Numerical Investigation of Funicular Liquid Bridges between Three Spherical Grains in a Bidisperse Particulate System.

Appropriate capillary effects are beneficial for controlling the wet powder performance and agglomerate formation. As water content rises, the funicular regime supplants the pendular regime as the predominant state in wet granular media. The displacement of grains leading to the stretching of funicular liquid bridges until rupture is an interesting and common phenomenon. Utilizing Surface Evolver software (an energy minimization approach), this work develops an efficient and accurate numerical model to describe liquid interactions among three spherical grains. The effects of liquid volume, contact angle, grain size ratio, grain-pair gap, and separation distance on the capillary forces and rupture distances are investigated. Notably, we present a modified closed-form equation for predicting the rupture distance of funicular bridges between three grains, which reflects the coupled effects of the contact angle, grain size, and liquid volume on rupture distance. This present study provides insights for incorporating capillary effects into mechanical models relying on microassembly composed of several grains in bidisperse particulate systems. Additionally, the numerical findings confirm some findings regarding the splitting of funicular bridges.

Discovery of anti-inflammatory agents from 3, 4-dihydronaphthalene-1(2H)-one derivatives by inhibiting NLRP3 inflammasome activation.

NLRP3 inflammatory vesicles are a polymer of cellular innate immunity composed of a pair of proteins. The continuous activation of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammatory vesicles induces the occurrence and enhancement of inflammatory response. In this study, a series of 3, 4-dihydronaphthalene-1(2H)-one derivatives (DHNs, 6a-u, 7a-e, 8a-n) were synthesized and characterized by NMR and HRMS. We evaluated the cytotoxicity and anti-inflammatory activity of all compounds in vitro, and selected 7a substituted by 7-Br in A-ring and 2-pyridylaldehyde in C-ring as effective lead compounds. Specifically, 7a can block the assembly and activation of NLRP3 inflammasome by down-regulating the expression of NLPR3 and apoptosis-associated speck-like protein containing a CARD (ASC), and inhibiting the production of reactive oxygen species (ROS) and other inflammatory mediators. In addition, 7a inhibits the phosphorylation of inhibitor kappa B alpha (IκBα) and NF-κB/p65 and the nuclear translocation of p65, thereby inhibiting nuclear factor kappa-B (NF-κB) signaling. Molecular docking analysis confirmed that 7a could reasonably bind the active sites of NLRP3, ASC and p65 proteins. Therefore, 7a is predicted as a potential NLRP3 inflammatory vesicle inhibitor and deserves further research and development.

Effects of plant residues on C:N:P of soil, microbial biomass, and extracellular enzyme in an alpine mea-dow on the Qinghai-Tibetan Plateau, China.

Plant residues can affect C:N:P of soil, microbial biomass, and extracellular enzyme, but the effects are still unclear. We conducted a field experiment in an alpine meadow on the eastern part of the Qinghai-Tibetan Plateau to explore the effects of removing aboveground plant or roots and adding plant residues on the C:N:P of soil, microbial biomass, and extracellular enzyme. The results showed that removing aboveground plant biomass significantly decreased soil C:N (the change was -23.7%, the same below) and C:P (-14.7%), microbial biomass C:P and N:P, while significantly increased microbial biomass C:N, and enzyme C:N:P compared with meadow without human disturbance. Removing all plant biomass (aboveground and roots) significantly reduced soil C:N (-11.6%), C:P (-24.0%), N:P (-23.3%) and microbial biomass C:N in comparison to removing aboveground plant, while significantly improved microbial biomass N:P and enzyme N:P. Adding plant residues after removing aboveground plant significantly increased microbial biomass C:N and C:P, enzyme C:N compared with removing aboveground plant, while significantly decreased enzyme N:P. Compared with removing all the plant, adding plant residues after removing whole plant significantly reduced soil C:N (-16.4%), microbial biomass C:P, N:P and enzyme N:P, while significantly increased enzyme C:N. Our results suggest that removal of plants could have a strong effect on C:N:P of soil, microbial biomass, and extracellular enzyme, and C:N:P of microbial biomass and that extracellular enzyme woule be more sensitive to plant residues. Roots could play a key role in stabilizing C:N:P of soil, microbial biomass, and extracellular enzyme under plant residues addition. Adding plant residues could be a suitable solution for restoring alpine meadows under the circumstance of intact roots, which was conducive to soil C storage, but might not be suitable for alpine meadows with serious root damage, which would increase soil CO2 emission.

A triple-source CT system for micro-scale investigation of geological materials: A simulation study.

A triple-source CT system is proposed for micro-scale testing of geological materials. This study aims at reducing the projection acquisition time by two-thirds compared to a conventional single-source CT system. The proposed system with different positioning errors in the source-to-object distance (SOD) was simulated and tested using the Shepp-Logan phantom model, as well as slices of sand, glass beads, and concrete samples. Furthermore, the imaging quality of a single-source and the triple-source CT system with different dead detector pixels was compared. The results showed that within the maximum allowable positioning error, the pixel differences between the simulated and the original images are close to zero, and the structural similarities are greater than 0.96. In the presence of dead detector pixels, the quality of the simulated images in the triple-source CT system is superior to that of a single-source CT system. The presented triple-source CT system performs well in high-quality image reconstruction.

Architectural Glazed Tiles Used in Ancient Chinese Screen Walls (15th-18th Century AD): Ceramic Technology, Decay Process and Conservation.

The glazed tile is an important building material used throughout the history of traditional Chinese architecture. Architectural glazed tiles used to decorate the screen walls of ancient China are studied scientifically for the first time. More than 30 glazed tile samples from the screen walls of the 15th to 18th century AD of the Hancheng Confucian Temple and Town God's Temple in Shaanxi Province were carefully investigated using SEM-EDS and XRD. Microstructure and chemistry indicated the raw materials, the recipes and the technological choices used to produce the paste and glaze of the glazed tile samples studied. The causes for the key degradation processes of these glazed tiles used as building materials in the screen walls have also been discussed. This work has clear implications for the restoration and conservation treatments on these kinds of ancient Chinese building materials.

The short- and long-term effects of formic acid on rapid nitritation start-up.

The feasibility of achieving stable nitritation inoculating with activated sludge by adding formic acid was studied in this work. Short-term batch effects of formic acid on nitrification showed that the nitrite accumulation ratio (NAR) significantly increased from 0.3% to 83.7% with an increase of formic acid concentration from 0 to 50 mM at an initial ammonia concentration of 75 mg·L-1, which was demonstrated to be due to the inhibition of nxrB transcription in nitrite oxidizing bacteria (NOB). The long-term effects of formic acid at 30 mM were constantly monitored in an aerobic sequencing batch reactor. During 27 days of operation, the NAR was rapidly raised and maintained approximately 90%. What's more, in the following 52 days without addition of formic acid, the NAR was kept above 91.3%. The sustained suppression of NOB genus Nitrospira coupling nxrB inhibition was the main reason to maintain stable nitritation. These results supported the feasibility of formic acid as an efficient nitritation regulator, thus providing a new approach for the development of the BNR process via nitrite pathway.

Juglanin inhibits lung cancer by regulation of apoptosis, ROS and autophagy induction.

Juglanin (Jug) is obtained from the crude extract of Polygonum aviculare, exerting suppressive activity against cancer cell progression in vitro and in vivo. Juglanin administration causes apoptosis and reactive oxygen species (ROS) in different types of cells through regulating various signaling pathways. In our study, the effects of juglanin on non-small cell lung cancer were investigated. A significant role of juglanin in suppressing lung cancer growth was observed. Juglanin promoted apoptosis in lung cancer cells through increasing Caspase-3 and poly ADP-ribose polymerase (PARP) cleavage, which is regulated by TNF-related apoptosis-inducing ligand/Death receptors (TRAIL/DRs) relied on p53 activation. Anti-apoptotic members Bcl-2 and Bcl-xl were reduced, and pro-apoptotic members Bax and Bad were enhanced in cells and animals receiving juglanin. Additionally, nuclear factor-κB (NF-κB), phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) and mitogen-activated protein kinases (MAPKs) activation were inhibited by juglanin. Further, juglanin improved ROS and induced autophagy. ROS inhibitor N-acetyl-l-cysteine (NAC) reversed apoptosis induced by juglanin in cancer cells. The formation of autophagic vacoules and LC3/autophagy gene7 (ATG7)/Beclin1 (ATG6) over-expression were observed in juglanin-treated cells. Also, juglanin administration to mouse xenograft models inhibited lung cancer progression. Our study demonstrated that juglanin could be a promising candidate against human lung cancer progression.

Iron oxide magnetic nanoparticles combined with actein suppress non-small-cell lung cancer growth in a p53-dependent manner.

Actein (AT) is a triterpene glycoside isolated from the rhizomes of Cimicifuga foetida that has been investigated for its antitumor effects. AT treatment leads to apoptosis in various cell types, including breast cancer cells, by regulating different signaling pathways. Iron oxide (Fe3O4) magnetic nanoparticles (MNPs) are nanomaterials with biocompatible activity and low toxicity. In the present study, the possible benefits of AT in combination with MNPs on non-small-cell lung cancer (NSCLC) were explored in in vitro and in vivo studies. AT-MNP treatment contributed to apoptosis in NSCLC cells, as evidenced by activation of the caspase 3-signaling pathway, which was accompanied by downregulation of the antiapoptotic proteins Bcl2 and BclXL, and upregulation of the proapoptotic signals Bax and Bad. The death receptors of TRAIL were also elevated following AT-MNP treatment in a p53-dependent manner. Furthermore, a mouse xenograft model in vivo revealed that AT-MNP treatment exhibited no toxicity and suppressed NSCLC growth compared to either AT or MNP monotherapies. In conclusion, this study suggests a novel therapy to induce apoptosis in suppressing NSCLC growth in a p53-dependent manner by combining AT with Fe3O4 MNPs.

Force Transmission Modes of Non-Cohesive and Cohesive Materials at the Critical State.

This paper investigates the force transmission modes, mainly described by probability density distributions, in non-cohesive dry and cohesive wet granular materials by discrete element modeling. The critical state force transmission patterns are focused on with the contact model effect being analyzed. By shearing relatively dense and loose dry specimens to the critical state in the conventional triaxial loading path, it is observed that there is a unique critical state force transmission mode. There is a universe critical state force distribution pattern for both the normal contact forces and tangential contact forces. Furthermore, it is found that using either the linear Hooke or the non-linear Hertz model does not affect the universe force transmission mode, and it is only related to the grain size distribution. Wet granular materials are also simulated by incorporating a water bridge model. Dense and loose wet granular materials are tested, and the critical state behavior for the wet material is also observed. The critical state strength and void ratio of wet granular materials are higher than those of a non-cohesive material. The critical state inter-particle distribution is altered from that of a non-cohesive material with higher probability in relatively weak forces. Grains in non-cohesive materials are under compressive stresses, and their principal directions are mainly in the axial loading direction. However, for cohesive wet granular materials, some particles are in tension, and the tensile stresses are in the horizontal direction on which the confinement is applied. The additional confinement by the tensile stress explains the macro strength and dilatancy increase in wet samples.

Mercury in forest mushrooms and topsoil from the Yunnan highlands and the subalpine region of the Minya Konka summit in the Eastern Tibetan Plateau.

This study aimed to investigate and discuss the occurrence and accumulation of mercury in the fruiting bodies of wild-growing fungi (Macromycetes) collected from montane forests in two regions of southwestern China with differences in soil geochemistry, climate and geographical conditions. Fungal mycelia in soils of the subalpine region of the Minya Konka (Gongga Mountain) in Sichuan and in the highlands of Yunnan efficiently accumulated mercury in fruiting bodies (mushrooms). The examined sites in Yunnan with highly mineralized red and yellow soils showed Hg contents ranging from 0.066 to 0.28 mg kg-1 dry biomass (db) which is roughly similar to the results obtained for samples collected from sites with dark soils relatively rich in organic matter from a remote, the subalpine region of Minya Konka. Due to the remoteness of the subalpine section of Minya Konka, as well as its elevation and climate, airborne mercury from long-range transport could be deposited preferentially on the topsoil and the Hg levels determined in soil samples taken beneath the fruiting bodies were up to 0.48 mg kg-1 dry matter. In Yunnan, with polymetallic soils (Circum-Pacific Mercuriferous Belt), Amanita mushrooms showed mercury in caps of fruiting bodies of up to 7.3 mg kg-1 dry biomass. Geogenic Hg from the mercuriferous belt seems to be the overriding source of mercury accumulated in mushrooms foraged in the regions of Yunnan, while long-range atmospheric transport and subsequent deposition are the mercury sources for specimens foraged in the region of Minya Konka.

Calcium-dependent protein kinase (CDPK) and CDPK-related kinase (CRK) gene families in tomato: genome-wide identification and functional analyses in disease resistance.

Calcium-dependent protein kinases (CDPKs) and CDPK-related kinases (CRKs) play multiple roles in plant. Nevertheless, genome-wide identification of these two families is limited to several plant species, and role of CRKs in disease resistance remains unclear. In this study, we identified the CDPK and CRK gene families in genome of the economically important crop tomato (Solanum lycopersicum L.) and analyzed their function in resistance to various pathogens. Twenty-nine CDPK and six CRK genes were identified in tomato genome. Both SlCDPK and SlCRK proteins harbored an STKc_CAMK type protein kinase domain, while only SlCDPKs contained EF-hand type Ca(2+) binding domain(s). Phylogenetic analysis revealed that plant CRK family diverged early from CDPKs, and shared a common ancestor gene with subgroup IV CDPKs. Subgroup IV SlCDPK proteins were basic and their genes contained 11 introns, which were distinguished from other subgroups but similar to CRKs. Subgroup I SlCDPKs generally did not carry an N-terminal myristoylation motif while those of the remaining subgroups and SlCRKs universally did. SlCDPK and SlCRK genes were differently responsive to pathogenic stimuli. Furthermore, silencing analyses demonstrated that SlCDPK18 and SlCDPK10 positively regulated nonhost resistance to Xanthomonas oryzae pv. oryzae and host resistance to Pseudomonas syringae pv. tomato (Pst) DC3000, respectively, while SlCRK6 positively regulated resistance to both Pst DC3000 and Sclerotinia sclerotiorum in tomato. In conclusion, CRKs apparently evolved from CDPK lineage, SlCDPK and SlCRK genes regulate a wide range of resistance and SlCRK6 is the first CRK gene proved to function in plant disease resistance.

Target-catalyzed hairpin assembly and intramolecular/intermolecular co-reaction for signal amplified electrochemiluminescent detection of microRNA.

Herein, a new electrochemiluminescence (ECL) strategy for enzyme-free microRNA-21 (miR-21) amplified detection was designed based on target-catalyzed hairpin assembly by combining the signal-amplification capability of both intramolecular and intermolecular ECL co-reaction. In this strategy, two hairpin DNA probes of H1 and H2 were designed as capture probes and detection probes, respectively. To be specific, the capture probes of H1 were immobilized on the multilayer interface of AuNPs and thiosemicarbazide (TSC) assembly on the single-walled carbon nanohorns decorated electrode, while the detection probes of H2 was anchored on the nanocarriers of gold nanoparticals functionalized reduced graphene oxide (Au-rGO) which were tagged with the self-enhanced ruthenium complex (PEI-Ru(ΙΙ)) in advance. Based on the target-catalyzed hairpin assembly, target miR-21 could trigger the hybridization of H1 and H2 to further be released for initiating the next hybridization process to capture a large number of H2 bioconjugates on the sensing surface. Herein, the TSC was used not only as a coupling reagent to attach the AuNPs via Au-S and Au-N bonds but also as a novel intermolecular coreactant to enhance the ECL intensity, and the PEI-Ru(ΙΙ) as emitters exhibited enhanced ECL efficiency. Therefore, a strong ECL signal was achieved by the dual amplification strategies of target recycle and the intramolecular/intermolecular co-reaction of PEI-Ru(ΙΙ) and TSC. The designed protocol provided an ultrasensitive ECL detection of miR-21 down to the sub-femtomolar level with a linear response about 6 orders of magnitude (from 1.0 × 10(-16)M to 1.0 × 10(-11)M) with a relatively low detection limit of 0.03 fM (S/N=3).

Phylogeny of Plant Calcium and Calmodulin-Dependent Protein Kinases (CCaMKs) and Functional Analyses of Tomato CCaMK in Disease Resistance.

Calcium and calmodulin-dependent protein kinase (CCaMK) is a member of calcium/calmodulin-dependent protein kinase superfamily and is essential to microbe- plant symbiosis. To date, the distribution of CCaMK gene in plants has not yet been completely understood, and its function in plant disease resistance remains unclear. In this study, we systemically identified the CCaMK genes in genomes of 44 plant species in Phytozome and analyzed the function of tomato CCaMK (SlCCaMK) in resistance to various pathogens. CCaMKs in 18 additional plant species were identified, yet the absence of CCaMK gene in green algae and cruciferous species was confirmed. Sequence analysis of full-length CCaMK proteins from 44 plant species demonstrated that plant CCaMKs are highly conserved across all domains. Most of the important regulatory amino acids are conserved throughout all sequences, with the only notable exception being observed in N-terminal autophosphorylation site corresponding to Ser 9 in the Medicago truncatula CCaMK. CCaMK gene structures are similar, mostly containing six introns with a phase profile of 200200 and the exception was only noticed at the first exons. Phylogenetic analysis demonstrated that CCaMK lineage is likely to have diverged early from a calcium-dependent protein kinase (CDPK) gene in the ancestor of all nonvascular plant species. The SlCCaMK gene was widely and differently responsive to diverse pathogenic stimuli. Furthermore, knock-down of SlCCaMK reduced tomato resistance to Sclerotinia sclerotiorum and Pseudomonas syringae pv. tomato (Pst) DC3000 and decreased H2O2 accumulation in response to Pst DC3000 inoculation. Our results reveal that SlCCaMK positively regulates disease resistance in tomato via promoting H2O2 accumulation. SlCCaMK is the first CCaMK gene proved to function in plant disease resistance.

[Preliminary Genetic and Functional Analyses of Schistosoma japonicum Chymotrypsin-like Protease].

OBJECTIVE: To investigate the gene expression, localization and potential functions of Schistosoma japonicum chymotrypsin-like protease (SjCTRL) in the host, and evaluate its potential immune-protection efficacy against S. japonicum infection in mice. METHODS: The physicochemical properties of SjCTRL and its phylogenetic relationship with homologous genes from other species were analyzed with bioinformatic software. The distribution of SjCTRL transcripts in 26-day-old worms was investigated using whole-mount in situ hybridization. The transcriptional levels of SjCTRL in male and female worms at four development stages (14, 18, 22, and 26 days after infection) were measured with quantitative real-time PCR. The SjCTRL-dsRNA was prepared and used to induce RNA interference (RNAi) in 26-day-old worms via soaking in vitro, and confocal microscopy was used to observe the morphological changes of worms after RNAi. Primers were designed to amplify the encoding sequence (excluding the transmembrane region) from the S. japonicum cDNA. The truncated gene was subcloned into the pET-28a plasmid, transformed into E. coli BL21 (DE3) for expression. Mice were immunized with the purified recombinant protein and challenged with cercariae. The worms and mouse liver were collected on day 35 after the challenge, and the worm-reduction rate and egg-reduction rate were calculated. RESULTS: The in situ hybridization results showed that SjCTRL mRNA was located in the posterior segment of intestinal tract of female worms, and having abundence only in 26-day-old female worms. After RNAi with SjCTRL-dsRNA, the mRNA expression was reduced to 25.7% (P< 0.05), without significant morphological changes. Using the recombinant plasmid pET-28a/SjCTRL, expression of insoluble SjCTRL protein was induced. Mice immunized with this protein gained a worm-reduction rate of 25.4% and an egg-reduction rate of 80.5% in liver after being challenged with cercariae. CONCLUSION: This study proves a high transcriptional level of SjCTRL in the posterior segment of intestinal tract in 26-day-old female worms, which can be reduced by RNAi treatment in vitro. Immunization with the SjCTRL protein can reduce adult worms and liver eggs.

Proteomic Analysis on Cercariae and Schistosomula in Reference to Potential Proteases Involved in Host Invasion of Schistosoma japonicum Larvae.

Schistosomiasis is a parasitic zoonosis posing great threat to human health. The infection is acquired by larval cercariae penetrating host skin and transforming into juveniles, schistosomula. Proteolytic enzymes secreted from the cercarial acetabular glands are known to aid to the skin penetration, but molecular mechanisms remain largely unclear. To profile the protein composition and identify potential invasive proteases, we developed a new method for simulating cercarial transformation and collecting schistosomula, and for the first time, we compared the proteomes of Schistosoma japonicum cercariae and schistosomula by using in-gel shotgun proteomic analysis. Totally, 1972 proteins were identified in association with ten main biological processes based on Gene Ontology analysis; 46 proteases were detected in cercariae, and among them, 25 proteases disappeared after penetrated. Notably, leishmanolysins and serine and cysteine proteases were found abundant but differentially expressed. Recombinant serine protease SjCE2b and cysteine protease SjCB2 were produced and used for validation of native proteins. Immunofluorescence and Western blotting assays detected SjCE2b and SjCB2 in cercariae but not in schistosomula, suggesting the two enzymes might be consumed upon skin migration. Our data comprehensively chart the proteomic changes during cercarial invasion, revealing the potential proteases involved, providing a platform for the development of molecular anti-infection strategy.

Triple Quenching of a Novel Self-Enhanced Ru(II) Complex by Hemin/G-Quadruplex DNAzymes and Its Potential Application to Quantitative Protein Detection.

Herein, a novel "on-off" electrochemiluminescence (ECL) aptasensor for highly sensitive determination of thrombin has been constructed based on the triple quenching of the effect of hemin/G-quadruplex DNAzymes upon the Ru(II) complex-based ECL system. First, a strong initial ECL signal was achieved by the dual amplification strategies of (i) intramolecular coreaction of a self-enhanced Ru(II)-based molecule (PTCA-PEI-Ru(II)) and (ii) intermolecular coreaction between PTCA-PEI-Ru(II) and nicotinamide adenine dinucleotide (NADH), which was named the signal-on state. Then, a novel triple quenching of the effect of multifunctional hemin/G-quadruplex DNAzymes upon the Ru(II) complex-based ECL system was designed to realize the desirable signal-off state, which was outlined as follows: (i) the hemin/G-quadruplex DNAzymes mimicked NADH oxidase to oxidize NADH and in situ generate the H2O2, consuming the coreactant of NADH; (ii) its active center of hemin could oxidize the excited state PTCA-PEI-Ru(II)* to PTCA-PEI-Ru(III), making the energy and electron transfer quench; (iii) it also acted as horseradish peroxidase (HRP) to catalyze the H2O2 for in situ producing the quencher of O2. Based on triple quenching of the effect of hemin/G-quadruplex DNAzymes, the highly sensitive "on-off" thrombin aptasensor was developed with a wide linear detection range of 1.0 × 10(-14) M to 1.0 × 10(-10) M and a detection limit down to the femtomolar level.

Cyclic nucleotide gated channel gene family in tomato: genome-wide identification and functional analyses in disease resistance.

The cyclic nucleotide gated channel (CNGC) is suggested to be one of the important calcium conducting channels. Nevertheless, genome-wide identification and systemic functional analysis of CNGC gene family in crop plant species have not yet been conducted. In this study, we performed genome-wide identification of CNGC gene family in the economically important crop tomato (Solanum lycopersicum L.) and analyzed function of the group IVb SlCNGC genes in disease resistance. Eighteen CNGC genes were identified in tomato genome, and four CNGC loci that were misannotated at database were corrected by cloning and sequencing. Detailed bioinformatics analyses on gene structure, domain composition and phylogenetic relationship of the SlCNGC gene family were conducted and the group-specific feature was revealed. Comprehensive expression analyses demonstrated that SlCNGC genes were highly, widely but differently responsive to diverse stimuli. Pharmacological assays showed that the putative CNGC activators cGMP and cAMP enhanced resistance against Sclerotinia sclerotiorum. Silencing of group IVb SlCNGC genes significantly enhanced resistance to fungal pathogens Pythium aphanidermatum and S. sclerotiorum, strongly reduced resistance to viral pathogen Tobacco rattle virus, while attenuated PAMP- and DAMP-triggered immunity as shown by obvious decrease of the flg22- and AtPep1-elicited hydrogen peroxide accumulation in SlCNGC-silenced plants. Additionally, silencing of these SlCNGC genes significantly altered expression of a set of Ca(2+) signaling genes including SlCaMs, SlCDPKs, and SlCAMTA3. Collectively, our results reveal that group IV SlCNGC genes regulate a wide range of resistance in tomato probably by affecting Ca(2+) signaling.

Downregulation of Kruppel-like factor 2 is associated with poor prognosis for nonsmall-cell lung cancer.

Kruppel-like factor 2 (KLF2) expression is diminished in many malignancies. However, its expression and role in nonsmall-cell lung cancer (NSCLC) remain unknown. In this study, we found that KLF2 levels were decreased in NSCLC tissues compared with adjacent normal tissues. Its expression level was significantly correlated with TNM stages, tumor size, and lymph node metastasis. Moreover, patients with low levels of KLF2 expression had a relatively poor prognosis. Furthermore, knockdown of KLF2 expression by siRNA could promote cell proliferation, while ectopic expression of KLF2 inhibited cell proliferation and promoted apoptosis in NSCLC cells partly via regulating CDKN1A/p21 and CDKN2B/p15 protein expression. Our findings present that decreased KLF2 could be identified as a poor prognostic biomarker in NSCLC and regulate cell proliferation and apoptosis.

[Prokaryotic expression and function analysis of Schistosoma japonicum calpain].

OBJECTIVE: To clone and express recombinant calpain of Schistosoma japonicum (Sjcalpain), observe the distribution of Sjcalpain in S. japonicum cercariae and analyze its role in skin invasion. METHODS: The primers were designed according to the full-length sequence of calpain (GenBank accession No. AB016726). The genes encoding catalytic domain and Ca2+ binding domain of Sjcalpain were amplified by PCR, and the target fragments were subcloned into pET-28a. The recombinant proteins were expressed in E. coli BL21 (DE3) and purified by Ni-NTA resin. The rabbit polyclonal antibodies were prepared with the two purified recombinant proteins by immunizing New Zealand white rabbits. ELISA was used to detect the titer of rabbit antiserum. Immunolocalization was used to investigate the distribution of Sjcalpain in S. japonicum cercariae. Cercariae were incubated with specific inhibitor before infection of mice and the worm reduction rate was calculated. RESULTS: The recombinant expression vector Sjcalpain catalytic domain/pET28a and Sjcalpain Ca2+ binding domain/pET28a were constructed and the recombinant proteins were successfully expressed in E. coli BL21 (DE3) (about M(r) 43 000 and M(r) 39 000, respectively). The two target proteins were expressed as inclusion bodies. The purified target proteins were obtained through Ni-NTA affinity purification. ELISA result showed that the titer of prepared rabbit polyclonal antibodies was higher than 1 : 80 000. Immunolocalization study demonstrated that Sjcalpain protein was mainly expressed in the head of cercariae. Inhibition assays suggested that the average number of adult worms in calpain inhibitor-incubation group and control group was 19 and 23, respectively, with a worm reduction rate of 17.4%. CONCLUSION: Sjcalpain is mainly expressed in the head of S. japonicum cercariae. Inhibition of Sjcalpain could reduce the number of invading cercariae in infected mice, which suggest that Sjcalpain may play a role in skin invasion by cercariae.

[Cloning, expression and transcription specificity analysis of two tyrosinases from Schistosoma japonicum].

OBJECTIVE: To clone and express the recombinant proteins based on the whole open reading frame of two tyrosinases (tyrosinase 1 and tyrosinase 2) from Schistosoma japonicum, and study the transcription specificity of the two tyrosinases in different sex and developmental stages of S. japonicum. METHODS: The full-length of SjTYR1 and SjTYR2 were amplified with specific primers and subcloned into pSJ2. The recombinant plasmids were transformed into E. coli Rosetta Gami strains and induced with IPTG for expression. The recombinant proteins were purified by Ni-NTA agarose. The recombinant proteins SjTYR1 and SjTYR2 were used to produce the specific antibodies by immunizing the rabbits. The immunogenicity of the recombinant proteins SjTYR1 and SjTYR2 were detected by Western blotting using sera of recombinant proteins-immunized rabbits and S. japonicum-infected rabbit serum as the primary antibody, respectively. The reactivity of sera from recombinant proteins-immunized rabbits was analyzed by Western blotting against the native protein of S. japonicum worm. Total RNA was extracted from 14, 16, 18, 20, 22, 24, 26, and 28-day male and female worms. Transcription levels of the two tyrosinases in different sex and different stage were determined via RT-PCR method. RESULTS: The expression vector of SjTYR1/pSJ2 and SjTYR2/pSJ2 were constructed and the recombinant pro teins SjTYR1 and SjTYR2 were expressed in inclusion body in E. coli (about M(r) 55 000 and M(r) 56 800). The sera of S. japonicum-infected rabbits reacted positively with the purified recombinant protein SjTYR1, but not with recombinant protein SjTYR2. The native protein of S. japonicum worm could be recognized by sera of rSjTYR1-immunized rabbits (M(r) 100 000), but not by sera of rSjTYR2-immunized rabbits. Transcription levels of the two tyrosinases in male worms were nearly zero. In female worms, the transcription levels of the two tyrosinases increased sharply from the 24th day post-in- fection and reached maximum on the 28th day. CONCLUSION: The recombinant proteins of SjTYR1 and SjTYR2 show immunogenicity and immunoreactivity. SjTYR1 and SjTYR2 are both expressed specifically in female worms and the transcription levels increase in 24-28 days after infection.