing that different coronaviruses and even different subtype strains differ in regulation of C3 expression. In addition, this study provides a deeper understanding of the mechanism of the PEDV variant in immune escape and enhanced virulence.

Kinematics and Aerodynamics of Dragonflies (Pantala flavescens, Libellulidae) in Climbing Flight.

This study presents a detailed analysis of dragonflies' climbing flight by integratinghigh-speed photogrammetry, three-dimensional reconstruction, and computational fluid dynamics. In this study, a dragonfly's climbing flight is captured by two high-speed cameras with orthogonal optical axes. Through feature point matching and three-dimensional reconstruction, the body kinematics and wing kinematics of 22 dragonflies in climbing flight are accurately captured. Experimental results show that the climbing angles (η) are distributed from 10° to 80° and are concentrated within two ranges, 60°-70° (36%) and 20°-30° (32%), which are defined as large angle climb (LAC) and small angle climb (SAC), respectively. In order to study the aerodynamic mechanism of the climbing flight based on the biological observation results, the kinematic parameters of the dragonfly during LAC and SAC are selected for analysis and numerical simulation. The results show that the climbing angle η and wing kinematics are related. There are considerable differences in wing kinematics during climbing with different η, while the wing kinematics are unchanged during climbing with similar η. With the increase in η, the phase difference (λ) between the forewing and the hind wing decreases and the amplitude of the positional angle (θ mean) of the hind wing increases, while θ mean of the forewing remains almost unchanged. Through numerical simulation of LAC and SAC, it can be found that during the climb with different η, the different wing kinematics have a significant influence on aerodynamic performance. During SAC, the increase in λ and the decrease in θ mean of the hind wing weaken the aerodynamic disturbance of the forewing by the vortex wing of the hind wing, thus improving the flight efficiency.

Global tissue transcriptomic analysis to improve genome annotation and unravel skin pigmentation in goldfish.

Goldfish is an ornamental fish with diverse phenotypes. However, the limited genomic resources of goldfish hamper our understanding of the genetic basis for its phenotypic diversity. To provide enriched genomic resources and infer possible mechanisms underlying skin pigmentation, we performed a large-scale transcriptomic sequencing on 13 adult goldfish tissues, larvae at one- and three-days post hatch, and skin tissues with four different color pigmentation. A total of 25.52 Gb and 149.80 Gb clean data were obtained using the PacBio and Illumina platforms, respectively. Onto the goldfish reference genome, we mapped 137,674 non-redundant transcripts, of which 5.54% was known isoforms and 78.53% was novel isoforms of the reference genes, and the remaining 21,926 isoforms are novel isoforms of additional new genes. Both skin-specific and color-specific transcriptomic analyses showed that several significantly enriched genes were known to be involved in melanogenesis, tyrosine metabolism, PPAR signaling pathway, folate biosynthesis metabolism and so on. Thirteen differentially expressed genes across different color skins were associated with melanogenesis and pteridine synthesis including mitf, ednrb, mc1r, tyr, mlph and gch1, and xanthophore differentiation such as pax7, slc2a11 and slc2a15. These transcriptomic data revealed pathways involved in goldfish pigmentation and improved the gene annotation of the reference genome.

Pressure Drop in Tortuosity/Kinking of the Internal Carotid Artery: Simulation and Clinical Investigation.

Background. Whether carotid tortuosity/kinking of the internal carotid artery leads to cerebral ischemia remains unclear. There is very little research about the hemodynamic variation induced by carotid tortuosity/kinking in the literature. The objective of this study was to research the blood pressure changes induced by carotid tortuosity/kinking. Methods. We first created a geometric model of carotid tortuosity/kinking. Based on hemodynamic boundary conditions, the hemodynamics of carotid tortuosity and kinking were studied via a finite element simulation. Then, an in vitro system was built to validate the numerical simulation results. The mean arterial pressure changes before and after carotid kinking were measured using pressure sensors in 12 patients with carotid kinking. Results. Numerical simulation revealed that the pressure drops increased with increases in the kinking angles. Clinical tests and in vitro experiments confirmed the numerical simulation results. Conclusions. Carotid kinking leads to blood pressure reduction. In certain conditions, kinking may affect the cerebral blood supply and be associated with cerebral ischemia.

Physicochemical properties of exopolysaccharide produced by Lactobacillus kefiranofaciens ZW3 isolated from Tibet kefir.

An exopolysaccharide (EPS) producing strain, ZW3, was isolated from Tibet kefir grain and was identified as Lactobacillus kefiranofaciens. FT-IR spectroscopy revealed the presence of carboxyl, hydroxyl, and amide groups, which correspond to a typical heteropolymeric polysaccharide. The GC analysis of ZW3 EPS revealed that it was glucogalactan in nature. Exopolymer showed similar flocculation stability like xanthan gum but better than guar gum with a melting point of 93.38 degrees C which is lower than xanthan gum (153.4 degrees C) and guar gum (490.11 degrees C). Compared with other commercially available hydrocolloids like xanthan gum, guar gum and locust gum ZW3 EPS showed much better emulsifying capability.

[Spectral analysis and synthesis of artificial antigen of the organophosphors pesticide methamidophos].

In the present paper, a hapten of methamidophos was synthesized and conjugated with KLH by active ester method, thus the first artificial antigen was obtained. By diazotization method methamidophos conjugated with BSA, and the second artificial antigen was obtained. The synthesized haptens were characterized by MS, IR and 1H NMR, and the two artificial antigens were determined by the method of IR spectrum. The result implied that both the artificial antigens have absorbance peaks of hapten and protein, indicating that they were prepared successfully. This could provide evidence that the method of IR spectrum can be used to determine whether the artificial antigens are synthesized successfully.

Structure of a king cobra phospholipase A2 determined from a hemihedrally twinned crystal.

An acidic PLA(2) (OH APLA(2)-II) from the venom of Ophiophagus hannah (king cobra) shows greater phospholipase A(2) activity and weaker cardiotoxic and myotoxic activity than a homologous acidic PLA(2) from the same venom. The crystal of the enzyme belongs to space group P6(3). The crystals are invariably hemihedrally twinned, exhibiting perfect 622 Laue symmetry. The structure was determined by molecular replacement and refined using a hemihedral twinning program at 2.1 A resolution. The final model has reasonable stereochemistry and a crystallographic R factor of 19.5% (R(free) = 21.5%). The structure reveals the molecular arrangement and the mode of twinning. There are six independent molecules in the asymmetric unit. Owing to the presence of a non-crystallographic twofold parallel to the hemihedral twinning twofold, the molecular packing in the twinned crystal is extremely similar to that in an untwinned crystal for four of the molecules. This unique molecular arrangement may be related to the difficulty in recognizing the twinning. The structure was compared with the previously determined structure of a homologous acidic PLA(2) from the same source. The comparison shows structural changes that might be implicated in the increased catalytic activity and weakened toxicity.

Structure of a cardiotoxic phospholipase A(2) from Ophiophagus hannah with the "pancreatic loop".

The crystal structure of an acidic phospholipase A(2) from Ophiophagus hannah (king cobra) has been determined by molecular replacement at 2.6-A resolution to a crystallographic R factor of 20.5% (R(free)=23.3%) with reasonable stereochemistry. The venom enzyme contains an unusual "pancreatic loop." The conformation of the loop is well defined and different from those in pancreas PLA(2), showing its structural variability. This analysis provides the first structure of a PLA(2)-type cardiotoxin. The sites related to the cardiotoxic and myotoxic activities are explored and the oligomer observed in the crystalline state is described.

Crystal structures of an acidic phospholipase A(2) from the venom of Naja Kaouthia.

A phospholipase A(2) purified from the venom of Naja kaouthia (Guangxi cobra) exhibits anticoagulant activities. The structures of two crystal forms were determined by X-ray crystallography at 2.8A resolution with the Naja naja (India cobra) PLA(2) as an initial model. The enzyme exhibits a trimer structure, which is similar to that of India cobra PLA(2). This reinforces the physiological relevance of the oligomer. The trimer has a wide cavity, which allows the substrate to enter and interact with the catalytic site. The formation of the trimer may serve as a storage method to improve the solubility at high concentration in the venom gland. The Ca2+ binding loop in the absence of the cation can exist in different conformations depending on its surroundings.

Structure of an acidic phospholipase A2 from the venom of Deinagkistrodon acutus.

An acidic phospholipase A(2) was purified from Deinagkistrodon acutus (Agkistrodon acutus) which displays an inhibitory effect on platelet aggregation. The three-dimensional structure of the enzyme was determined by molecular replacement at 2.6 A resolution with a crystallographic R factor of 18.40% (R(free) = 22.50%) and reasonable stereochemistry. Two molecules in the asymmetric unit form a dimer and the dimer formation accompanies a significant conformational adaptation of segment 14-23, a constituent of the 'interface recognition site' (IRS). This probably reflects the inherent structural flexibility of the IRS. The possible expansion of the site for inhibiting platelet aggregation as proposed previously [Wang et al. (1996), J. Mol. Biol. 255, 669-676] is discussed.

Preliminary Crystallographic Analysis of Phospholipase A(2) from Naja naja kaouthia Lesson Venom.

An acidic phospholipase A(2) isolated from the venom of Naja naja kaouthia Lesson in Guangxi exhibits anticoagulative and hemolytic activities. In this work, the enzyme was crystallized by the method of hanging drop vapor diffusion. Two crystal forms were obtained and characterized by X-ray diffraction. One of them belonged to space group P 4 ( 3 ) 2 ( 1 )2 or P4(1)2(1)2 with unit cell parameters a b 8.797 nm, c 10.831 nm and there were three molecules per asymmetric unit the other belonged to space group P2(1)3 with unit cell parameters a b c 6.840 nm and there was one molecule per asymmetric unit. The diffraction data were collected up to 0.28 nm for each crystal form. The crystal properties of Naja naja verom phospholipase A(2) from different geographical regions are compared.

Structure Determination of Basic Phospholipase A(2) from the Venom of Agkistrodon halys Pallas in A New Monoclinic Crystal Form.

Basic phospholipase A(2) from the venom of Agkistrodon halys Pallas ( Agkistrodin blomhoffii brevicaudus ) exhibits hemolytic and anti-coagulant activities. A new monoclinic crystal form with four molecules per asymmetric unit was grown in the absence of n-octyl beta-o-glucopyranoside (beta-OG). The enzyme structure was determined by the molecular replacement method. The combined analysis of self- and cross- rotation function was used and non-crystallographic symmetry restraints were imposed to the structure refinement. The final model gave an acceptable crystallographic R factor and reasonable stereochemistry. Two molecules formed an interfacial-recognition-site linked dimer and two such dimers constituted a tetramer having pseudo 222 symmetry. Structural comparison with previously reported monoclinic forms, in which beta-OG was bound, showed that the variation of crystallization conditions had effects on the crystal packing, leading to significant changes of the cell parameters. Nevertheless, the structures of both the dimer and tetramer in the two crystal forms closely resembled to each other, indicating that the oligomers found in the monoclinic crystal forms were stable.