The Wnt signaling pathway is mediated by a family of secreted glycoproteins through canonical and noncanonical mechanism. The signaling pathways are regulated by various modulators, which are classified into two classes on the basis of their interaction with either Wnt or its receptors. Secreted frizzled-related proteins (sFRPs) are the member of class that binds to Wnt protein and antagonizes Wnt signaling pathway. The other class consists of Dickkopf (DKK) proteins family that binds to Wnt receptor complex. The present review discusses the disease related association of various polymorphisms in Wnt signaling modulators. Furthermore, this review also highlights that some of the sFRPs and DKKs are unable to act as an antagonist for Wnt signaling pathway and thus their function needs to be explored more extensively. On the contrary, the absence of Wnt ligand leads to constant degradation of cytoplasmic β-catenin protein via AXIN complex [β-catenin at amino terminus followed by β-catenin recognition and ubiquitination by β-transducing repeats-containing proteins (β-TrCp), an E3 ubiquitin ligase subunit directing proteasome mediated degradation [β-catenin is unable to reach the nucleus and subsequently Wnt targeted genes are repressed by DNA-bound T-cell factor/lymphoid enhancer factor (TCF/LEF) family of proteins. In noncanonical signaling pathway, Wnt ligand binds to the receptors of FZD family and receptor tyrosine kinase-like orphan receptor 2/receptor tyrosine kinase (ROR2/RYK) coreceptors to form a complex. Upon stimulation by the receptors, DVL is activated leading to sequential activation of the Rho family of GTPases  and JNKs. Another noncanonical Wnt signaling pathway involves activation of Wnt/Ca++ signaling cascade.The Wnt family, a group of secreted glycoproteins, directs cell proliferation and polarity as well as determining the fate of a cell during embryonic development through a series of signaling transduction pathways . The mem cascade , 4. The  complex . Scaffolradation . As a reVarious natural inhibitors of Wnt signaling pathway have been identified till date, which can antagonize or regulate Wnt signaling pathway . The mecAs mentioned earlier Wnt signaling is one of the most critical pathways for cellular development, even in adult tissues; henceforth, the polymorphism in genes of Wnt signaling modulators can be a foremost cause for various types of associated diseases. The present review highlights disease associated polymorphism studies in some of the Wnt signaling modulators. Summarizing the role of polymorphism in Wnt signaling modulators can open a new spectrum to understand the origin of various related diseases and to trace out novel pathways for potential therapeutics. SOST gene encoded secreted glycoprotein containing cysteine knot which acts as a Wnt antagonist and regulates skeletal mineralization [α) [β-catenin signaling antagonist by binding to LRP5/6 receptors and thus inhibiting bone formation [ SOST genes have been associated with osteoporotic fractures and bone mineral density (BMD) [ SOST gene region was not found associated with high or low BMD at lumbar spine [ SOST polymorphism [ SOST genotype in correspondence to the allele dose effect was also observed concluding the necessity for large sample size, Li et al., 2008, showed increases in BMD in SOST knockout mice [ SOST gene leads to monogenic bone disorder, sclerosteosis (Online Mendelian Inheritance in Man ID: 269500), characterized by hyperostosis all over the skeleton (massive bone overgrowth) [ SOST gene causes van Buchem disease with similar disease phenotype [ SOST gene region influences the function of sclerostin causing variations in BMD in a subset of the population. Further, to verify their hypothesis they observed eight polymorphisms within the region of the SOST gene, which were in relation to BMD observed in 1,939 elderly Dutch white. According to their results, a decrease in BMD of spine/femoral neck in Dutch white females was associated with a 3 bp insertion in the SOST promoter region (SOST gene region polymorphism 3) and increase in BMD of spine/femoral neck in Dutch white males was associated with G variant of SOST-region polymorphism 9, that is, van Buchem deletion region. Recently, two BMD associated SNPs (rs851054 and rs851056) were also identified in 5′ region of SOST gene [ SOST gene region between 23 kb and 57 kb  [ SOST gene region was also proved by a gene wide tag SNP association study performed on Chinese population (1243 subjects) with low and high BMD [Sclerostin is alization , 12. Reclization , though lization  and cemelization . Even dution [α) . In osteormation . SOST gety (BMD) –20. Howear spine . Similarmorphism . On the morphism . Althougout mice . It has rgrowth) , 26. Addhenotype , 28. Uithenotype , hypotheOST gene . Styrkar1107748) . Evidenchigh BMD . in vitro, at low concentration [ sFRP3 in the etiology of osteoarthritis has been discussed in many reports. Replication studies among Caucasian women showed two SNPs in sFRP3/FrzB gene, R200W (rs288326) and R324G (rs7775), may elevate knee osteoarthritis [ sFRP3 results in structurally occult hip dysplasia leading to osteoarthritis. Further, they revealed that the G allele of R324G variant is in close association with hip replacement in elderly females. Furthermore, Min et al., 2005, also showed increased incidence of G allele of this variant in individuals with hip radiographic osteoarthritis. According to them individuals as G allele carrier have a high risk for hip radiographic osteoarthritis [ sFRP gene has been shown to exhibit a strong association with breast cancer. SNP rs7775 was found in exon 6 and was observed to encode arginine (CGC) or glycine (GGC) in Saudi women [Based on sequence homology, the five members of sFRP family (sFRP1 to sFRP5) are divided into two subgroups. sFRP1, sFRP2, and sFRP5 constitute one subgroup while sFRP3 and sFRP4 are in another subgroup. These proteins are soluble cysteine rich and bind directly to Wnt molecules antagonizing Wnt signaling pathway . sFRP3, ntration  while sFntration . The rol00W rs2886 and R32rthritis . In addirthritis  displayerthritis . Similarrthritis . Besidesdi women .Another class of Wnt antagonist is cysteine rich secreted protein known as Dickkopf (DKK-1 to -4) which binds to LRP5/6 receptor and Kremen 1/2 (transmembrane protein) and inhibits Wnt signaling pathway. In human the gene for DKK-1 is located in 10q11.2 position of chromosome (NM_012242.2). As a result, the canonical pathway of Wnt signaling is inhibited , 45. Stu DKK-1 gene containing chromosomal region 10q21 with BMD in men through genome wide linkage scan. The influence of genetic variation in DKK-1 on hip geometry, BMD, and bone turnover has been studied earlier by Piters et al., 2010 [ DKK-3 gene and rs3763511 in DKK-4 with breast cancer risk was studied by Alanazi et al., 2013 [ DKK-3 gene SNP. Furthermore, GG genotype and AG genotype showed enhanced protection against estrogen receptor positive tumor and estrogen receptor negative tumor progression, respectively. Conversely, SNP in DKK-4 gene was strongly associated with age independent increased breast cancer risk of estrogen receptor negative tumor. In order to determine the relationship between polymorphism in DKK genes and renal cancer Hirata et al., 2009 [ DKK-2, rs3206824, rs11022095, rs1472189, rs7396187, and rs2291599 in DKK-3, and rs2073664 in DKK-4 were analyzed. Results demonstrated significant association of DKK-3 rs1472189 C/T with renal cell carcinoma and SNP rs17037102 G DKK-2 may contribute to increased survival rate in patients, after radical nephrectomy.Ralston et al.  suggestel., 2010 . They sel., 2010 . For the Wif 1 gene promoter region was found to downregulate Wif 1 gene in prostate cancer cell lines. Further, a decreased motility and invasiveness of prostate cancer cells were reported after restoration of Wif 1 expression [ Wif 1 promoter hypermethylation and increase in bladder tumor has also been reported by other research groups [ Wif 1 gene has also been reported to be involved in osteosarcoma. Study performed by Kansara et al. revealed that in primary human osteosarcoma promoter hypermethylation leads to Wif 1 gene silencing, which results in loss of differentiation and increased cell proliferation [ Wif gene in regulation of tumorigenesis and any probability of existence of polymorphism in Wif gene, which may result in deleterious effect, makes it a probable candidate for requiring urgent studies for verifying its role in regulation of Wnt signaling pathway.Another evolutionary conserved modulator protein of Wnt signaling is Wnt inhibitory factor 1 (Wif 1). It is similar to sFRPs and inhibits Wnt signaling by binding to Wnt proteins. Wif 1 contains epidermal growth factor like repeats (EGF repeats) and Wif domain which binds to Wnt proteins , 63. As pression . Correlah groups , 70. Wifferation . Taking β-catenin [ Rspo4 have been reported and were found associated with the absence of finger and toe nails  (OMIM 206800) in humans [ RSPO4 mutations related with anonychia/hyponychia to 17 and all of them were found located in the first three exons encoding a signal peptide and the highly conserved furin-like cysteine-rich domains. Study concluded and suggested that p.M1I variant is a recurrent mutation among Pakistani patients and can be considered as a polymorphism, having no effect on the observed disorder [R-spondins are a newly discovered, cysteine rich and containing a thrombospondin type 1 domain/repeat-1, secreted protein family consisting of 4 members (Rspo1-4). Recently, Rspo has been reported to act as modulators of Wnt signaling pathway. Rspo has been demonstrated to upregulate the Wnt signaling pathway by stabilizing cytosolic -catenin , 73. Thi-catenin , 74. All-catenin . Recentl-catenin , 77. Til-catenin . Studies-catenin , 80. Alt-catenin , 82. Latn humans –86. Khandisorder .The functional complexity of Wnt signaling pathway has driven the interest of leading researchers towards in-depth analysis of Wnt signaling controlling factors. The extracellular antagonists and agonists provide defined regulation of signal and their further transmission. Herein, we have tried to summarize the various reported polymorphisms in known antagonists  and agonists (Rspo) of Wnt signaling pathway leading to the observed diseased state in humans . As disc


Mycobacterium tuberculosis (Mtb) has acquired resistance andconsequently the antibiotic therapeutic options available against this microorganismare limited. In this scenario, the use of usnic acid (UA), a natural compound,encapsulated into liposomes is proposed as a new approach in multidrug-resistanttuberculosis (MDR-TB) therapy. Thus the aim of this study was to evaluate the effectof the encapsulation of UA into liposomes, as well as its combination withantituberculous agents such as rifampicin (RIF) and isoniazid (INH) against MDR-TBclinical isolates. The in vitro antimycobacterial activity of UA-loaded liposomes(UA-Lipo) against MDR-TB was assessed by the microdilution method. The in vitrointeraction of UA with antituberculous agents was carried out using checkerboardmethod. Minimal inhibitory concentration values were 31.25 and 0.98 µg/mL for UA andUA-Lipo, respectively. The results exhibited a synergistic interaction between RIFand UA [fractional inhibitory concentration index (FICI) = 0.31] or UA-Lipo (FICI =0.28). Regarding INH, the combination of UA or UA-Lipo revealed no marked effect(FICI = 1.30-2.50). The UA-Lipo may be used as a dosage form to improve theantimycobacterial activity of RIF, a first-line drug for the treatment of infectionscaused by Mtb. Mycobacterium tuberculosis (Mtb). The treatment for susceptibleMtb isolates is based on a combination of isoniazid (INH), rifampicin (RIF), ethambutol(EMB) and pyrazinamide (PZA) (Tuberculosis (TB) is a chronic bacterial infection caused by an airborne microorganismknown as de (PZA) . Patientde (PZA) .In 2010de (PZA) . The thede (PZA) . Since tde (PZA) , there iIn the last few years, many new synthetic or natural agents have been tested against Mtb inorder to discover compounds capable of replacing or complementing established drugs used inTB therapy . Usnic aMycobacterium against well-known antibioticsusually prescribed in clinical practice  was furnished by Lipoid GMBH . Allsolvents and other chemicals were supplied by Merck .INH, RIF, (+)-UA, cholesterol (Chol), stearylamine (SA) and Middlebrook 7H9 medium wereobtained from Sigma-Aldrich . Middlebrook OADC Enrichment waspurchased from Becton Dickinson . Soybean phosphatidylcholine (PC)  and UA (2 mg/mL) by the thin film method. Briefly, lipidconstituents and UA were dissolved in a mixture of chloroform and methanol (3:1 v/v)under magnetic stirring. The solvents were removed under pressure  and thethin film formed was hydrated with 10 mL of phosphate buffer solution (7.4 pH). Theliposomal dispersion was then sonicated  at 200 W and 40 Hzfor 300 s in order to obtain small unilamellar vesicles.The mean particle size and polydispersity index of liposomes were measured by photonautocorrelation spectroscopy using a laser particle analyser Delsa TM Nano-S . The zeta potential of the liposomes was determined by electrophoreticmobility using a ZetasizerNano-ZS90 . Analyses wereperformed using samples diluted with deionised water (2:1) at 25ºC. Moreover, theencapsulation efficiency of UA into liposomes was determined usingultrafiltration/centrifugation by a previously validated method .Antimycobacterial activity - Mycobacterium strains and growthconditions - The Mtb H37Rv ATCC 27294 strain and six MDR-TB clinicalisolates were studied . MDR-TB isolateswere obtained from the Central Public Health Laboratory of Pernambuco . Drug susceptibility of MDR-TB isolates to first-line drugs  was verified using standard agar methods . Mtbstrains were cultured in Middlebrook 7H9 broth supplemented with 10% (v/v) OADC, 0.05%(v/v) Tween 80 (Sigma-Aldrich) and 0.2% (v/v) glycerol (Sigma-Aldrich) and incubated at37°C for approximately 10 days.Determination of the minimum inhibitory concentration (MIC) - Theantimycobacterial activity of the tested samples (UA and UA-Lipo) and the referencedrugs, INH and RIF, was determined in triplicate through the Microplate Alamar BlueAssay  as follows:AB equals the MIC of drug A in combination with drug B;MICA is the MIC of drug A alone; MICBA equals the MIC of drug Bin combination with drug A and MICB is the MIC of drug B alone. Theinteraction is considered synergistic for FICI < 0.5; additive (0.5 ≤ FICI ≤ 1);indifferent (1 < FICI ≤ 4) and antagonistic (FICI > 4) (Where MICICI > 4) .Physicochemical characterisation of liposomes - UA-Lipo prepared with a14:1 lipid:drug molar ratio (80 mM of lipids) exhibited a mean particle size of 146.46 ±1.91 nm, PDI of 0.32 ± 0.01, surface charge of + 21.30 ± 0.51 mV and the drug efficiencyratio was practically 100% (99.56 ± 0.74%).Antimycobacterial activity - MIC - Theantimycobacterial activity of UA, UA-Lipo, as well as reference drugs against MDR-TBclinical isolates is shown in Checkerboard method - The in vitro interactions were evaluated in twoMDR-TB isolates (1619 and 1411) as these isolates presented the MIC values set for thereference and tested drugs. The interaction results of UA and UA-Lipo with INH and RIFagainst MDR-TB isolates are shown in In this paper, conventional liposomes were selected as vehicles for UA because they aresuitable carriers for antibiotics used in the treatment of intracellular pathogens, suchas Mtb .Initially, the resistance of all the clinical isolates used in the present paper wasthus confirmed, given that 37Rv and 1.56-12.5µg/mL for Mtb clinical isolates with monoresistance to INH, SM or RIF. Regarding theUA-Lipo, all isolates exhibited MIC values (0.98 µg/mL) more than 30-fold lower than UAalone. This phenomenon observed in MIC for all isolates may be explained in the sense ofthese strains have never come into contact with UA and UA-Lipo earlier, and thus have acertain degree of susceptibility and uniformity.The antimycobacterial activity of UA is in agreement with those reported in theliterature. One possible explanation for the enhancement of antimicrobial activity owing theencapsulation of UA into lip- osomes may be attributed to electrostatic interactionsbetween negatively-charged carboxyl groups of mycolic acids, which are the maincomponents of the Mtb cell wall , and posMycobacterium bovis was 0.2 µM, whereas the MIC of RIF washigher (0.8 µM), thus indicating that liposomes were more efficient. Recently, M.bovis was assessed. The antimycobacterial activity of LEV-proliposomes washigher than LEV . These in vitro resultsdemonstrate that the encapsulation of drugs into liposomes may boost theirantimycobacterial activity.Other authors also have found that the encapsulation of antibiotics into liposomesenhances the antimycobacterial activity as compared with pure drugs. In 2009, The checkerboard method was used to verify the possible interaction effects of thecombination of drugs on the antimicrobial activity of the compounds tested against Mtb.The drug combination regimens could lead to successful therapeutic schemes for TBtreatment, considering MDR-TB isolates can rapidly develop resistance to new drugs,especially those redesigned from the existing scaffolds of the currently used anti-TBdrugs .In synergism, the drugs involved can affect different targets in the microorganisms. Thus thEuclea natalensis, reduced MIC values four fold and eightfold,respectively. Results showed a synergistic effect for both combinations against Mtbisolates.In 2013, Thus, UA-Lipo may be used as a dosage form to improve the antimycobacterial activity ofRIF, a first-line drug for the treatment of infections caused by Mtb.


Giardia duodenalis on dendritic cell (DC) functions may contribute to the pathogenesis of chronic giardiasis. G. duodenalis lysate has been shown to inhibit the activation of murine DCs through the ligands of various Toll-like receptors (TLRs), including TLR2 and TLR4. Our study aimed at translating these findings to human DCs.Effects of G. duodenalis lysate produced less IL-12/23p40 (p = 0.002), IL-12p70 (p = 0.011), and IL-23 (p = 0.004), but more IL-10 (p = 0.006) than cells incubated in the absence of the parasite. Concomitantly, the expression of CD25, CD83, CD86, and HLA-DR was reduced on G. duodenalis-incubated DCs as compared to control cells. In contrast, human DCs stimulated through TLR2 in combination with TLR1 or TLR6 and G. duodenalis lysate secreted significantly more IL-12/23p40 (p = 0.006), IL-23 (p = 0.002), and IL-10 (p = 0.014) than cells stimulated through TLR2 ligands alone. Ligands for TLR2/TLR1 or TLR2/TLR6 also induced enhanced extracellular expression of CD25, CD83, and CD86 (p < 0.05).As described previously for murine DCs, also human DCs were only weakly activated by the parasite itself. LPS-stimulated DCs incubated in the presence of G. duodenalis and stimulated through TLR2 show increased activation as compared to cells incubated in the absence of the parasite. Thus, TLR2 ligands, e.g., delivered by probiotic lactobacilli, might be beneficial in human giardiasis through an adjuvant effect on the induction of cellular immune responses against G. duodenalis.In contrast to murine DCs, human DCs incubated in the presence of  Giardia duodenalis is the most frequent parasitic agent of gastroenteritis worldwide and has been targeted as part of the WHO “Neglected Disease Initiative” since 2006 [nce 2006 . In Eastnce 2006 . Notablynce 2006 .+ T helper cells are involved in protection against G. duodenalis[Giardia immune responses as they are able to reach with their cellular protrusions into the gut lumen and sample antigen [G. duodenalis lysate may interfere with the activation of DCs through TLR2 or TLR4 in vitro[Giardia immune responses because G. duodenalis by itself only weakly activates murine DCs [In vivo, bacteria belonging to the gut flora might deliver the required stimuli to G. duodenalis-processing DCs since TLR2 may recognize lipoproteins derived from Gram-positive while TLR4 binds LPS of Gram-negative bacteria [The pathogenesis of chronic giardiasis is poorly understood and both parasitic and host factors might be involved. The parasite is non-invasive but stays in the intestinal lumen where it adheres to epithelial cells. Since CD4uodenalis, chronic antigen . Followi antigen . A recen in vitro. Signalsrine DCs . In vivobacteria .The present study aimed at translating the previous findings from murine to human DCs. To monitor DC activation, we focused on the expression of typical surface markers and cytokine production . RegardiG. duodenalis strain WB-C6 (ATCC 50803) trophozoites were propagated in TYI-S-33 medium as previously described [8 parasites/ml. Lysates were prepared by subjecting the suspensions in three consecutive freezing-thawing cycles.escribed . Parasit+ monocytes were separated with magnetic microbeads  and cultured at 3x106 cells/well in 6-well cell culture dishes  for six days in medium consisting of RPMI 1640 supplemented with 2 mM L-glutamine, 10 mM HEPES, penicillin -streptomycin  , 50 μM 2-mercaptoethanol , 10% fetal calf serum , 1000 U/ml human rGM-CSF , and 100 U/ml human rIL-4 . On days 2 and 4, fresh medium and cytokines were added to the wells.DCs were generated as described previously . Briefly5 cells/well. The TLR stimuli Pam2CSK4 and Pam3CSK4  or LPS  were added and the cells incubated in the presence or absence of G. duodenalis lysate . Immature cells kept in medium were equally incubated in the presence or absence of G. duodenalis lysate.On day 6, DCs were harvested and transferred to 96-well round-bottom trays (Nunc) at 10The phenotype of DCs was monitored by flow cytometry with PE- or FITC-labeled anti-human mAbs against HLA-DR, CD14, CD25, CD86 , CD83 , or the appropriate isotype controls, as previously described [Cell-free supernatants of cell cultures were harvested after 48 h and stored at -80°C until analysis by sandwich ELISAs for IL-12p40, IL-12p70, and IL-10 , or IL-23 (eBioscience). Samples with known cytokine concentrations provided by the manufacturers of the ELISA kits were included in each analysis and used to create standard curves. Only values within the linear section of these curves were considered. The level of detection was 5 pg/ml for all assays.Data were analyzed statistically with the nonparametric Wilcoxon matched-pairs signed rank test using GraphPad Prism version 6.0a for Mac OS X. Differences were considered statistically significant for p < 0.05.G. duodenalis lysate with the TLR4 ligand LPS or with ligands binding to TLR2/TLR1 or TLR2/TLR6, i.e., Pam3CSK4 and Pam2CSK4, respectively, since TLR2 is expressed together with either TLR1 or TLR6 [We incubated human immature monocyte-derived DCs in the presence or absence of  or TLR6 . ControlG. duodenalis lysate by itself only weakly activated human DCs (data not shown). On LPS-activated DCs, it reduced the expression of CD25 and CD83, typical DC surface markers indicative of cellular activation, and of CD86 and HLA-DR, which are involved in antigen-presentation . In contrast, among DCs stimulated through either TLR2/TLR1 or TLR2/TLR6 and incubated in the presence of G. duodenalis lysate, significantly more cells expressed CD25, CD83, and CD86 than in DC populations incubated in the absence of the parasite-derived stimuli (Table Flow cytometry revealed that similar to murine DCs, on Table . SimilarG. duodenalis lysate dependent on the TLR agonist involved. When cells were activated through TLR4, G. duodenalis significantly reduced the production of IL-12/23p40, IL-12p70, and IL-23 while the IL-10 secretion was enhanced (Figure G. duodenalis lysate enhanced the secretion of IL-12/23p40, IL-23, and IL-10 by DCs activated through TLR2 – although the cells produced comparably less cytokines than LPS-stimulated DCs (Figure G. duodenalis lysate in the cell cultures. As noted before for LPS-stimulated human monocytes [We next determined the concentrations of IL-12/23p40, IL-12p70, IL-23, and IL-10 in all supernatants and again observed distinct effects of onocytes , we alsoG. duodenalis to the human cell system, we observed the opposite for DCs stimulated through TLR2. Exposure of TLR2-activated DCs to G. duodenalis considerably enhanced the expression of crucial surface molecules and the secretion of cytokines belonging to the IL-12 family.Thus, while we could translate the previous murine data regarding the inhibition of LPS-stimulated DCs through G. duodenalis on human DC functions. In contrast, G. duodenalis apparently may interfere with immune responses induced by human DCs activated through TLR4 agonists.In contrast to TLR4 ligands and in contrast to murine DCs, TLR2 ligands may augment the antigen-presenting functions of human DCs in giardiasis. Although the LPS-induced cytokine responses were comparably stronger than those induced by the TLR2 ligands, which might be due to the concentrations of the stimuli used, we feel that the data together, i.e., opposite results for cytokine secretion and also surface molecule expression for the stimuli tested, may argue for an additive effect of TLR2 and Lactobacillus spp. are recognized by human DCs through TLR2 and NOD2 [G. duodenalis infection in vivo[G. duodenalis can augment TLR2-mediated DC function suggests that TLR2 ligands might be of considerable interest in the management of clinical giardiasis. Since an anti-giardial effect by Lactobacillus spp. on murine giardiasis is also evident when the bacteria are administered together with anti-giardial drugs [Probiotic and NOD2 , and mayand NOD2 . In a Gen in vivo, and thial drugs  or in maal drugs , clinicaThe authors declare that they have no competing interests.G. duodenalis lysate, designed the study, and wrote the manuscript; RI designed and supervised the study and wrote the manuscript. All authors have approved the final version of the manuscript.JO and PRV performed the experiments, analyzed the data, and wrote the manuscript; MF analyzed the data and wrote the manuscript; CK and TA prepared the


Interaural Level Difference (ILD) provides an important cue for the location of a sound source in the azimuthal plane. Typically, ILD decoding in the brainstem is modeled as a subtraction of spike rates, with inhibitory inputs from one ear subtracted from the excitatory inputs from the other -3. The iRecent experimental evidence suggests that inhibitory input for ILD doesn’t provide subtraction, but instead provides a gain adjustment . In addiThe auditory periphery was modeled as a gammatone filterbank which provided inputs directly to a leaky integrate-and-fire model representing the cells of the cochlear nucleus. These cells are known to lock to the envelope of the sound stimulus, and this behavior was recreated by low-pass filtering of the gammatone filterbank inputs to the cells, and use of a dynamic spike threshold mechanism . The ILD


Monthly average temperature humidity index (THI) values were estimated. Threshold THI value affecting fertility traits was identified by fixed least-squares model analysis. Three zones of non-heat stress, heat stress and critical heat stress zones were developed in a year. The genetic parameters heritability (h2) and repeatability (r) of each fertility trait were estimated. Genetic evaluation of Murrah buffaloes was performed in each zone with respect to their expected breeding values (EBV) for fertility traits.Fertility data on 1379 records of 581 Murrah buffaloes spread over four lactations and climatic parameters Effect of THI was found significant (p<0.001) on all fertility traits with threshold THI value identified as 75. Based on THI values, a year was classified into three zones: Non heat stress zone(THI 56.71-73.21), HSZ (THI 75.39-81.60) and critical HSZ (THI 80.27-81.60). The EBVfor SP, PR, CR were estimated as 138.57 days, 0.362 and 69.02% in non-HSZ while in HSZ EBV were found as 139.62 days, 0.358 and 68.81%, respectively. EBV for SP was increased to 140.92 days and for PR and CR, it was declined to 0.357 and 68.71% in critical HSZ.The negative effect of THI was observed on EBV of fertility traits under the non-HSZ and critical HSZ Thus, the influence of THI should be adjusted before estimating the breeding values for fertility traits in Murrah buffaloes. Accordik census , the popk census . Buffalo% share tk census . Ravagnok census  found a The estimation of expected breeding value for the traits defines the total genetic worth of the individual. The prediction of breeding values constitutes an integral part of most breeding programmes for genetic improvement of economic traits of Murrah buffaloes. The accuracy of estimating the breeding value of an animal is a major concern affecting the genetic progress due to selection . The estThe hypothesis of the study was to assess the role of environment, as well as genotype on the fertility traits of Murrah buffaloes. Until today, there is no report available on the assessment of EBV s for fertility traits of Murrah buffaloes under the non-heat stress, heat stress and critical heat stress zones (CHSZ) of a year. This investigation, therefore, is aimed to find out the expected breeding values (EBV) of fertility traits in three different defined zones of a year.This study is based on animal breeding data and it does not include animal experiment. So the ethical approval was not required.viz. winter (December-March), summer (April-June), rainy (July-September) and autumn (October and November). The average temperatures in the four seasons were 15.6°C, 35.8°C, 28.7°C and 22.5°C and the average RH were 71.34%, 48%, 79% and 65%, respectively. Buffaloes were maintained in a loose housing system with brick on edges flooring under group management practice. The nutritional requirements of buffaloes were fulfilled through a standardized balanced ration of seasonal green fodders along with concentrates. Oestrus detection was carried out with the help of teaser Murrah bull (vasectomised bulls). Females detected in oestrus were inseminated with frozen semen of progeny tested bulls and pregnancy was confirmed after 45 days of insemination through rectal palpation as per standard management practices of the herd. Only healthy buffaloes were included while abortion, metritis, still birth, retained placenta and dystocia records were considered abnormal, and these were not included in the present study.The present study was conducted on Murrah buffaloes maintained at Livestock Research Centre, National Dairy Research Institute (NDRI), Karnal, Haryana, India, located at 29° 42’ N latitude and 72° 02’ E longitudes in the bed of Indo-Gangetic alluvial plain. The climate of Karnal is subtropical in nature. There are four major seasons in the year st, 2nd, 3rd and 4th parity spanned over a period of 20 years from July 1993 to December 2012 were collected. Three traits were generated such as service period (SP), PR and CR. The SP of buffaloes with more than 350 days was excluded from the study. The data were normalized with mean ± 3 standard deviation (SD) for SP. PR is a new and recent method of measuring fertility in buffaloes. It is defined as the percentage of non-pregnant buffaloes to become pregnant during each 21 days after voluntary waiting period (VWP)[The information related to fertility of Murrah buffaloes was collected from records maintained by dairy cattle breeding division, NDRI, Karnal. A total of 1379 records of 581 Murrah buffaloes under 1iod (VWP).PR=21/(SP−VWP+11)Where, VWP is the days in milk when buffaloes were not inseminated and VWP of Murrah Buffaloes has been standardized as 63 days at NDRI herd . The CR Where, N=No. of inseminations required for pregnancy. A summary of the edited and normalized data sets is presented in db) and RHwith the corresponding period of study were obtained from Central Soil and Salinity Research Institute, Karnal. Monthly average Tdb (°C) and RH (%) were computed from daily weather information and subsequently the information were used to calculate the monthly average dew-point temperature (Tdp) by the method given by Jensen et al. [dp =116.9+237.3×ln(e)/16.78−ln(e), where, e (Kpa) = ambient vapour pressure.Meteorological data pertaining to daily dry bulb temperature  by using the formula given by Yousef .db+(0.36×Tdp)+41.2THI=TThe data were distributed up to fourth parity in the present study. A total of 13 periods of calving were taken with an interval of 18 months artificial insemination cycle of a set of Murrah bulls for each period according to progeny testing programme under network project on Buffalo improvement. Ages of Murrah buffaloes at first calving were classified into t 3 Groups i.e., age Group 1: < 37 months; Age Group 2: 37-53 months and age group 3: > 53 months, using mean and one standard deviation in the population. The effect of non-genetic factors like parity, period of calving and age group at first calving on normalized traits viz; SP, PR and CR were assessed by using a fixed model least-squares analysis with least-squares maximum likelihood software (LSML) as suggested by Harvey . The staijkl=µ+pai+pj+(AG)k+eijklYijkl is the observation on lth Murrah buffalo belonging to kth age group at first calving calved in jth period of calving in ith parity, and eijkl is random error ~NID . The analysis of variance for the non-genetic factors affecting different fertility traits was computed and the difference of means between significant sub-classes was tested by using Duncan’s multiple range test (DMRT) as modified by Kramer [where, Yy Kramer . The datThe effect of THI on fertility traits of Murrah buffaloes was assessed by using a fixed Least-squares model with software LSML. The genThe model used wasij = µ+THIi+eijYij is the observed fertility trait of jth Murrah buffalo under ith THI subclasses, µ=overall mean, THIi=fixed effect of ith THI (1−8) and eij is random error ~NID . Least-squares means along with standard errors for fertility traits under different THI subclasses were estimated. The significant effect of THI on fertility traits was assessed, and traits were adjusted with the significant effect of THI. The difference of means between THI sub-classes was tested using DMRT as modified by Kramer [Where, Yy Kramer . The thrij=a+b xi+eijYij is random residual ~NID . The zone where maximum increase in SP and decline in PR and CR were observed with per unit rise in monthly average THI was determined as the critical heat stress within the HSZ.Where, a is intercept, b is regression coefficient and e2) of different fertility traits. Similarly, (r) of each fertility trait was estimated by intra class correlation method [Murrah buffaloes were genetically evaluated in NHSZ, HSZ and CHSZ based on their EBV for SP, PR and CR. Paternal half-sib correlation method  was usedn method . The EBV=Herd average of each fertility trait, n=number of records, h2=heritability estimate of fertility trait, r=repeatability estimate of fertility trait, =average of each fertility trait.Where, The month wise average dry bulb temperature varied from 12.43°C in the month of January to -32.54°C in may while RH ranged between 42.01% in April and 80.72% in August during the period. The lowest monthly average THI was 56.71 in January, and the highest mean THI was 81.60 in June over 20 years period as shown in The parity (p<0.01) and period of calving (p<0.05) had significant effect on SP, but THI had highly significant effect (p<0.001) on all the fertility traits of Murrah buffaloes  The leasIn our study, monthly average THI value<75 were found for the months from October to March while mean monthly THI value>75 was observed during April to September over 20 years period. Accordingly, two zones in a year were identified such as NHSZ with average THI 56.71-73.21 and HSZ with average THI 75.39-81.60. May and June were thus identified as CHSZ with THI 80.27-81.60.2) of SP, PR and CR was estimated as 0.06±0.03, 0.024±0.01 and 0.04±0.03, while the (r) estimates for the above traits were 0.13±0.04, 0.059±0.04 and 0.08±0.04, respectively , stressful (71-78) and extreme distress (>78)[Our findings classified a whole year into three different zones ess (>78). Armstroess (>78) categori2) and (r) estimates of SP in Murrah buffaloes were 0.06 and 0.13 in the present study. Thiruvenkadan et al. [2) of SP in Murrah buffalo cows. The variation may be due to the difference in number of sires used for breeding, differences in management practices and environmental conditions such as ambient temperature, humidity and rainfall. Jamuna [2) and (r) of PR in Murrah buffaloes as 0.02 and 0.09±0.05, which are in agreement with our findings. Boichard and Manfredi [2) and (r) estimates of CR of French Holstein dairy cattle as 0.02 and 0.032.The (hn et al.  reported. Jamuna  obtainedManfredi  reportedet al. [The EBV for SP was found increased from NHSZ to CHSZ, while the decline in EBV for PR and CR was observed from NHSZ to CHSZ in Murrah buffaloes. There was no report available regarding the estimation of breeding values for fertility traits of Murrah buffaloes in three different zones in a year under subtropical climate. The breeding value of CR and PR of Angus heifer cattle ranged between −0.2582 to 0.3401 and −0.4821 to 0.7793 . Bermannet al.  also repThe result revealed that May and June months were the CHSZ for SP and PR, The months June, July and August months were the CHSZ for CR in Murrah buffaloes. The breeding value for fertility traits was influenced by THI. The EBV for fertility traits differed in three zones. In CHSZ the EBV of SP was found around 2 days higher than NHSZ. A decline of −0.5% in EBV of PR and −0.31% in the EBV of CR was observed in CHSZ as compared to NHSZ. As an overall conclusion, climatic factors seem to influence on fertility and in order to enhance the fertility performance of Murrah buffaloes proper management should be followed during the CHSZ.AKC designed the work. SD conducted study and analyzed the data. PRS and AU helped in the compilation of data. AKC and AS reviewed and edited the manuscript. VS and KMS helped in writing the manuscript. All authors read and approved the final manuscript.


Human T cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T cell leukemia. We observed previously that depletion of dendritic cells (DCs) in CD11c-diphtheria toxin receptor transgenic mice followed by infection with cell-free virus led to greater proviral and Tax mRNA loads and diminished cellular immune response compared with mice infected with cell-associated virus. To understand the significance of these in vivo results and explore the host-pathogen interaction between DCs and cell-free HTLV-1, we used FLT3 ligand-cultured mouse bone marrow-derived DCs (FL-DCs) and chimeric HTLV-1. Phenotypically, the FL-DCs upregulated expression of surface markers  on infection, however, the level of MHC class I remained unchanged. We performed kinetic studies to understand viral entry, proviral integration, and expression of the viral protein Tax. Multiplex cytokine profiling revealed production of an array of proinflammatory cytokines and type 1 IFN (IFN-α) by FL-DCs treated with virus. Virus-matured FL-DCs stimulated proliferation of autologous CD3(+) T cells as shown by intracellular nuclear Ki67 staining and produced IFN-γ when cultured with infected FL-DCs. Gene expression studies using type 1 IFN-specific and DC-specific arrays revealed upregulation of IFN-stimulated genes, most cytokines, and transcription factors, but a distinct downregulation of many chemokines. Overall, these results highlight the critical early responses generated by FL-DCs on challenge with cell-free chimeric HTLV-1.


Rheum palmatum L., Aloe barbadensis Miller, Cassia angustifolia Vahl., and Polygonum multiflorum Thunb. Rhein has various pharmacological activities, such as anti-inflammatory, antitumor, antioxidant, antifibrosis, hepatoprotective, and nephroprotective activities. Although more than 100 articles in PubMed are involved in the pharmacological mechanism of action of rhein, only a few focus on the relationship of crosstalk among multiple pharmacological mechanisms. The mechanism of rhein involves multiple pathways which contain close interactions. From the overall perspective, the pathways which are related to the targets of rhein, are initiated by the membrane receptor. Then, MAPK and PI3K-AKT parallel signaling pathways are activated, and several downstream pathways are affected, thereby eventually regulating cell cycle and apoptosis. The therapeutic effect of rhein, as a multitarget molecule, is the synergistic and comprehensive result of the involvement of multiple pathways rather than the blocking or activation of a single signaling pathway. We review the pharmacological mechanisms of action of rhein by consulting literature published in the last 100 years in PubMed. We then summarize these pharmacological mechanisms from a comprehensive, interactive, and crosstalk perspective. In general, the molecular mechanism of action of drug must be understood from a systematic and holistic perspective, which can provide a theoretical basis for precise treatment and rational drug use.Rhein is a major medicinal ingredient isolated from several traditional Chinese medicines, including  Rheum palmatum L., Aloe barbadensis Miller, Cassia angustifolia Vahl., and Polygonum multiflorum Thunb.; rhein also has a relatively high content in R. palmatum L.  expression is enhanced after rhein treatment in NPC cells. The significant increase in CHOP protein levels is observed after 12 or 24 h of rhein treatment (Lin et al., PPAR is a nuclear hormone receptor, and it can be activated by fatty acids and their derivatives. PPAR has three subfamilies, which are involved in the regulation of the major metabolism, inflammation, and the important process of controlling cell fate (Feige et al., As a multitarget molecule, rhein exerts its therapeutic effect via multipathways. The pharmacological action of rhein is the comprehensive regulation result on the crosstalk among signaling pathways. As shown in Figure In the pathways involved in the pharmacological pathways of rhein, MAPK signaling pathway can be considered one of the most interactive pathways. The activation of MAPK is affected by four pathways, including VEGF, TGF-β, Wnt, and PI3K-AKT signaling pathways. Figure The influence of MAPK signaling pathway on other pathways is extensive and complex. Figure The rhein action on MAPK signaling pathway was reported in many articles, but the findings were controversial. Although the majority of researchers believe that rhein inhibits the phosphorylation of ERK1/2, p38, and JNK, a few studies determined that rhein increases the activity of these proteins. Figure PI3K-AKT signaling pathway is another signal transduction pathway with a wide interaction with other signaling pathways. Figure As the hub molecule in this pathway, AKT is the bridge of the extracellular signal and the nuclear gene expression. The activation of AKT affects some of its downstream pathways. In addition to its parallel MAPK signaling pathway, Wnt, NF-κB, and FOXO signaling pathways are regulated by the activated AKT. The activated AKT can inhibit the important molecule GSK3 of Wnt signal pathway, and it then promotes Wnt signaling pathway (Cohen and Frame, The effect of rhein on AKT is similar to the effect on the interaction of the three MAPK cascades. The activation or inhibition of AKT may also be the result of the comprehensive effects of rhein on the PI3K-AKT signaling pathway and on its interaction pathways. In addition, rhein affects several downstream targets of AKT and some of its downstream pathways. This phenomenon occurs probably because of the indirect influence of rhein through hub AKT protein mediation or the direct action to the AKT downstream protein. Likewise, the uncertainty and variousness of rhein regulation result in the diversity of pharmacological activities.As an important cellular process, cell cycle is affected by several upstream pathways. Cell cycle mainly manifests the performances for the regulation on cyclins and CDKs. By comparing the regulations of p42/p44MAPK and p38/HOGMAPK cascades on the activity and expression of cyclin D1 in CCL39 cells, researchers determined that p42/p44MAPK cascade can increase the activity and expression of cyclin D1, whereas the result of p38/HOGMAPK cascade is the opposite (Lavoie et al., Apoptosis is another important cellular process. Cell cycle arrest is often accompanied by apoptosis, and apoptosis can cause cell growth arrest (Rubin et al., In addition to the wide range of interactive pathways, such as MAPK and PI3K-AKT signaling pathways, some certain relationship exists among other signaling pathways. The Smad3/4 complex in TGF-β signaling pathway can bind to the β-catenin of Wnt signaling pathway with high affinity to block the transcriptional activity of TCF/LEF (Li et al., The rhein-mediated biological network is vast and complex. As shown in Figure Rhein is a multi-target molecule regulating multi-pathways at the molecular level. The therapeutic effect of rhein is the synergistic and comprehensive result of the involvement of multiple pathways. If one signal pathway is blocked in two parallel pathways, the other one may become a compensatory pathway. This phenomenon is a possible reason for the poor efficacy of single target drugs. For example, rhein can block the multiple sites of MAPK signaling pathway, thereby resulting in anti-inflammatory and antitumor effects (Martin et al., It is notable that the impact of drug concentration on the therapeutic effect is also essential. For example, the low concentration of rhein inhibits cell proliferation by blocking MAPKs, whereas the result of rhein with high concentration is on the contrary (Lin et al., Rhein has extensive pharmacological activities, and its mechanisms are complex and interrelated. The mechanisms of rhein are involved in the multipathways which contain close interactions. The therapeutic effect of rhein is the synergistic and comprehensive result of the involvement of multiple pathways rather than the blocking or activation of a single signaling pathway. The effects of rhein with different concentrations on the pathway may vary. In this review, the pharmacological mechanisms of rhein are summarized from a systematic and holistic perspective, thereby providing an important reference for precise treatment and individualized drug administering.Conceived and designed the experiments: ZX and DC. Performed the experiments: HS, GL, and DC. Wrote the paper: HS and ZX.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The primate T Lymphotropic viruses, which include HTLV-1/STLV-1, HTLV-2/STLV-2, HTLV-3/STLV-3 and HTLV-4/STLV-4 constitute a group of related human and simian retroviruses which share some common epidemiological, biological and molecular features. While HTLV-1 and HTLV-2 are relatively widespread, HTLV-3 has only been observed in few African individuals living in close contact with infected non-human primates (NHPs) and HTLV-4 has been identified in only one hunter living in South Cameroon. Recently, a molecular study conducted in Cameroon on 1107 NHPs of 21 different species detected and characterized STLV-4 infection in 6 gorillas. Therefore HTLV-4 could have emerged from a gorilla reservoir. In the context of our current ongoing studies on PTLV in Cameroon, we decided to look for PTLV-4 in a large series of blood DNA originating from persons at risk for such infection (hunters of NHPs), living in the same region than the index case, and also from several gorillas. HTLV WB serology was available for all the persons. We searched for HTLV-4/STLV-4 infection in humans and gorillas using two different nested PCR, one targeting the pol gene, the other one targeting the LTR. These methods were able to detect 1 copy in a background of 250 ng of DNA. We included in the first preliminary study: 1) 228 individuals from Cameroon that had reported a contact (mostly bite) with a NHP ; 2) 21 gorillas . Out of the 21 gorillas, one originating from Cameroon was found positive for both STLV-4 pol and LTR PCR. The screening of the human samples is still in progress and will be presented.


Drosophila JNK homolog) induced cell death is mediated by the canonical Wg signaling. First, loss of Wg signaling abrogates Bsk-mediated caspase-independent cell death. Second, activation of Wg signaling promotes cell death in a caspase-independent manner. Third, activation of Bsk signaling results in upregulated transcription of wingless (wg) gene. Finally, Wg pathway participates in the physiological function of Bsk signaling in development. These findings not only reveal a previously undiscovered role of Wg signaling in Bsk-mediated cell death, but also provide a novel mechanism for the interplay between the two important signaling pathways in development.Cell death is an essential regulatory mechanism for removing unneeded cells in animal development and tissue homeostasis. The c-Jun N-terminal kinase (JNK) pathway has pivotal roles in the regulation of cell death in response to various intrinsic and extrinsic stress signals. The canonical Wingless (Wg) signaling has been implicated in cell proliferation and cell fate decisions, whereas its role in cell death remains largely elusive. Here, we report that activated Bsk (the  This pathway is initiated by various intrinsic and extrinsic signals including the tumor necrosis factor (TNF) family ligands, and is mediated through a mitogen-activated protein (MAP) kinase cascade.β-catenin pathway, the Wnt/Ca2+ pathway, and the Wnt/Frizzled (Fz)-Planar Cell Polarity (PCP) pathway.15 The canonical Wnt signaling represents one of the best investigated pathways and has crucial roles in regulating cell proliferation, cell migration, and cell fate decisions.17 Deregulated Wnt signaling is frequently associated with human diseases, especially cancers.20 Genetic studies of the Drosophila Wnt protein Wingless (Wg) have made great contributions to our understanding of Wnt signaling.23 In the absence of Wg signal, the β-catenin homolog Armadillo (Arm) is phosphorylated and degraded by a multi-protein 'destruction complex' that includes the Adenomatous Polyposis Coli protein (APC), Shaggy (Sgg)/Zeste-white-3, Axin (Axn), and Casein Kinase1 (CK1). In the presence of Wg signal, the receptor dFrizzled (dFz) and co-receptor Arrow (Arr) bind to Wg, recruit and phosphorylate the scaffold protein Dishevelled (Dsh), which antagonizes the 'destruction complex' and prevents Arm from degradation. As a consequence, accumulated Arm translocates to the nucleus and stimulates target genes expression by binding to the transcription factor Pangolin (Pan).25Wnt proteins are a family of highly conserved secreted molecules that regulate cell–cell interactions through distinct intracellular signal transduction pathways, including the canonical Wnt/28 Yet little is known about the interaction between JNK and the canonical Wnt/Wg pathways in development. A collaboration between JNK and the canonical Wg signaling was reported to promote dorsal closure and ventral patterning during Drosophila embryogenesis, but the two pathways appear to function in parallel.29 An interaction between AP-1 and β-catenin/TCF complex was also reported to have a role in carcinogenesis, though the underlying mechanism remains elusive.30Crosstalk between different signaling pathways occurs extensively and forms a complex signaling network that enable cells to interpret multiple inputs and execute different responses in a context-dependent manner. The interplay between JNK and the non-canonical Wnt/Frizzled (Fz)-PCP pathways has been extensively studied, in which JNK signaling acts downstream of Dsh to promote PCP establishment.Drosophila. Loss of Wg signaling specifically suppresses Egr or Hep-induced Bsk-dependent cell death in eye, wing, and thorax. Stimulation of Wg signaling phenocopies Bsk activation and induces caspase-independent cell death. Furthermore, Wg signaling is both necessary and sufficient for the expression of Bsk signaling reporter puckered (puc). Epistasis experiments indicate that Wg signaling acts downstream of Bsk to regulate cell death and other physiological functions. Finally, activated Bsk signaling upregulates wg transcription, providing a molecular mechanism for the genetic interaction between Bsk and Wg pathways. These findings not only expand our existing knowledge about the molecular basis of complex signaling network in regulating cellular activities, but also provide additional strategy and approach for cancer therapy.In the present study, we have identified from a genetic screen that the canonical Wg signaling has a crucial role in Bsk-mediated cell death in GMR-Gal4 (GMR>Egr) triggers massive cell death and produces a Bsk-dependent small eye phenotype  animals  induces vast cell death and produces small and rough eyes with fused ommatidia  encoding the Drosophila JNK kinase. Expression of HepCA in the eye (GMR>HepCA) induces Bsk-dependent cell death and generates a small eye phenotype  triggers Bsk-dependent cell death in the thorax and produces a small scutellum phenotype  compartment boundary driven by ptc-Gal4 results in elevated cell death in third instar wing discs as revealed by acridine orange (AO) staining  and Bsk activation (puc-LacZ expression) than Hep in the wing disc  (Df(3 L)H99 , expression of the caspase inhibitor Diap1  . As a po (Cas3*) . Further (Cas3*)  are not (3 L)H99  that delor Diap1 , a domin1 .41 In wild-type wing discs of third instar larva, puc is only expressed in the dorsal tip  presented only in the wing disc and salivary gland, but not in the eye disc, is required for Wg signaling to induce puc expression.To examine whether Wg signaling is sufficient to elicit ignaling . Consistignaling . Howeverye discs , suggestpuc expression in both the wing disc and the salivary gland.Together, the above data provide compelling evidence that the canonical Wg pathway is both necessary and sufficient for Bsk-induced DN  boundary and in two concentric rings in the wing pouch  that resulted in a dramatic reduction of the posterior compartment , lethal giant larvae (lgl) and disc large (dlg),43 results in Bsk-mediated cell death and elimination from the tissue.45 Knocking-down of lgl along the A/P compartment boundary (ptc>lgl-IR) in the wing pouch induces Bsk-dependent cell death  by expressing P35, induce compensatory proliferation in neighbor cells by secreting growth factors like Wg, Dpp and Hh.50 Both secretion of growth factors and compensatory proliferation depends on Bsk signaling,52 yet the mechanism has not been fully illustrated. In mammal, a connection between c-Jun and TCF4, transcription factors of JNK and Wnt pathways respectively, is reported to have a role in intestinal tumorigenesis.30The interplay between JNK signaling and the non-canonical Wnt/Fz-PCP pathway has been extensively studied, in which JNK acts downstream of Dsh through Rho family small GTPase, and regulates the key effectors such as Myosin II and other cell adhesion components.Drosophila, and obtain the following results: (1) loss of Wg signaling suppresses Bsk-mediated caspase-independent cell death and puc expression; (2) activation of Wg signaling induces caspase-independent cell death and puc expression, which challenges the previous opinion that puc is a direct transcription target and readout of Bsk signaling. We cannot rule out the possibility that a context-dependent feed-back loop may exist between Wg and Bsk signaling; (3) Wg signaling acts downstream of Bsk in promoting cell death; (4) Wg pathway participates in the physiological function of Bsk signaling; (5) activated Bsk signaling results in upregulated wg transcription. Thus, we not only deliver compelling evidences for the conclusion that activated Bsk signaling promotes Wg pathway-dependent cell death, but also provide the underlying mechanism for the interplay of the two pathways that have crucial roles in development. However, since Wg signaling is necessary for cell proliferation and viability, complete loss of Wg signaling would result in developmental defect and animal lethality, we used heterozygous mutants or RNAi-mediated knock-down approach to examine the effect of loss-of-Wg signaling on Bsk-mediated cell death. In such backgrounds, Wg signaling is effectively reduced, but not completely abolished. Consequently, Bsk-mediated cell death and resulted phenotypes are significantly, but not fully, suppressed H99 and expression of Diap1, a dominant-negative form or knockdown of dronc, or knockdown of drice and dcp-1 . Constitutively active Wnt signaling has been associated with tumor progression in many cancers, yet in this study we characterize a role of Wg signaling in promoting cell death, which implies that Wnt signaling may also possess a tumor suppressor function. Meanwhile, a substantial body of evidence also suggests that JNK signaling is closely related with tumor formation and metastasis,64 thus the present study shed new light on the crosstalk and involvement of JNK and Wnt signaling in cell death and cancer development.Since the discovery of the first wg1–17;65Sp;34dsh6;66wgIG22, dshV26, armXM19;67arm1, pan13a;68 SggEP1576;69UAS-Wg, UAS-Dsh, UAS-wg-IR and UAS-arm-IR;70UAS-Pan;71bsk1; 72UAS-EgrRegg1;10UAS-HepCA, UAS-Egr, UAS-BskDN, UAS-Puc, Df(3 L)H99, DIAP1, DRONCDN and GUS-Dp53259H;32UAS-HepWT;31pucE69, pnr-Gal4;12GMR-Gal4, ptc-Gal4 and sd-Gal4;73UAS-Dp53H159N.35 The wg-LacZ, UAS-Rpr, UAS-Dp53, UAS-Arm, UAS-Apc2, UAS-Axn, and UAS-dsh-IR lines were obtained from the Bloomington stock center. The UAS-pan-IR and UAS-lgl-IR lines were obtained from the Vienna Drosophila RNAi Center (VDRC). The UAS-wg-IR, UAS-arm-IR, UAS-dronc-IR, UAS-drice-IR, UAS-dcp-1-IR lines were got from Fly Stocks of National Institute of Genetics (NIG-FLY).The following stocks have been described previously: y w1118hs-Flp; act>CD2>Gal4 UAS-GFP/Cyo strain. Clones were induced by heat shock at 37 °C for 10 min, late third-instar larvae were dissected after recovering for 3 days.Fluorescently labelled clones were produced in larval imaginal discs using the β-Gal (DSHB). The second antibodies were used as follows: 1 : 1000 anti-mouse CY3 (CST), 1 : 1000 anti- rabbit CY3 (CST).The following primary antibodies were used: 1 : 400 rabbit-anti-Caspase 3 , 1 : 300 mouse-anti-Wingless , 1 : 500 mouse-anti-32The discs were dissected at the third instar larva stage, and stained for AO as described.The discs were dissected at the third instar larva stage, and stained for TUNEL using the Fluorescein Cell Death Kit produced by Boster Company. Imaging of prepared sample was conducted by a Leica confocal microscope .β-galactosidase as described.74The discs were dissected at the third instar larva stage, and stained for 75 primers pairs of rp4975 and puc76 were used as previously described.Wing discs were dissected at third instar larva, for each genotype, more than 200 discs were collected and total RNA was isolated using TRIzol . qRT-PCR was performed as previously described,P-value of <0.05 was considered as significant.For loss of ACV phenotype, statistics were analyzed using chi-square test. For AO stanining and area of disc clones, one-way analysis of variance test followed by the post Dunnett test or Kruskal–Wallis text followed by the post Dunns text was used. A


There are various fertility control methods  currently available that aim to reduce the abundance of problematic free-ranging mammalian wildlife. Here, we propose that dissimilarities in the mechanism of action indicate these methods produce great variation in animal welfare outcomes. We present a framework to assist managers in minimising animal welfare risks.Investigation of fertility control techniques to reduce reproductive rates in wildlife populations has been the source of much research. Techniques targeting wildlife fertility have been diverse. Most research into fertility control methods has focused upon efficacy, with few studies rigorously assessing animal welfare beyond opportunistic anecdote. However, fertility control techniques represent several very different mechanisms of action , each with their own different animal welfare risks. We provide a review of the mechanisms of action for fertility control methods, and consider the role of manipulation of reproductive hormones (“endocrine suppression”) for the long-term ability of animals to behave normally. We consider the potential welfare costs of animal manipulation techniques that are required to administer fertility treatments, including capture, restraint, surgery and drug delivery, and the requirement for repeated administration within the lifetime of an animal. We challenge the assumption that fertility control modalities generate similar and desirable animal welfare outcomes, and we argue that knowledge of reproductive physiology and behaviour should be more adeptly applied to wild animal management decisions. We encourage wildlife managers to carefully assess long-term behavioural risks, associated animal handling techniques, and the importance of positive welfare states when selecting fertility control methods as a means of population control. Equus caballus); [Animal welfare has been steadily gaining importance and society now recognises it has an obligation to consider the welfare of all animals, including free-ranging species that are perceived to be overabundant or pests. As community and ethical opposition to the killing of animals has risen, especially for charismatic species ; ), more fballus); . Fertiliballus); ,4,5, or ballus); ,7. Theseballus); ,9, incluballus); .Despite the importance of animal welfare, there are other aspects of fertility control methods that cannot be ignored. Efficacy will always need to be considered because if a method is chosen that has a reduced welfare impact on the individual, but does not achieve its animal management goals, then arguably doing nothing at all would have resulted in better animal welfare outcomes . The oveOnce it has been decided that a fertility control program will be employed, there is considerable uncertainty when choosing a specific fertility control product or technique. Several authors have attempted to address the broad side-effects associated with fertility control of wildlife, including animal welfare impacts ,14,15, bThis review attempts to demonstrate the differences between broad categories of fertility control techniques, with a view to facilitating future selection of practices. Fertility control has been applied to non-mammalian wildlife  but thiThe central aim of this study is to specifically provide a framework for assessing animal welfare risks for various fertility control methods for mammalian wildlife. It cannot be disputed that efficacy and financial costs are important considerations when management decisions are being made, however, these issues have been thoroughly addressed by other reviews  and so wversus remote injection or “darting”). Odocoileus virginianus)), multiple fertility control methods, representing several mechanisms of action, have been employed. These methods, and many more, have previously been reviewed in detail [versus chemical), but given the inextricable association between any method’s mechanism of action and its effect, we argue that classification according to mechanism of action is the most logical approach.A wide diversity of methods has been applied to wildlife fertility control over several decades. n detail ,15. SeveOwing to the diversity of fertility control techniques currently available  or the use of implants that physically irritate the female reproductive tract  means to disrupt the normal functioning of the reproductive system, and do not alter naturally occurring reproductive hormone levels. These methods encompass surgical techniques that devices; ). Surgicdevices; ,43. We tChemical non-endocrine methods rely on inducing an autoimmune response through vaccination against naturally occurring ovum proteins, most commonly the zona pellucida (ZP) , to prevThis first principles approach of classifying fertility control methods according to modalities should enable more meaningful comparisons and improved assessments of welfare risks at an individual animal level. This approach should also help to clarify some misconceptions arising from the oversimplification that has been used in the past to describe classes of methods . We thead hoc basis, and there remains much variability in how modalities are chosen and applied.For some time, many contentious lethal  and non-lethal  wildlife management methods have been closely scrutinised, with strong focus placed upon animal welfare impacts, rather than efficacy in isolation ,48. QuanIt is proposed that any animal treatment can have short-term adverse effects on welfare (for example those caused by an injection) followed by long-term effects, which may be very different . Both shin situ [Most stages of wildlife management techniques that involve animal manipulation are likely to impose welfare costs. This is recognised for lethal wildlife control techniques, where animal welfare assessment frameworks require consideration of stages of animal manipulation prior to the killing method . Some wiin situ . ClassesIn contrast, other fertility control programs have recognised the potential animal welfare costs of administration procedures and have chosen methods that allow for remote delivery of a single dose of an agent , effectiThere are variations in the durations of action for different fertility control approaches. Some methods  produce permanent infertility while some exogenous endocrine methods may affect fertility for less than one year . Some cMany remotely delivered immunocontraceptive modalities, being adjuvanted vaccines that rely on stimulating the immune system, have been shown to induce granulomatous inflammation at the site of injection ,65. SomeO. virginianus; [Macropus eugenii; [M. eugenii) also resulted in increased fat deposition [Significant or prolonged physiological changes may be considered an animal welfare concern as they may alter an animal’s ability to perform natural behaviour  and/or pinianus; ) and tameugenii; ). Endocrposition . Changesposition . Especiaposition ,72. SuchThe importance of natural behaviour, and the ability of animals to experience positive welfare states, is a central tenet of modern animal welfare science . BehavioE. caballus) subjected to endocrine suppression (GnRH immunocontraception) included observations that treated animals fed less, rested more, and travelled less [M. eugenii) resulted in decreased agonistic (related to fighting) behaviour with intact males, lower dominance, and higher sexual interest from intact males [Macropus giganteus) prefer to be associated with females that have not been subjected to endocrine suppression [For example, behavioural effects in free-ranging feral horses  with the extension of a breeding season through the presence of sterile, but hormonally competent, females. The average body condition of these males was significantly poorer than that of males in control areas. Similar effects have been observed in male deer [E. caballus; [Loxodonta africana; [Beyond any welfare impact suffered by individual animals subjected to fertility control treatment, there are also often non-intuitive welfare costs to other animals of the same species, through social interactions. Specifically, fertility control modalities that prevent pregnancy occurring without preventing oestrus behaviour  often induce a prolonged breeding season. This phenomenon has been observed for white-tailed deer  aaballus; ). The pr [et al.  demonstrale deer . It has aballus; ) and Afrfricana; ). In addfricana; .Despite the central intended effects of fertility control agents in preventing the birth of offspring, and hence the opportunity for animals to express parental behaviour, the loss of this positive welfare state has not commonly been cited as a welfare concern for treated wildlife. In comparison, the welfare risks of deprivation of this natural behaviour have been recognised in zoo animals. The European Association of Zoos and Aquaria  stated: The impact of this animal welfare risk is not easily demonstrated with empirical evidence. However, deprivation of freedom to express natural behaviour is universally considered an animal welfare concern  as it alO. virginianus) males subjected to an endocrine suppression modality (GnRH immunocontraception) display very few mating attempts with untreated  females [Another positive welfare state that may be deprived of animals is the freedom to express natural reproductive behaviour, including courtship and mating. Endocrine suppression modalities often reduce or eliminate courtship and mating behaviour , through females . CourtshMany fertility control modalities currently applied in wildlife management were initially developed to meet the economic and emotional needs of owners of domesticated animals, such as companion or production animals. Some endocrine suppression modalities  were developed for livestock species to intentionally alter physiology  and behaviour (to reduce aggression) to benefit production, rather than to prevent reproduction . FertiliIn livestock management, hormonally incompetent animals have been viewed as “modified” by many scientists, with concerns about the welfare of treated animals as well as human health from consuming treated animals . Many stAnimal welfare is complex and guidelines for how it should be assessed have evolved over time ,92. ModeAlthough animal welfare is a complex entity, the Five Domains model was proposed over 20 years ago as a conceptual framework to simplify welfare considerations for research animals , but it etc.), their welfare is considered by a large majority of the general public to be severely impaired through impedance of natural behavior, and an absence of positive experiences, that is imposed by small, restrictive housing [The majority of studies that have addressed animal welfare during fertility control programs have effectively restricted their assessments to domain 3 of the model; health, as typically only measures of injury rate, the presence of disease, and body condition have been examined . The hig housing ,96,97.i.e., 2–3 years [For the majority of wildlife fertility control studies, the length of time that any side-effects have been monitored for has been restricted to short time frames . How–3 years . The pot–3 years , and ter–3 years ). These –3 years , it is lSimilar approaches have been used for fertility control in domestic and free-ranging animals, but there are important differences in society’s perception of our duty of care for animals in each context. There is broad acceptance that the maximisation of health should be the focus of animal welfare for the keeping of companion animals , while tF. catus); [ad libitum access to resources (water and feed) for study animals [or wild  for com catus); ,104). We catus); , or anim catus); ). For th animals ,106. In et dogs” ,61), somE. caballus) fertility control, approaches that impact upon androgenic  behaviour [Canis latrans) should be those that allow the animals to remain hormonally competent, such is the importance of the endocrine system for the maintenance of natural behaviour [The same logic should not apply for all wild animals, as the duty of care provided by humans often ends for managed wildlife at the point of release from restraint or administration of treatment . For thehaviour  are no lehaviour . We arguE. caballus) in North America and the second describes the relative lack of refinement in modalities considered suitable for Eastern grey kangaroos (M. giganteus) in Australia.To illustrate the dissimilarities in approaches that have been taken to fertility control in different species and jurisdictions, we present two case studies. The first describes refinement of modalities used for feral horses  [The management of Australian eastern grey kangaroos contrasts strongly with the management of American feral horses. In the USA, management goals have included minimising animal welfare impacts of fertility control on wildlife, particularly feral horses , by usininereus) ,115,118.These case studies have shown the different approaches taken towards fertility control in two wildlife contexts and highlight the need for increased consideration for the welfare risks of current practices. For this reason, we propose a framework to better enable researchers and managers to understand the animal welfare risks of any fertility control modality and to improve the balance between meeting animal interests with management needs.carte blanche approach towards a “best practice” approach for refining methods to minimise animal welfare impacts. It is our contention that methods that induce endocrine suppression, and requiring repeated or multi-stage interventions, are likely to represent poorer animal welfare outcomes than those methods that do not  are likely to be the most humane fertility manipulation methods currently available  represent poorer animal welfare outcomes when compared to remotely deliverable methods . Given tvailable . While wOn the same mechanistic basis, we contend that gonadectomy represents the least humane of the currently available fertility control technique for wildlife. Gonadectomy requires capture, often major  surgery, anaesthesia, and induces complete and irreversible reproductive endocrine suppression, and accompanying permanent changes in natural behaviour and physiology ,90. We scarte blanche and this represents the antithesis of scientific reasoning. We have attempted to address the short-term and long-term animal welfare risks, as per Broom [(1)It should be recognised that all fertility control methods impose animal welfare impacts. The deprivation of the opportunity to display parental behaviour in nurturing species is an animal welfare impact,(2)Methods should be chosen that pose the lowest cumulative animal welfare cost, including capture, administration and frequency of treatment,(3)All domains of animal welfare should be considered, including behaviour and the opportunity for animals to have positive experiences,(4)Methods that maintain hormonal competence, thus promoting natural behaviour, should be chosen over methods that induce endocrine suppression, and(5)Where uncertainty exists regarding welfare impacts, fertility control modalities should not necessarily be chosen over alternative lethal or non-lethal methods.In an attempt to improve the present approach to the choice of fertility approaches, some authors have suggested the use of formal criteria for the selection of methods that are appropriate for field applications . While wer Broom , in a frer Broom ). Table Any decisions regarding wildlife management will involve seeking a balance between the interests of the wildlife and other stakeholders. The suggested use of our framework in ethical decision-making has some unavoidable subjectivity relating to the difficulty in comparing various types of risks , which value-based ranking systems  may addWe recognise that the range of species and contexts for which wildlife fertility control is desired makes the proposal of a single preferred method impossible . We acceEfforts to apply fertility control to wildlife have now been performed for over 30 years but there has been little standardisation of procedures over this time. Research into this area has primarily focused on the important aspect of efficacy but this has meant that the associated impacts on animal welfare have only seldom been addressed. Consequently, there is a growing need for more meaningful interchange between animal welfare scientists, wildlife biologists and wildlife managers . Underst


The Wnt signaling in the stably transfected cell line was proved to be very sensitive to (−)-epigallocatechin-3-gallate (EGCG) and lithium chloride (LiCl) treatment, respectively. Natural compounds were screened and a couple of novel inhibitory modulators of the Wnt signaling pathway were obtained.Abnormal activation of canonical Wnt signaling has been associated with various types of cancer. Inhibitory reagents targeting the Wnt signaling have great potential to inhibit the growth of relevant tumors. Here we generated a cell-based screening strategy for identification of antagonists of the Wnt/β-catenin signaling pathway. Stable expression


Molecular dynamic simulations show that this hydrophobicity can be further enhanced by the atomic-scale roughness. Consequently, the hydrophobic gap width is enlarged to about 0.6 nm for roughened CF3-terminated solid surfaces. In contrast, the hydrophobic gap width does not increase too much for a rough CH3-terminated solid surface. We show that the CF3-terminated surface exists in a microscopic Cassie–Baxter state, whereas the CH3-terminated surface exists as a microscopic Wenzel state. This finding elucidates the underlying mechanism for the different widths of the observed hydrophobic gap. The cage structure of the water molecules (with integrated hydrogen bonds) around CH3 terminal assemblies on the solid surface provides an explanation for the mechanism by which the CH3-terminated surface is less hydrophobic than the CF3-terminated surface.It is well known that the close-packed CF When the cosine of the apparent contact angle θ* is plotted as a function of the cosine of the Young contact angle, the slope yields the roughness factor.In the Wenzel model, the contact angle is described by the following equation13637ϕs is the ratio of solid in contact with the liquid. In this state, air is trapped between the solid and liquid. The Cassie–Baxter model works well for the large contact angle regime, while the Wenzel model works for intermediate contact angles36et al.θ cannot be predicted by only a roughness factor because large pinning effects are observed for the intermediate contact angle regime. By adding a term (to the Wenzel model), the pinning effect on the contact angle can be taken into account:dS is the magnitude of the pinning effect (the range of θ is approximately from 40° to 90°). The hemi-wicking state is just a counterpart of Cassie–Baxter state of the wetting phaseIn the Cassie–Baxter model, the contact angle is described by the following equation2374) and carbon tetrafluoride (CF4) molecules, without losing hydrogen bonds. However, an assembly of many apolar units (as they are on the solid surface) will inevitably break hydrogen bonds at the surface53-terminated surfaces better than at CF3-terminated surfaces. This, in turn, explains why a CF3-terminated surface is more hydrophobic than a CH3-terminated surface. The strong hydrophobic nature of the CF3-terminated surface favors a microscopic Cassie–Baxter state at the rough surface, which enhances the observed hydrophobic gap width.MD simulations162329304344454647484953- and CF3-terminated 2D rough silica/water system interface at 3 ns, respectively. “Vapor phase” water was observed within the concave surface regions the CF3-terminated 2D rough silica surface (n = 4). This implies an increase in the hydrophobic gap relative to that of the flat surface. We counted the number of water molecules within the concave regime  as a function of time and found the systems were equilibrated at most 1.5 ns. To evaluate changes in the hydrophobic gap, the density profile was calculated from 2.0 ns to 3.0 ns and plotted in 3-terminated 2D rough silica surface (n = 4). It corresponds well to the various hydrophobic gap values obtained by X-ray reflectivity measurements222326273-terminated surface show only a modest change over the same range of surface roughness. This also corresponds well to the consensus regarding the hydrophobic gap for the CH3-terminated surface162021243-terminated 2D rough silica surface (n = 4), but from the density profiles, we can confirm that the hydrophobic gap gradually increases depending on the concave number. It indicates that the water molecules have opportunities to enter and leave the concave, as determined by a dynamic equilibrium.Inspired by the pioneering work of Kulinich and Farzanhet al. by integrating the density deficit at the solid–water interface for similar systems3-terminated 2D rough silica surface is again confirmed to gradually increase from 0.3 Å for the flat case to 2.8 Å for the n = 4 CF3-terminated 2D rough surface. In X-ray or neutron reflectivity experiments, the multilayer structure is assumed and only one roughness parameter is introduced in the analysis. Our results show that at least, and perhaps more than, two parameters – not only the amplitude but also the number of grooves (i.e. the roughness geometry) – can influence the hydrophobicity . We therefore argue that the surface geometry (among others) is responsible for the different hydrophobic gap results reported previously22232627The depletion layer was also calculated by Mezger 3-terminated silica surface, the contact angle is ~119° on the flat surface and ~141° on the 2D roughened surface, which is similar to the experimental results ranging from 111° to 133° from previous studies223-terminated silica surface, the contact angle is ~81° on the flat surface and ~119° on 2D roughness surface. The contact angle on methylated SiO2 is reported as 80° to 112° in experiments33We studied the wetting behavior of a cylindrical drop on a silica surface . The cyl52533-terminated silica surfaces embody a microscopic Cassie–Baxter state (3 case (ϕs in 3-terminated surface.We suggest that CFer state . This caer state . With th (3 case . The slo3-terminated silica surfaces are in a microscopic Wenzel state (r as shown in Lx × Ly). For the 2D rough silica surface, the roughness factor is 1.37 and 1.59 for n = 3 and n = 4, respectively. In the case of the 3D rough silica surface (n = 4), the slope is 1.62. The green line passing through the origin  is drawn in the range from around −0.5 to around 0.5 with a slope of 1.6 . Spori et al.3-terminated silica surfaces causes the different equilibrium contact angles, which results from the pinning and depinning effect.The droplet contact angle for the CHzel line . Instead3-terminated surface system is in microscopic Cassie–Baxter state. The CH3-terminated surface system is in a microscopic Wenzel state, besides the pinning effect is observed. In fact, the “vapor phase” within the concave (as mentioned above) will be thermodynamically stable if the energy of the Cassie–Baxter state is lower than that in the Wenzel statecosθc = (Φs − 1)/(r − Φs)37Φs = 0.47 and 0.77, we obtained θc as 118.2° and 105.7° for 2D and 3D rough surface (n = 4), respectively. The contact angle for the CF3-terminated silica surface is ~119°, which is indeed larger than, and in proximity of, this critical angle.In summary, the CF3-terminated surface case, while a symmetrical droplet (with a circular contact line) was observed for the CF3-terminated silica. The anisotropic droplet behavior indicates that the CH3-terminated surface is indeed in a microscopic Wenzel state, while the CF3-terminated silica is in a microscopic Cassie–Baxter state. The “vapor phase” water enables the line-pattered (i.e. 2D rough) CF3-terminated silica surface to impart isotropic-like behavior (because the droplet sits on a cushion of vapor phase). It is reasonable that the contact angles would be different for a CH3-terminated silica substrate, since the effective contact lengths (i.e. contact areas if a stripe with a finite thickness is assumed) are different along the different directions. To corroborate this hypothesis, we measured the contact angles along the parallel and perpendicular directions of the 2D rough surface. The θ┴ is ~132°, whereas the θ|| is ~94°. The measured degree of wetting anisotropy, defined as the difference of the contact angle values for the two directions is about 38°. In contrast, the difference of the contact angle values for the CF3-terminated surface is only 6°. Here, we caution that these values should be read qualitatively as the contact angle of spherical droplet depends on the size of droplet because of the line tension effect5253Previous experiments showed that droplets in contact with a line-patterned surface elongated along the concave (micro-groove) and the contact line deviated from an ideal circle3-terminated surface. Furthermore, we found that this phenomenon is tightly connected with a microscopic Cassie–Baxter state induced by atomic-scale roughness. In particular, the CF3-terminated surface is more hydrophobic (with water contact angle ~119°) than a CH3-terminated surface (with water contact angle ~81°). To investigate the mechanism responsible for the different hydrophobicity of CH3- and CF3-terminated surfaces, the interfacial water structures were compared, including the radial distribution function (RDF) between C atoms of CH3/CF3 unit and O atoms of water, the angle distribution function (ADF) between the C…O interatomic vector and the dipole moment vector of the water molecule, and the hydrogen bond number distribution (between water molecules) surrounding each individual CH3 and CF3 unit. These values were calculated in each case by using the trajectory from 2.0 to 3.0 ns . The first minimum in the RDF is 0.54 and 0.57 nm for the CH4 and CF4 molecules, respectively. These values were used as the cutoff distance for the calculation of ADFs. As shown in 4 and CF4 molecule-in-water systems, which indicates a preferred orientation of water molecules within the cage (like methane clathrate hydrate)5649θ = −1.0 is almost diminished for CF3-terminated surface, while it is essentially the same for the CH3-terminated surface and CH4 molecule-in-water systems. The water coordination number of CX4 molecule is roughly 23.6 for CF4 and 20.4 for CH4, respectively. It is reduced more than half to a range with a maximum value 8.0 for CF3 terminals (the average is 4.0) and to a less extent 12.0 for CH3 terminals (the average is 6.4) on the surface. Remarkably, the hydrogen bond number  on the CH3-terminated surface is much higher that on the CF3-terminated surface . A simple schematic explanation (for better water “cage structure”) would be that CH3 group is smaller than CF3 group  provides the underlining mechanisms for weaker van der Waals interactions.All RDFs of the CH surface . This inF3 group . In a pr3-terminated rough surface) in contact with the liquid water phase is about 47%. This means almost half of the solid is in contact with the “vapor phase” indicating that water molecules do not exceed the middle points of the rough surface. The distance between the middle points of adjoining convexes d is 1.21 nm in the case of the 2D rough surface with n = 4; the RDF between the oxygens of water molecules has a peak at 0.28 nm3 and O of water is about 0.42 nm. This means that roughly three water molecules are in line with the middle points of the concave with n = 4  could have already played a significant role.We investigated the enhanced hydrophobic gap using 2D or 3D rough, and CH3 terminated silica surface have an ordered, ice-like structure3 unit can thus be responsible for the ice-like structure3-terminated) surface exhibits both hydrophobic and oleophobic character1011121314It has been suggested that interfacial water against a hydrophobic solid surface can have a structure similar to that of the liquid–vapor interface. Recent vibrational sum frequency spectroscopy studies have shown, however, that water molecules at CHA is the amplitude of the surface function, B is the average of the surface function, n is the number of concaves within L, and L is the length of the simulation box in the x-direction. We refer to this surface as the 2D rough surface. We proposed another surface function as follows:Lx and Ly are the length of the simulation box in the x- and y-directions, respectively. We call this the 3D rough surface. In the case of the 2D rough surface, the rms roughness becomes A/2. In this simulation, we assumed A = 7.07 Å for all calculations. When A = 7.07 Å, the rms roughness was 5.00 and 3.53 Å for the 2D and 3D rough surfaces, respectively, which correlate with the same roughness magnitude as in experimentFor simplicity, we assumed a surface function as follows:3 or CF3 groups were attached to unbonded terminals of both the upper and bottom surfaces. The percentages of single Si-CX3 is ~70–80% and the rest are germinal Si-(CX3)2.A large well-relaxed model of vitreous silica3 termini, and CF3 termini were described by the CLAYFF593 unit was attached to an unbonded Si, its charge was adjusted according to the number of termini to keep the local charge neutral. We used the Si-C bond length proposed by Sun593)2. The CH3 and CF3 groups were found to be re-distributed due to their repulsive forces between CX3 groups. The nearest inter-molecule C-C distances were 0.35 and 0.40 nm for CH3- and CF3-terminated surfaces, respectively. That is, the larger size of CF3 terminals62All MD simulations were performed using the GROMACS packageThe simulations were performed at a constant temperature of 300 K using the Berendesen thermostat3 or CF3 and 2D or 3D rough and 0 ≤ n ≤ 4). 16000 water molecules  were used to construct the interface system. We defined the width of the hydrophobic gap to account for the shift amount of the water boundary on the rough side of the silica. The bottom of the solid surface was defined as zero position (z = 0) of the density profile. The intersection position of the averaged density profiles of rough solid cases (1 ≤ n ≤ 4) and flat solid density profile was determined as the solid boundary. The water boundary (on the side of solid surface) was determined by the position which water density profile has half of the bulk density. The hydrophobic gap was defined as the difference in the position of the solid boundary and water boundary. The rough solid density profile basically follows the same arcsine function. Therefore, the averaged density can be used. However, the intersection position of the 2D rough surface and flat surface system is slightly different from the intersection position for the 3D case. Therefore, a minor difference of the hydrophobic gap width is anticipated for the flat case when the hydrophobic gap as function of concave number is shown x-direction and 19452 water molecules  were set above the substrate . The solid boundary was determined by the uppermost position that has 1% of the maximum local density of the solid. The contact angles were calculated every 0.1 ns by least-square fitting to a circle with water boundary points above the solid boundary .3-terminated 2D rough surface, an additional simulation using a CH3-terminated silica surface which has 3 concaves in 9.68 nm was conducted.To confirm the pinning effect in the case of the droplet on CHx- and y- directions. 11417 water molecules  were set above the substrate  or 4 concaves were used. The substrate was doubled in both ubstrate . 3 ns siHow to cite this article: Katasho, Y. et al. Mechanisms for Enhanced Hydrophobicity by Atomic-Scale Roughness. Sci. Rep.5, 13790; doi: 10.1038/srep13790 (2015).