Nicotiana tabacum Samsun nn. This hypersensitive-like response (HLR) blocked amplification of recombinant TMVFN20 in tobacco and limited the utility of recombinant TMV vaccines against FMDV.By using tobacco mosaic virus (TMV)-based vectors, foreign epitopes of the VP1 protein from food-and-month disease virus (FMDV) could be fused near to the C-terminus of the TMV coat protein (CP) and expressed at high levels in susceptible tobacco plants. Previously, we have shown that the recombinant TMV vaccines displaying FMDV VP1 epitopes could generate protection in guinea pigs and swine against the FMDV challenge. Recently, some recombinant TMV, such as TMVFN20 that contains an epitope FN20 from the FMDV VP1, were found to induce local necrotic lesions (LNL) on the inoculated leaves of a susceptible tobacco, N/N′-gene) was able to be induced by the recombinant TMV at both 25°C and 33°C, whereas the hypersensitive response (HR) in the resistant tobacco plants induced by wt TMV through the N/N′-gene pathways only at a permissive temperature (below 30°C). Furthermore, we reported for the first time that some of defense response (DR)-related genes in tobacco were transcriptionally upregulated during HLR.Here we investigate the molecular mechanism of the HLR in the susceptible Samsun nn. Histochemical staining analyses show that these LNL are similar to those induced in a resistant tobacco Samsun NN inoculated with wild type (wt) TMV. The recombinant CP subunits are specifically related to the HLR. Interestingly, this HLR in Samsun nn (lacking the N/N′-gene independent pathways. Induction of the HLR is associated with the expression of the recombinant CP subunits and upregulation of the DR-related genes.Unlike HR, HLR is induced in the susceptible tobacco through  Solanaceae. In a susceptible host, Nicotiana tabacum Samsun nn, systemic infection by wild-type (wt) TMV leads to a high accumulation of TMV coat protein (CP) Tobacco mosaic virus (TMV) is a plus sense single stranded RNA virus that infects plants of the family Foot-and-mouth disease virus (FMDV) is a viral pathogen that causes a severe epidemic of foot-and-mouth disease. Various vaccine products were developed in an attempt to protect the animals from the FMDV infection. In our laboratory we have utilized the TMV-based vector to express two B-cell epitopes (F11 and F14) of the VP1 protein from FMDV in tobacco plants for the purposes of new vaccines against FMDV N. benthamiana inoculated by the recombinant TMVs with a insertion of an entire foreign protein Based upon the above results, a T-cell epitope FN20 in FMDV VP1 was selected to develop a new FMDV vaccine using the TMV-based vector. However, instead of the systemic mosaic symptoms appearing on leaves infected by TMVF11 or TMVF14 1-2 weeks after inoculation N/N′-gene independent pathways.Our similar observations with recombinant TMVFN20 and TMVSC1754 Besides TMVFN20, another recombinant TMVSC1754 was introduced in this study as a parallel control to better understand the HLR occurring in susceptible tobacco plants. Unlike TMVFN20, TMVSC1754 has been reported to be related to the necrotic response in Samsun nn N)   was alsoN) .2O2 and autofluorescence of phenolic compounds were detected between typical HR lesions and HLR lesions  gene to generate TMVΔcpGFP, TMVΔcpGFPFN20 and TMVΔcpGFPSC1754, respectively . The 5′-At 4 dpi with each recombinant virus, the recombinant GFP protein in the inoculated leaves of Samsun nn was detected by western blotting analysis using a rabbit antibody to GFP . HoweverN. tabacum plants harboring the resistance gene N (or N′). HR is elicited through the specific interaction of the N gene product with the TMV 126-kDa replicase N′ gene product with the TMV CP The local necrotic response or hypersensitive response to the TMV infection was commonly seen in the resistant N or N′ gene  N/N′ gene. In addition, more LNL were induced by recombinant TMVs in Samsun NN than in Samsun nn at 25°C probably due to a synergistic action of HLR and HR   : The HMGectively . Howeverectively . Interesectively . Consideectively , signaliHarpin-induce 1 gene (HIN1) (HIN1 gene is directly related to leaf senescence HIN1 was greatly upregulated by inoculation of recombinant TMVs in both Samsun nn and Samsun NN at two different temperatures (25°C and 33°C) (HIN1 to a similar extent (HIN1 in HLR.e (HIN1) : The HINnd 33°C) . Comparer extent , suggestN gene  and a viral effector p50 of the TMV 126 kDa replicase in resistant tobacco to elicit the N-mediated HR N gene does not contributed to induction of HLR in susceptible tobacco. Our data suggested that the HLR is controlled by a mechanism different from the HR associated with N protein. Obviously, the HLR has greatly limited the application of TMV-based expression vectors in vaccine research.Our results show that fusion of TMV CP with different foreign peptides induced a similar HLR at both the biochemical and molecular level in in vitro by purified TMV CP has been developed To avoid this restriction on foreign epitope expression via recombinant TMV vector in tobacco, a novel peptide-display system in which the RNA genome of Semliki Forest virus (SFV) was trans-encapsidated N/N′-gene independent pathways in the susceptible tobacco Samsun nn. The recombinant CP subunits are specifically involved in the HLR. Given that some DR-related genes are greatly induced during HLR, our findings provide new insight into the local resistance response against recombinant TMVs in susceptible tobacco and could be helpful in the expression of foreign epitopes through TMV-based vectors in tobacco.In summary, our study shows that the recombinant TMVFN20 or TMVSC1754 induces a HLR through Nicotiana tabacum Samsun nn and Samsun NN) were cultivated in a growth chamber at 25°C with a 16 h light/8 h dark photo cycle. In vitro transcription of the recombinant TMV or wt TMV plasmid was rub-inoculated with infection buffer  onto maturely detached leaves of 6-week-old tobacco seedlings as mentioned previously Tobacco plants (153 and Trp152 in the TMV CP as described previously KpnI/XhoI/EcoRV/NotI) was firstly introduced downstream the stop codon of the CP gene in pTMV to generate a new plasmid, pTMV-KXEN. pTMVΔcp was then generated from pTMV-KXEN by partially deleting the CP gene from downstream +55 bp of the start codon of CP. The green fluorescent protein (GFP) gene was amplified by using primers 5′-AAGGATATCATGAGTAAAGGAGAAGAAC-3′ and 5′-TTTTCCTTTTGCGGCCGCTCATTTGTAGAGCTCATCC-3′ from pGFPuv  and sub-cloned into EcoRV/NotI sites in pTMVΔcp to obtain pTMVΔcpGFP. Foreign peptides FN20 and SC1754 were then inserted upstream the stop codon of the GFP gene in pTMVΔcpGFP to construct pTMVΔcpGFPFN20 and pTMVΔcpGFPSC1754.Plasmids pTMVFN20 and pTMVSC1754 were constructed from pTMV by inserting foreign peptide coding sequences of FN20 (ETQVQRRQHTDVSFILDRFV) or SC1754 (FFFVSYIIISFLVVVNMY) between codons of ThrN. tabacum Samsun nn and Samsun NN were processed for histochemical analysis and observed by microscopy. Evans blue staining was used to indicate cell death as described previously 2O2 was detected as described previously 2O2 was detected under a light microscope. Formation of phenolic compounds was detected as described previously Infected leaf tissues of Total proteins from 1 leaf disk (1 cm in diameter) of wt TMV-infected leaves or recombinant TMVs-induced necrotic lesions were extracted in 100 µl of SDS-sample buffer  by incubating at 100°C for 5 minutes and then centrifuging at 15,000 g for 15 min. 20 µl of soluble proteins were separated by 12% SDS-PAGE. After electrophoresis, the proteins on the gel were electro-transferred onto a PVDF membrane . After being incubated in a blocking buffer  for 1 h, the PVDF membrane was incubated with rabbit anti-TMV CP serum (1∶500 dilution in the blocking buffer) or with rabbit anti-GFP serum  for 1 h, followed by goat anti-rabbit IgG conjugated to horseradish peroxidase  for 1 h, and then developed with a chemiluminescence system .Total RNA was extracted from 1 leaf disk with 500 µl Trizol™ reagent according to the protocol . 5 µg of DNase I-treated total RNAs were used as template for reverse transcription (RT) in 20 µl volume by using SuperScript™ III First-Strand Synthesis System . The RT reaction was carried out by following the product manual. 1 µl of the RT product was amplified in 15 µl of real-time PCR reaction. PCR parameters were: 95°C for 10 min, 40 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 30 s. Real-time PCR was performed on a DNA engine opticon 2 system  using the SYBR Green Real-time PCR Master Mix as mentioned in the protocol . Expression level of each investigated gene was normalized by the ubiquitin mRNA. Primers were listed in Figure S1DR-related gene expression profiles in tobacco plants infected with recombinant TMVs. 6-week-old tobacco seedlings (Samsun nn and NN) were inoculated with in vitro transcripts of TMV (Red), TMVFN20 (Yellow) and TMVSC1754 (Green) as mentioned in Experimental Procedures. Infection buffer was used as negative control (Blue). Infected seedlings were incubated at 25°C or 33°C and sampled for real-time PCR assay at different time points  as listed on X-axis. The DR-related genes were evaluated in transcription regulation according to various virus challenges. Data are shown as the mean of at least two biologically repeated experiments, and the error bar is the standard error (SE). The expression value of each gene is presented as the percentage of the reference gene ubiquitin. At 0 dpi, average expression values of the genes in Samsun plants are 0.26±0.05 (PR-1b), 0.23±0.07 (PR-1c), 0.01±0.00 (PR-2d), 60.92±18.64 (PR-2e), 47.57±6.09 (PR-5c), 0.04±0.01 (PR-6), and 0.06±0.01 (PR-8) in terms of mean ± SE percent of ubiquitin, respectively. Signals of each gene at different time points  were compared to that at the initial time point (0 dpi) by t test, *p<0.01.(DOC)Click here for additional data file.Table S1Primer sequences used for real-time PCR.(DOC)Click here for additional data file.


La Crosse (LAC) virus, a California serogroup bunyavirus, is the leading cause of pediatric arboviral encephalitis in the United States and an emerging disease in Tennessee, West Virginia, and North Carolina. Human cases of LAC encephalitis in Tennessee and North Carolina have increased above endemic levels during 1997 to 1999 and may represent an expansion of a new southeastern endemic focus. This report describes the isolation of LAC virus from the exotic mosquito Aedes albopictus. The discovery of LAC virus in wild populations of Ae. albopictus coupled with its expanding distribution in the southeastern United States, suggests that this mosquito may become an important accessory vector, potentially increasing the number of human cases in endemic foci or expanding the range of the disease.


Snap25 gene provides additional SNAP-25-function required for complex neuronal processes in higher eukaryotes.Alternative splicing is an evolutionary innovation to create functionally diverse proteins from a limited number of genes. SNAP-25 plays a central role in neuroexocytosis by bridging synaptic vesicles to the plasma membrane during regulated exocytosis. The SNAP-25 polypeptide is encoded by a single copy gene, but in higher vertebrates a duplication of exon 5 has resulted in two mutually exclusive splice variants, SNAP-25a and SNAP-25b. To address a potential physiological difference between the two SNAP-25 proteins, we generated gene targeted SNAP-25b deficient mouse mutants by replacing the SNAP-25b specific exon with a second SNAP-25a equivalent. Elimination of SNAP-25b expression resulted in developmental defects, spontaneous seizures, and impaired short-term synaptic plasticity. In adult mutants, morphological changes in hippocampus and drastically altered neuropeptide expression were accompanied by severe impairment of spatial learning. We conclude that the ancient exon duplication in the  Snap25 gene with an additional SNAP-25a copy. We show that SNAP-25b is required for early postnatal development and that a balanced expression of the two proteins is a prerequisite for maintaining an operational neuronal network during adulthood. Mice that only have SNAP-25a develop seizures, and show learning deficits and anxiety. Synaptic plasticity is impaired, and structural changes are observed in areas that are connected to such behavioral functions. In man, SNAP-25 function has been linked to behavioral and neuropsychiatric disorders, including attention deficit hyperactivity disorder, ADHD. Our present findings using genetic elimination of SNAP-25b suggest that even small alterations in the regulation of the Snap25 gene, resulting in a disturbed balance between SNAP-25a and SNAP-25b, lead to nervous system dysfunction.In evolution, duplication of genes or gene segments appears to be an efficient way to add diverse functions in more complex organisms. The SNAP-25 protein plays an important role in mediating the release of neurotransmitters and hormones. SNAP-25 exists as two variants: SNAP-25a, which is present in early development, and SNAP-25b, which is most abundant from early adulthood and onwards. We have developed mouse mutants that only express SNAP-25a, but retain normal SNAP-25 levels by replacing the SNAP-25b segment in the  Caenorhabditis elegansThe evolution of more advanced organisms has required adaptation of genomes to be able to generate new gene functions. The original genome sequence analyses of different organisms surprisingly revealed that the number of genes in the human genome is only around 30,000, versus 20,000 in much simpler organisms such as the nematode soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complex synaptosomal-associated protein of 25 kD, SNAP-25, that associates with membranes through palmitoylation. The three SNARE proteins are held together by strong protein-protein interactions, whereby the cytoplasmic domains form a four α-helix coiled-coiled bundle Regulated membrane fusion forms the basis for synaptic transmission but is also fundamental for appropriate release of hormones and modulatory neuropeptides Snap25 gene has demonstrated that complete removal of SNAP-25 results in total absence of evoked neuroexocytosis and embryonic lethality 2+-evoked catecholamine release differed In higher vertebrates SNAP-25 is expressed as two developmentally regulated and complementary distributed splice variants termed SNAP-25a and SNAP-25b Snap25 gene we used a novel approach by developing SNAP-25b KO/SNAP-25a knock-in mouse mutants. The exon 5b was genetically eliminated and replaced with a second supplementary exon 5a, to maintain alternative splicing and normal expression levels, but allowing only SNAP-25a to be expressed. The SNAP-25b deficient mouse mutants exist in two versions, neo-containing with a Tkneo marker retained and neo-excised with the selection gene removed. Based on results from electrophysiological, behavioral and immunohistochemical experiments we conclude, that under physiological conditions, mice deficient in SNAP-25b have developmental defects, impaired short-term synaptic plasticity, a seizure-prone phenotype and malfunctioning cognitive performance. We suggest that the ancient duplication in the Snap25 gene followed by regulated alternative splicing between two similar but distinct exon 5 sequences is required for accurate synaptic function during development. Furthermore, a balanced expression of the two isoforms is a prerequisite for maintaining an operational neuronal network also during adulthood in advanced organisms.To specifically explore the physiological importance of the exon 5 duplication in the Snap25 gene, exon 5a and 5b are closely spaced and differ in only nine of 39 amino acids Snap25 gene also contained a Tkneo selection cassette flanked by loxP recombination sites  littermates at PN14. Semi-quantitative RT-PCR analysis demonstrated that neo-containing SNAP-25b deficient mice expressed approximately 50% of the SNAP-25 mRNA levels present in WT littermates . Unlike neo-containing homozygous SNAP-25b deficient mutants, the growth of neo-excised homozygous mutants between PN5 and 15 was not severely affected. No difference in body weight was observed between mutants and WT littermates when genders were mixed. However, when females were analyzed separately they demonstrated a small but significant reduction in body weight  or predominantly SNAP-25b (WT) were incubated at different temperatures, either 4°C or heated, prior to gel electrophoresis and Western blotting. In non-heated samples, several immunoreactive bands were observed in SNAP-25b deficient and in WT brain homogenates ; at 85°C 59.8±7 and 36.9±3.4 (*p<0.05), and at 90°C 48.3±12.3 and 4.5±2.2 (**p<0.01)]. The ternary complex almost disappeared in SNAP-25b deficient brain preparations treated at 90°C while the WT samples still showed a strong immunoreactive signal containing 48.3% of total SNAP-25 in non-disassociated complex  at the Schaffer collateral-CA1 pyramidal neuron synapses of the hippocampus Neo-containing SNAP-25b deficient mouse mutants (n = 8 cells in 6 mice) showed a reduction (*p<0.05) in PPF at 0.2 Hz compared to WT mice  compared to WT littermates  or 0.5 Hz . All recorded neurons were responsive to the presynaptic stimulation and there was no obvious rundown or difference in baseline responses, in contrast to what is seen in complete SNAP-25 KO mice Finally, we compared the PPF-ratios obtained from homozygous Our results demonstrate that absence of SNAP-25b reduces PPF at Schaffer collateral-CA1 synapses in PN12-16 mice during low-frequency stimulation.eo-excised SNAP-25b deficient mutants spent less time in the open arms compared to the corresponding control group  in the latency to navigate to the visible platform compared to the corresponding control group . Spatial acquisition was examined during five days of training and revealed highly significant differences between the groups with regard to escape latency .Acquisition and retention of spatial learning, which involve hippocampal mechanisms, were studied in the Morris water maze task. In the pre-training phase, there was no overall significant effect of genotype  locomotor activity than WT controls. Female mutants demonstrated a low activity level already during the first 10 min of recording, i.e. during the initial exploration of the locomotor cage (data not shown).Young neo-excised SNAP-25b deficient mice, irrespective of gender demonstrate a higher anxiety index, a severe impairment in spatial learning with females being most severely affected, and a lower locomotor activity when compared to WT controls.In summary, neo-containing and neo-excised SNAP-25b deficient mouse mutants no obvious difference in structural and morphological appearance of the brain was observed by histological and immunohistochemical analysis  mossy fibers had expanded and formed large bundles . Thus, the absence of SNAP-25b results in progressing pathological changes in brain areas important for cognitive functions.In young  compare , separatructures . These mneo-excised SNAP-25b deficient mutants and WT littermates. Numbers of doublecortin (DCx)+ migrating neuronal precursor cells were also inspected, as seizures may increase neurogenesis.Epileptic activity causes dramatic changes in peptide expression in the hippocampal formation (HF), in particular affecting the granule cell-mossy fiber system, termed ‘epilepsia peptidergic profile’ + terminals partially overlapped with SNAP-25-ir in SLu , while DCx+ processes had an even localization in the molecular and granular cell layers .WT mice displayed a wide expression of CCK immunoreactivity (-ir) in the HF, being strongest in SLu and supragranular layer of the dentate gyrus (DG) . Double-r in SLu ; however mutants , but was mutants . NPY+ fimouse HF , but NPY mutants . NPY+ teribution . Diffuseent mice . BDNF-irent mice , but incent mice . In WT ml layers . The numar layer . No seizneo-excised SNAP-25b deficient mutants.In conclusion, deletion of SNAP-25b results in dramatic alterations in neuropeptide levels in the HF, which together with increased levels of BDNF and DCx may provide an environment that promotes neuroprotection/neuroproliferation in response to disrupted synaptic transmission and seizure activity in Torpedo marmorata or in the fruit fly Drosophila melanogasterTorpedo SNAP-25 protein shares 81% and the Drosophila protein 61% amino acid identity with the mouse SNAP-25b protein, respectively Snap25 gene must have occurred during early bony fish development more than 400 million years ago, when cartilaginous and bony fish divided and arose from the Placoderms, the first primitive jawed fishes The protein sequences of the two splice variants of SNAP-25, SNAP-25a and SNAP-25b, are remarkably well conserved in higher eukaryotes and demonstrate 100% amino acid identity in human, mouse and chicken neo-containing SNAP-25b deficient mutants SNAP-25 protein levels were reduced by 20%, indicating that the Tkneo gene suppressed gene expression. Reduced SNAP-25 protein levels in absence of SNAP-25b are lethal, as demonstrated in the homozygous neo-containing SNAP-25b deficient mutants. The phenotype becomes evident already in the second PN week with characteristic tremors and increased activity, including hyperactive episodes and reduced growth; all signs of uncontrolled neurotransmitter and hormonal release. The neo-excised SNAP-25b deficient mutants, with restored levels of SNAP-25 expression, escaped the critical early PN period with minor defects. Thus, the increased vulnerability in neo-containing SNAP-25b deficient mutants is the result of both lack of the SNAP-25b isoform and reduced levels of protein, underlining the importance of a balanced expression of SNAP-25 isoforms.We asked ourselves if the ancient duplication of exon 5, followed by mutually exclusive splicing, and resulting in increased protein diversity of the SNAP-25 protein was a prerequisite for the development of more intricate neuronal functions found in higher vertebrates. We now show that expression of SNAP-25b is important for early postnatal development and synaptic plasticity. Moreover, genetic elimination of SNAP-25b results in progressive morphological and neurochemical changes in CNS and impairment of cognitive function in adult animals. However, in brains of homozygous neo-excised SNAP-25b deficient mutants only express SNAP-25a instead of predominantly SNAP-25b as in adult WT mouse brain. We have now been able to show for the first time that ex vivo SNARE complexes in brain homogenates from neo-excised SNAP-25b deficient mutants dissociated at lower temperatures than WT SNARE complexes (mostly holding SNAP-25b). In this respect our attention was drawn to a SNAP-25b mouse mutant named the “blind-drunk mouse” (Bdr) with retinal degeneration (caused by choice of the background strain) and a mild phenotype with ataxia and impaired sensorimotor gating due to a dominant mutation in SNAP-25b In the SNARE complex with syntaxin and VAMP, SNAP-25 contributes with two amphipathic α-helices to the four-helix-coiled-coiled SNARE structure neo-excised SNAP-25b deficient mutants (only expressing SNAP-25a) and WT littermates (predominantly expressing SNAP-25b). In agreement with our assumption the mutants only had around 80% of the SNAP-25 protein levels found in WT plasma membrane fractions. Instead, in mutant brain more SNAP-25 was detected in low-density fractions, representing soluble protein and/or protein associated with small intracellular organelles. This difference in subcellular localization of the two SNAP-25 isoforms might contribute to the phenotype of the mutants. Whether that depends on the ability of the two isoforms to associate with membranes due to the altered organization of the cysteine residues that are substrates for palmitoylation, or, whether SNAP-25a and SNAP-25b reside in complex with additional secretory factors that guide the subcellular localizations needs to be further investigated.SNAP-25a and SNAP-25b also exhibit differences in the linker region between the N- and C-terminal SNARE motifs, where a quartet of cysteines, implicated in membrane anchoring of the protein, exists in two different contexts neo-containing and neo-excised SNAP-25b deficient mutants during low-frequency stimulation (0.2 Hz), suggesting a specific effect of SNAP-25b-deficiency. During higher frequency stimulation (0.5 Hz), when Ca2+ may be expected to be elevated during longer time periods in the stimulated presynaptic terminals, a reduction of the PPF-ratio was observed in neo-containing experiments while there was only a non-significant tendency to a reduction in neo-excised SNAP-25b deficient mice. Whether this is a result of the presumed higher average Ca2+-concentration or due to other factors will need to be investigated in future studies.To explore a possible effect on presynaptic mechanisms we studied PPF at Schaffer collateral-CA1 pyramidal synapses. We observed a reduction of PPF in young 2+-dependent maturation steps in order to be mobilized to the fusion-competent state 2+ in chromaffin cells et al. 2+-dependent types of facilitation including PPF. At physiological levels of SNAP-25 expression and elevated Ca2+-concentrations the replacement of SNAP-25b by SNAP-25a does not affect synaptic release enough to result in a statistically significant effect. However, there was a tendency to a reduction that needs to be investigated further.Normally, only few synaptic vesicles are “fusion-competent” and require no further modifications prior to exocytosis. Remaining vesicles close to the site of fusion need to be “primed”, that is undergoing ATP- and CaNeo-excised SNAP-25b deficient mutants demonstrated a higher anxiety index compared to WT mice, however, without alterations in overall locomotor activity in the elevated plus maze. This finding is consistent with the profound thigmotaxis observed in the computerized measurements obtained in the water maze task. In the Morris water maze task, homozygous neo-excised SNAP-25b deficient mutants were severely impaired in their ability to acquire the spatial learning task. This impairment cannot simply be explained by visual or motor disturbances although there was a trend for female neo-excised SNAP-25b deficient mutants to perform less well in the visible platform test. The failure of the mutants to consistently improve their performance in the visible platform test is probably related to the same mechanisms as those underlying the deficiency in spatial learning. It is important to stress that the mutants did not display impaired swim performance since they did not differ from WT in their swim speed. However, unlike the WT mice, the mutants displayed a high level of thigmotaxis in the water maze task. This suggests that the deficiency in spatial learning in the mutants could partly be related to their anxious phenotype evidenced by their profound thigmotaxis and/or related to attention deficits observed in the visible platform test.SNAP-25b is normally highly expressed in brain areas involved in cognitive function, such as the HF. Therefore we examined mutants in behavioral tasks dependent on hippocampal mechanisms. neo-containing and neo-excised SNAP-25b deficient mutants demonstrate spontaneously occurring convulsive seizures and freezing behavior. The neo-containing SNAP-25b mutants are most severely affected with frequent attacks debuting around PN12-13, whereas in neo-excised SNAP-25b deficient mutants seizure activity is rare prior to young adulthood. These behavioral impairments were paralleled by increasingly pronounced anatomical and immunohistochemical changes, which are only observed after the debut of seizure activity. In neo-excised SNAP-25b deficient mice, mossy fibers appear notably enlarged and swollen with certain areas almost devoid of synaptophysin immunoreactivity, suggesting a locally decreased density of functional nerve endings. Increased NPY expression in mossy fibers after seizures is believed to be neuroprotective by dampening excitatory activity, while BDNF can regulate NPY expression + migrating neuronal precursor cells observed in our mutants. It has been reported that cells generated in the adult dentate gyrus mature into functional neurons that integrate into hippocampal circuitry Both Snap25 gene. The functions of SNAP-25a and SNAP-25b appear to complement each other in tuning the presynaptic exocytotic machinery towards different modes of release. For complex neuronal circuitries this modulation of release is necessary, and is also instrumental in protecting highly plastic brain areas from accumulating degenerative morphological changes with age. The development of higher brain functions during evolution has been complemented with expanding number of gene products, sometimes by gene duplications but primarily by increasing protein diversity and complexity via alternative splicing. The importance of alternatively spliced isoforms from other genes has previously been extensively analyzed in vivo, by hindering expression of selected exons using gene targeted mouse models We here demonstrate the physiological importance of the ancient exon 5 duplication in the Interestingly, a growing amount of reports are connecting SNAP-25 function with a wide variety of behavioral and neuropsychiatric disorders, as well as linking it with cognitive capability in humans Snap25 gene, was substituted with an additional exon 5a. A Tkneo gene surrounded by loxP repeats was introduced into the vector neo-containing” as the Tkneo gene inserted into the targeting vector was still present in the modified Snap25 gene. Two of the neo-containing lines were crossed with the Protamine-Cre recombinase transgene Prm1Cre in vivo excision of the Tkneo gene. The neo-excised lines were bred independently onto B6 background until congenic, and the PrmCre1 transgene crossed out. Genotyping was routinely performed by PCR and by Southern blotting when necessary. In vivo expression of the introduced chicken exon 5a was demonstrated by RT-PCR  using the GenElute Mammalian Total RNA kit (Sigma). For quantification of total SNAP-25 mRNA; 1 µg RNA, 10 µµ of each primer and a trace of [α32P] dCTP  were used in 25 µl reactions with the SuperScript III RT-PCR System (Invitrogen). Semi-quantitative RT-PCR was performed with 20 cycles of amplification and SNAP-25 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) primers in the same reaction , pH 7.4. For whole cell homogenates cells were lyzed with 1% NP-40 (Sigma) and 5 µg protein was run on 10% Tris-glycine/NU-PAGE gels  followed by Western blotting. Primary antibodies used were a rabbit polyclonal antibody against SNAP-25 from Synaptic Systems , a rabbit polyclonal anti-SNAP-23 , mouse monoclonals anti-syntaxin 1, HPC-1  and anti-α-tubulin, clone DM 1A , both from Sigma. Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit and anti-mouse immunoglobulins (IgGs) from Dako Corporation and Rockland. Statistical analyses were made using Wilcoxon's signed-rank test for paired data.Mouse brains were homogenized in (in mM): 20 HEPES, 1 MgClneo-excised SNAP-25 deficient (KO) mouse brain tissues were homogenized in buffer described for whole cell homogenates. SDS sample buffer  was added to equal amounts (20–40 µg) of WT and KO homogenate before heating treatment at 70, 75, 80, 85, 90 and 100°C (boiling), or kept at 4°C, for 20 min. Treated samples were immediately loaded and separated on 10% Bis-Tris NU-PAGE gels (Invitrogen). SMI81 antibody  was used to detect immunoreactivity of bands migrating as ternary SNARE complex or solitaire SNAP-25 protein. To measure the grade of disassembly of heat-resistant SNARE complex in WT and SNAP-25b KO tissue, percentage monomeric SNAP-25 protein of total SNAP-25 in ternary complex at 4°C was calculated and compared with the value calculated at a certain temperature. All experiments were performed at least three times. Results were analyzed with unpaired Student's t-test.Pulverized adult WT and homozygous For histological analysis, mice were anesthetized with isoflurane and transcardially perfused with PBS followed by freshly prepared 4% paraformaldehyde in PBS. Organs were post-fixed overnight, dehydrated in graded ethanol and embedded in paraffin according to standard procedures. 4 µm sections were stained with hematoxylin-eosin or cresyl violet.For immunohistochemistry of paraffin embedded tissue, sections were collected on Superfrost Plus slides, deparaffinized (xylene), dehydrated (ethanol) and boiled by microwaving for antigen unmasking see also . AntibodNeuropeptide immunohistochemistry was performed as described Hind limb specimens were fixed in 4% formaldehyde in phosphate buffer and embedded in paraffin. Sections, 4–5 µm thick, were stained with hematoxylin and eosin. Growth plates of tibia and femur were scanned using a Zeiss Axiovert 35M microscope fitted with a LSR Astro Cam type TE3/A/S digital camera. Images were analyzed using Concord software from Life Science Resources Ltd. The width of the different zones was determined at least in triplicate per section. Per sample, at least two cross sections were measured. Data was analyzed with unpaired Student́s t-test.2 and sacrificed by decapitation. Brains were quickly removed and placed in ice-cold low Ca2+/ high Mg2+ artificial cerebrospinal fluid (aCSF) containing (in mM): 124 NaCl, 5 KCl, 1.24 NaH2PO4, 0.5 CaCl2, 10 MgSO4, 26 NaHCO3, and 10 glucose, and oxygenated with 95% O2/5% CO2 (pH 7.4). Coronal hippocampal slices (350 µm thick) were cut on a vibratome  and transferred to regular aCSF (same composition as above but with 2.4 mM CaCl2 and 1.3 mM MgSO4) at RT. Patch-clamp electrodes (6–10 MΩ) were filled with (in mM): 135 Cs-methane sulphonate, 10 HEPES, 1 EGTA, 4 Mg-ATP, 0.3 Na-GTP, 2–5 QX-314, and 8 NaCl . Whole-cell voltage clamp (−70 mV) recordings of evoked excitatory postsynaptic currents (EPSCs) were obtained at RT from CA1 pyramidal neurons of the hippocampus, visualized with differential interference microscopy (DIC) using an Olympus BX50WI microscope. 50 µM picrotoxin (Sigma-Aldrich) was added to the aCSF to block GABAA receptor-mediated synaptic transmission. EPSCs were evoked by stimulation of Schaffer collaterals with fine concentric Pt/Ir bipolar stimulation electrodes (Fredrik Haer and Co.) placed in the stratum radiatum. Paired stimuli were delivered at IPIs from 40 to 300 ms at 0.2 and 0.5 Hz and data was collected after at least 5 minutes of baseline recording to make sure responses were stable. Statistical comparisons of PPF ratios between WT and SNAP-25b deficient mice were made with two-way repeated measurements analysis of variance (ANOVA). For additional information, see PN12-16 mice were terminally anesthetized with 100% COThe elevated plus maze analysis was performed as described previously Data from the behavioral testing was analyzed by non-parametric statistics using Kruskal-Wallis ANOVA followed by Mann-Whitney U as the Post-hoc test.Figure S1neo-excised SNAP-25b deficient mouse mutants from PN5 to PN15. (A) Weight gain was not reduced for neo-excised male mutants compared to male WT littermates . (B) Neo-excised female SNAP-25b mouse mutants at the second PN week weigh less than their WT littermates . Data was analyzed with two-way repeated measures ANOVA.Weight curves of (1.30 MB TIF)Click here for additional data file.Figure S2neo-excised SNAP-25b deficient and WT mice for comparison of subcellular localization of SNAP-25a (KO) and SNAP-25b (WT) proteins. More SNAP-25a than SNAP-25b protein could be found in low-density fractions, as shown in the representative immunoblots. Neo-excised SNAP-25b deficient mice (KO) had 80.4±3.4% (*p = 0.05) SNAP-25 in plasma membrane (PM)-associated fractions using a polyclonal antibody, whereas monoclonal antibody resulted in 86.5±5.5% PM-associated protein . Mean±S.E.M., n = 3 for each genotype of animals, and each gradient was run in triplicate for each antibody, data was analyzed using Mann-Whitney U test.Sucrose density gradient fractionation of brain homogenates from 2–5 months old (1.21 MB TIF)Click here for additional data file.Figure S3neo-excised SNAP-25b deficient (KO) mutants. Number of cells was 298±28 in the homozygous neo-excised SNAP-25b deficient KOs, compared to 125±20 in WT .Doublecortin-ir cells in the hippocampal dentate gyrus. Number of DCx+ migrating neuronal precursor cells was increased by 140% in the (1.39 MB TIF)Click here for additional data file.Text S1Supplementary information.(2.98 MB DOC)Click here for additional data file.


The Drug Abuse Resistance Education (D.A.R.E.) program is a widespread but controversial school-based drug prevention program in the United States as well as in many other countries. The present multivariate meta-analysis reviewed 20 studies that assessed the effectiveness of the D.A.R.E. program in the United States. The results showed that the effects of the D.A.R.E. program on drug use did not vary across the studies with a less than small overall effect while the effects on psychosocial behavior varied with still a less than small overall effect. In addition, the characteristics of the studies significantly explained the variation of the heterogeneous effects on psychosocial behavior, which provides empirical evidence for improving the school-based drug prevention program. Drug abuse is a prevalent problem affecting young generations worldwide . In respdrug use, which includes tobacco, alcohol, marijuana, and other illicit drugs; and (b) the improvement of psychosocial behavior, which includes social skills , self-esteem, attitudes towards drug use, attitudes towards police, and family bonding. The program is normally taught by a police officer; and the core curriculum has 17 lessons, usually offered once a week for 45 to 60 minutes [et al. [The D.A.R.E. program was designed to help elementary and junior high school students resist the peer-pressure of experimenting with drugs, tobacco, and alcohol. The D.A.R.E. program aims to reduce drug abuse among children by providing them with information that encourages them to make healthy decisions. Its effectiveness has been assessed by its two major outcomes: (a) the reduction of  minutes . This tyAlthough there seems to be great support for the D.A.R.E. program, the literature revealed inconsistent research results concerning the program’s effectiveness. For example, some studies found that the D.A.R.E. program did not work , 8–15; wet al. [Considering the tremendous investment of time and money in the D.A.R.E. program, these inconsistent findings necessitate a conclusive synthesis of the research to assess the effectiveness of the program. To date, only two published research syntheses or meta-analytic reviews exist that solely focused on evaluating the effectiveness of the D.A.R.E. program , 10. Unfet al. , 37. Weset al. , on the et al. . Becauseet al.  serves aThe purpose of this multivariate meta-analytic review was to: (a) quantitatively synthesize updated evaluation studies of the D.A.R.E. program, and (b) simultaneously synthesize all the outcomes of the D.A.R.E. program. Specifically, this review addressed the following three research questions: (a) Did the effects of the D.A.R.E. program on the outcomes vary across the studies? (b) What was the overall effect of the D.A.R.E. program on the outcomes? (c) What study characteristics explained the variation of the effects of the D.A.R.E. program on the outcomes?Using the terms “Drug Abuse Resistance Education,” “D.A.R.E.,” and “school-based drug prevention program” as keywords, an extensive literature search in the PsycINFO, MEDLINE, ERIC, PUBMED, SOCIAL SCIENCE INDEX, SOCIAL SCIENCE INDEX CITATION, and DIGITAL DISSERATIONS databases produced 198 relevant studies published between January 1983 and December 2005. Of the 198 studies, 73 were found to be quantitative studies on the D.A.R.E. program. After applying the criteria for inclusion described below, 20 final studies were selected for this meta-analysis.d [d is a standardized mean difference between the treatment group and the control group. That is,x̄t is the mean of the treatment group, x̄c is the mean of the control group, and sp is the pooled standard deviation. Whether the study utilized an experimental or quasi-experimental design was the second criterion because these designs are more rigorous and provide more valid research results than other less scientific designs. The third criterion necessitated that the study evaluated at least one of the outcomes on drug use and psychosocial behavior. The fourth and final criterion called for studies where the effect of the D.A.R.E. program could be independently evaluated. That is, whether the studies provided a D.A.R.E. treatment group and a comparable control group.The first criterion for inclusion required the study to have sufficient quantitative information for calculating the outcome measure or the effect size of the outcome: Cohen’s d . Cohen’sOutcome measures. The outcome measures for the present review were two sets of effect sizes: one was for drug use and the other for psychosocial behavior. Each effect size in the former set was the average Cohen’s d for all the available drug use outcomes , and the latter for all the available psychosocial behavior outcomes . Different effect size measures calculated from various statistical methods in the studies were converted to Cohen’s d. In line with the previous reviews [ds of the outcomes across the studies were calculated at the longest follow-up, which ranged from 0  to 10 years. reviews , 10, theStudy characteristics. The following study characteristics were recorded for the analysis: Name of first author, year of publication, sample size, statistical method , year of D.A.R.E. curriculum, follow-up time, proportion of female participants, and proportions of ethnic groups. The selection of the study characteristics was partially guided by the pervious reviews and partially based on common information available in the studies.Each value of the variables of the study characteristics and outcome measures needed to be recorded or coded from the 20 studies. A concurrent double coding was performed independently by the researchers. Each researcher spent more than forty hours, equivalent to five full-time work days, on coding the 20 studies. Then, the researchers engaged in extensive discussions to compare every coded item. No variable was finalized until reaching an agreement.d = 0.20, 0.50, and 0.80 are considered small, medium, and large effect, respectively. 95% confidence intervals of the effect sizes were also computed. The confidence intervals showed whether the effect sizes were heterogeneous across the studies.Descriptive analysis was first conducted for each of the two outcomes, drug use and psychosocial behavior, by calculating the unweighted mean effect size of the outcome. According to Cohen’s guideline , d = 0.2Q-statistic was computed for each of the two outcomes, drug use and psychosocial behavior. The test for the Q-statistic provided statistical evidence for the heterogeneity of the 20 studies. If the test was significant, a random-effects model was tested, and the weighted mean effect size was calculated to provide a more valid estimate for the mean effect size than the unweighted mean effect size from the descriptive analysis.In terms of inferential analysis, a Hedges and Olkin’s  Q-statisweighted regression model to explain the variation in the heterogeneous effect sizes. Following Hedges’ [t-test for individual regression coefficient was adjusted as follows:s.e. is the original standard error given by common computer programs, and MSError is the mean square value for errors from the analysis of variance for the regression given by the computer programs. Note that some study characteristics had missing values that resulted from unavailable information, and they were replaced by means [In the case of heterogeneous effect sizes, the study characteristics were entered into a  Hedges’  suggestiby means  because by means –45.Test of homogeneity. Under the null hypothesis of H0: θ1 = … = θ20 = θ, the Hedges and Olkin’s [Q-statistic values of QTotal were 13.34 with df = 17 (p = 0.71) and 96.61 with df = 12 (p < 0.0001) for drug use and psychosocial behavior, respectively. The homogeneity test results showed that the effect sizes across the 20 studies were statistically heterogeneous for psychosocial behavior, but not for drug use. This inferential finding was consistent with the descriptive finding demonstrated in the confidence interval plots above. By testing a random-effects model for psychosocial behavior under H0: θ = 0, a z = 2.92 (p < 0.01) indicated that the weighted average effect size of the 20 studies from the random-effects model was statistically different from zero but was still 0.10, a less than small effect. Olkin’s  Q-statisWeighted regression analysis. Because the effect sizes were heterogeneous for psychosocial behavior, a weighted regression analysis was conducted to identify the study characteristics that explained the heterogeneity. R2 = 89.8%). Specifically, the longer follow-up time  and the more rigorous statistical method  the study used, the less effect of the D.A.R.E. program would be found for psychosocial behavior; whereas the later D.A.R.E. year , the more White students , and the more Black students  the study had, the more effect of the D.A.R.E. program would have on psychosocial behavior.By including more updated studies and analyzing the study characteristics related to the outcomes of the D.A.R.E. program on both drug use and psychosocial behavior, this present multivariate meta-analysis provided a more comprehensive review than previous ones on the effectiveness of the D.A.R.E. program; and therefore, the present review helps us to better understand the widespread, expensive, but controversial D.A.R.E. program. The results of the present review revealed that the effects of the D.A.R.E. program on drug use were homogeneous but less than small, which confirmed the findings in the literature –11, 13. For the heterogeneous effects of the D.A.R.E. program on psychosocial behavior, the present review found that the study characteristics explained most of the variation of the effects. The heterogeneous effects suggest that some studies showed larger effects than others. By examining the specific characteristics of the studies that had larger effects, which was executed in the weighted regression analysis, future program implementations can learn from those effective studies for improving the program effects. Among the significant study characteristics, follow-up time and statistical method were negatively related to the effects; and D.A.R.E. year, percent of White participants, and percent of Black participants positively related to the effects.et al. [These findings provided some important implications. First, the validity of long-term effects might be threatened by maturity and history. This point was also noted in the previous reviews , 10 but et al. . The newet al. . Some otet al. . As suchd = 0.05); on the other hand, the school-based drug intervention program also had a less than small effect on improving psychosocial behavior (Cohen’s d = 0.10). The analysis from this review also identified areas in the new versions of the D.A.R.E. program that need improvement. It would be, however, more important if the new versions of the D.A.R.E. program could transform the improved psychosocial behavior into the students’ actions of reducing drug use—the ultimate outcome.In sum, the effects of the D.A.R.E. program appear to be different on drug use and psychosocial behavior. The results of the present review provide an evidence-based interpretation to the inconsistent conclusions found in the previous research that was conducted on the D.A.R.E. program. This study found that, on one hand, the D.A.R.E. program had a less than small effect on reducing drug use (Cohen’s


Ergosterol biosynthesis mutants of S. cerevisiae failed to acidify the vacuole and exhibited multiple vma− phenotypes. Extraction of ergosterol from vacuolar membranes also inactivated V-ATPase without disrupting membrane association of its subdomains. In both S. cerevisiae and the fungal pathogen C. albicans, fluconazole impaired vacuolar acidification, whereas concomitant ergosterol feeding restored V-ATPase function and cell growth. Furthermore, fluconazole exacerbated cytosolic Ca2+ and H+ surges triggered by the antimicrobial agent amiodarone, and impaired Ca2+ sequestration in purified vacuolar vesicles. These findings provide a mechanistic basis for the synergy between azoles and amiodarone observed in vitro. Moreover, we show the clinical potential of this synergy in treatment of systemic fungal infections using a murine model of Candidiasis. In summary, we demonstrate a new regulatory component in fungal V-ATPase function, a novel role for ergosterol in vacuolar ion homeostasis, a plausible cellular mechanism for azole toxicity in fungi, and preliminary in vivo evidence for synergism between two antifungal agents. New insights into the cellular basis of azole toxicity in fungi may broaden therapeutic regimens for patient populations afflicted with systemic fungal infections.Ergosterol is an important constituent of fungal membranes. Azoles inhibit ergosterol biosynthesis, although the cellular basis for their antifungal activity is not understood. We used multiple approaches to demonstrate a critical requirement for ergosterol in vacuolar H +-ATPase function. V-ATPase plays essential roles in diverse cellular processes, and is required for fungal virulence. Concomitant ergosterol feeding restores vacuolar acidification and growth in cells treated with fluconazole. These results suggest that the critical requirement for ergosterol in V-ATPase function may underlie the antifungal activity of azoles. Moreover, we show in a mouse Candidiasis model that combining an ion homeostasis-disruptive drug with azole is an effective approach to treat fungal infections.Systemic fungal infections impose a significant threat to public health and therapeutic options to treat these diseases remain limited. Azoles represent the largest category of anti-fungal drugs and repress fungal growth by inhibiting biosynthesis of ergosterol, an important constituent of fungal membranes. Despite the wide use of azoles in the clinic for decades, the cellular basis for their antifungal mechanism remains elusive. In this study, we use a range of genetic, cellular and biochemical approaches to reveal a requirement for ergosterol in vacuolar H Aspergillus, Candida, Histoplasma and Cryptococcus among others, cause infections ranging from mucocutaneous disorders to life-threatening invasive diseases that can involve any organ. In the past two decades, expanding populations of immunocompromised patients and increased use of invasive devices and implants have led to an increase in the incidence of fungal infections Pathogenic fungal species, including Ergosterol is an important constituent of membrane lipids, similar to vertebrate cholesterol, and modulates the fluidity, permeability and thickness of the membrane. These sterols preferentially associate with sphingolipids in microdomains that have been postulated to have important roles in membrane organization and function + and Ca2+ into the cytoplasm vma genes encoding subunits of the vacuolar membrane H+-ATPase vma mutants was ascribed to defects in ion homeostasis. Notably, deletion mutants of several erg genes in the ergosterol biosynthesis pathway were also identified in the screen erg mutants, like vma mutants, had severely alkaline vacuoles . A separate study showed that sphingolipid, the other major membrane lipid component found associated with ergosterol in detergent-resistant microdomains, was required for the structural integrity of V-ATPase domains The limited categories of antifungal agents and emergence of resistance to existing antimycotics have prompted a search for compounds with alternative modes of action. The anti-arrhythmia drug, amiodarone, was recently documented to exhibit fungicidal activity S. cerevisiae haploid deletion collection for hypersensitivity to the antifungal agent amiodarone revealed multiple vma and erg mutants 2+ and H+ influx leading to fungal death from ion stress erg null mutants  revealed multiple −vma phenotypes, with erg24Δ displaying the most severe defects. In addition to hypersensitivity to amiodarone  cells were significantly more alkaline, around pH 7, as would be expected for loss of proton pump capacity . Taken together, these results provide evidence for a role for ergosterol in V-ATPase activity.V-ATPase hydrolyzes ATP and acidifies vacuolar compartments. To assess a possible requirement for ergosterol in V-ATPase function, we first measured the vacuolar pH in capacity . Vacuolar erg24Δ . Next, wn erg24Δ . Examina+-ATPase Pma1 (Entrez GeneID: 852876) has been documented to associate with ergosterol enriched domains erg24Δ cells. As shown in erg24Δ, while a previously characterized PMA1 mutant, pma1-105, reported to have a 65% reduction in activity, exhibited slower acidification rate as expected The P-type Herg24Δ mutant. The V-ATPase is made up of 14 subunits organized into the Vo sector, integral to the membrane, and the cytoplasmic V1 sector that reversibly dissociates from the membrane o sector (Vph1-GFP) and the V1 sector (Vma5-GFP) colocalized with the vacuolar membrane stain (FM4-64) in erg24Δ cells, similar to the isogenic wild type. It was possible that the ergosterol biogenesis defect significantly decreased V-ATPase expression or caused the dissociation of V1 from Vo domain. However, analyses of representative V-ATPase subunits in vacuolar vesicles purified from the wild type and erg24Δ showed similar expression levels and V1/Vo ratios that were identical between the two strains  or Vo (Vph1p) subunits, suggesting that the two sectors remain associated after ergosterol extraction. In contrast, a previous study pointed to a role for sphingolipids in maintaining structural integrity of the V-ATPase enzyme complex The oligosaccharide Methyl-β-Cyclodextrin (MβCD) extracts sterols from cellular membranes. We observed a dose dependent loss of V-ATPase activity following treatment of purified vacuolar vesicles with MβCD, which could be blocked by preloading cholesterol into MβCD . Both ATreatment : we did S. cerevisiae strain WPY361 with a gain-of-function mutation in UPC2 (Entrez GeneID: 851799), upc2-1, that allows overexpression of ATP-binding cassette transporters required for uptake of exogenously added sterol under aerobic condition upc2-1 cells after six hours of exposure to fluconazole and exogenous ergosterol. As expected, fluconazole alone caused reduction of ergosterol content (nine-fold) and accumulation of lanosterol and a major derivative, likely to be 14-methyl 3,6-diol , which is the enzymatic step immediately upstream from Erg24p. Despite the wide spread use of azole antifungals, our understanding of the specific cellular pathways disrupted by azoles is limited. In addition to ergosterol depletion, fluconazole treatment results in accumulation of lanosterol (substrate of Erg11p) and its derivative 14-methyl-3,6 diol 3,6-diol . Compare3,6-diol . These d+ pumping rates and ATPase activity  to monitor changes in cytosolic pH in wild type, vma2Δ and erg24Δ strains upon exposure to amiodarone vma2Δ mutant consistent with a loss in the ability to transport H+ from the cytosol to the vacuole. Depletion of ergosterol in erg24Δ mutant or by fluconazole treatment also exacerbated cytosolic acidification, relative to wild type, upon amiodarone addition . In the absence of FLC, amiodarone did not confer a significant antifungal activity at the concentrations tested . Yet in combination with fluconazole, the two doses of amiodarone significantly reduced C. albicans infection above and beyond the dose-dependent effect of fluconazole   . These d2+ and metal ion stress, and are unable to grow on high concentrations of non-fermentable carbon sources −vma phenotypes. Furthermore, we showed a reduction of vacuolar acidification by ergosterol depletion, restoration of vacuolar acidity by ergosterol feeding, and used biochemical assays of H+ pumping with purified vacuolar vesicles to collectively demonstrate the requirement of ergosterol for optimal V-ATPase function. This functional link explains simultaneous identification of multiple ergosterol biosynthesis genes (erg) and V-ATPase subunit genes (vma) in a number of genome-wide screens, including sensitivity to low Ca2+, alkaline stress, and acid stress vma and erg mutants are avirulent In fungal cells, V-ATPase acidifies intracellular compartments including the vacuole, endosomes, and late-Golgi. Mutants lacking V-ATPase exhibit characteristic phenotypes of growth sensitivity to alkaline pH, calcofluor white, Caerg24Δ. Fluorescent microscopy and immunoblot analysis ruled out possible mislocalization and reduced abundance of V-ATPase in erg24Δ vacuolar membrane. In yeast, the V-ATPase complex undergoes rapid reversible dissociation into non-functional V1 and Vo sectors in response to glucose withdrawal 1/Vo along the endocytic pathway effectively increased acidity along the compartments in the pathway 1 and Vo domains are still associated, and V1/Vo ratio remains unchanged upon ergosterol deprivation by treating cells with fluconazole and treating vacuolar vesicles with MβCD. These data rule out dissociation of V1 from Vo domain as the cause of reduced V-ATPase function, and indicate that ergosterol directly modulates the activity of V-ATPase. Sphingolipid, another major component of lipid raft, was thought to affect V-ATPase function by maintaining the structural integrity of V-ATPase because V1 subunits  dissociate from Vo domain during Ficoll gradient procedure in the sphingolipid mutant sur4Δ. In contrast, Vma2p remained associated with Vph1p after Ficoll gradient procedure in erg24Δ mutant and after ergosterol extraction by MβCD in the wild type. Thus, these two key membrane lipid components play distinct roles in maintaining V-ATPase function.To investigate the underlying basis for the requirement of ergosterol in V-ATPase function, we first checked the localization and abundance of V-ATPase in 2+-ATPase and Na+,K+-ATPase by membrane lipids include lowering of free energy of activation and proper packing at protein-protein interfaces +-ATPase, Pma1, is a major efflux mechanism for protons, and is also found associated with ergosterol-rich microdomains erg24Δ, in contrast to the pronounced effect seen on the V-ATPase. This suggests that membrane proteins have specific lipid requirement for their functions.The precise molecular basis of V-ATPase regulation by ergosterol remains to be determined. In mammalian cells, V-ATPase has been shown to associate with cholesterol-rich microdomains, with loss of vesicular acidification reported upon treatment with β-methylcyclodextrin erg3 mutants, 14-methyl fecosterol accumulates upon treatment with fluconazole upc2-1 mutation that allows uptake of ergosterol under aerobic conditions to distinguish between the effects of byproduct accumulation and ergosterol depletion on cell growth. The ability of exogenous ergosterol to reverse growth inhibition by fluconazole supports a plausible alternative hypothesis that antifungal activity of azoles is due to ergosterol depletion. Although we ruled out a corresponding decrease in lanosterol and other derivatives in the ergosterol fed cells, we cannot exclude the possibility that a specific ratio of ergosterol to other sterols may counter potential toxic effects. This possibility could be tested by varying ratios of sterols in upc2-1 feeding experiments; however, 14-methyl-3,6 diol is not commercially available and its potential toxicity cannot be directly assessed at this time. Furthermore, our results warrant more careful examination of the role of 14-methyl fecosterol in the potential compensation of ergosterol function in membranes.While the molecular target of azole drugs, Erg11p, has been extensively characterized, not much is known about the cellular basis of fungal growth inhibition. Inhibition of Erg11p by fluconazole results in accumulation of lanosterol and its derivative 14-methyl-3,6 diol. In azole resistant Some reports suggest a role for ROS in the toxicity of azoles to fungal cells The far-reaching effect of V-ATPase is illustrated by diverse phenotypes exhibited by V-ATPase mutants. By disrupting the function of V-ATPase through ergosterol deprivation, azole drugs can affect a wide range of cellular processes, including cation homeostasis, protein sorting, processing and degradation. Importantly, V-ATPase function and vacuolar processing of virulence factors are required for pathogenesis + and Ca2+ homeostasis in vitro studies showed azoles were synergistic with amiodarone against fungal pathogens, e.g. C. albicans and C. neoformansA. fumigatus and C. albicansT. cruzi both in vitro and in vivoAmiodarone exhibits antimicrobial activity against a wide range of fungi and protozoa through disruption of H+ and Ca2+ homeostasis. Therefore, depletion of ergosterol would be expected to exacerbate the disruption of H+ and Ca2+ homeostasis upon amiodarone treatment. Indeed, upon ergosterol depletion either by erg mutations or by azole treatment, 45Ca uptake by purified vacuolar vesicles was reduced while cytosolic H+ and Ca2+ surges increased upon exposure to amiodarone. Thus, we conclude that disruption of V-ATPase function in maintaining cation homeostasis by azoles contributes to the synergy between azoles and amiodarone.Data in this study show that ergosterol is required for the optimal function of V-ATPase, a central player in maintaining HC. albicans resulted in dampening of the transcriptional response to either drug alone VMA and ERG, determine phenotype of growth sensitivity to amiodarone. These genes tend to be constitutively expressed, have a non redundant function and represent pathways upstream of the transcriptional response. On the other hand, genes that are differentially regulated in response to drug appear to play a collective, rather than individual, response to adaptation to stress. Taken together, these mechanisms contribute to a more complete picture of the complex cellular response to drug toxicity. Finally, in this study, we evaluated the clinical potential of combining fluconazole and amiodarone in treating fungal infections in a murine Candidiasis model. The synergy demonstrated in this experiment is proof-of-principle that combining azoles with agents disruptive to cation homeostasis is a promising approach to better manage fungal infections.Recently, we demonstrated that a combination of amiodarone and fluconazole in −S. cerevisiae erg and −vma mutant strains are from MATα deletion library . WPY361  was kindly provided by Dr. Will Prinz (NIDDK). Yeast cells were grown in standard SC (synthetic complete) medium or YPD  medium at 30°C with shaking at 250 rpm unless specified otherwise. Media with non-fermentable carbon source contains 1% Bacto-yeast extract, 2% Bacto-peptone, 3% glycerol (v/v) plus 2% ethanol (v/v), or 2% sodium lactate. Antibodies against Vph1p and Vma2p were purchased from Invitrogen  or provided by Dr. Patricia Kane .TEF1 promoter was constructed to measure yeast cytosolic pH. Briefly, TEF1 promoter sequence was amplified from BY4742 genomic DNA with primers XbaITEF1 and BamHITEF1, which incorporate restriction sites of XbaI and BamHI. CYC1 terminator sequence was amplified with primers BamHICYC1 and EcoRICYC1, which incorporate restriction sites of EcoRI and BamHI. pHluorin gene sequence was amplified from pCB190YpHc plasmid with primers BamHIPhluo and BamHIPhluoR, which incorporate BamHI restriction site. The amplicons were digested with corresponding restriction enzymes and ligated sequentially with the backbone of pYEplac181, resulting in the gene for pHluorin being flanked by TEF1 promoter and CYC1 terminator. pZR4.1 was transformed to yeast strains to monitor cytosolic pH change upon exposure to amiodarone.Plasmid pZR4.1 with pHluorin gene under erg24Δ to replace the endogenous VPH1 and VMA5 genes by homologous recombination. Primer sequences are available upon request.Wild type (mating type a) Vph1-GFP and Vma5-GFP strains in which GFP was fused to the C-terminus of the two proteins were purchased from Invitrogen. The Vph1-GFP and Vma5-GFP fragments were amplified from these strains with primers Vph1L2 + Vph1R1, and Vma5L2 + vma5R1, respectively. The amplicons were transformed to 7 log-phase cells after washing twice with water and analyzed with an Agilent 6850 gas chromatograph with an HP-1 column and FID as described previously Ergosterol (Sigma) was dissolved in Tween 80∶ethanol (1∶1) as 5 mM stock. Stocks of ergosterol, fluconazole or their combination were added at the same time to WPY361 cells in YPD. For growth assay, stationary cultures were grown in 96-well plates for 30 hours at 30°C. For vacuolar pH measurement and sterol analysis, log phase cells were treated with ergosterol and fluconazole for 6 hours. Total sterols were extracted from ∼5×10erg24Δ and fluconazole treated cells 45Ca uptake assay, vacuolar vesicles were incubated in reaction buffer containing 5 µM CaCl2 with tracer quantities of 45CaCl2. After 5 min of incubation, vacuolar vesicles were filtered onto nitrocellulose membranes. The filters were washed and processed for liquid scintillation counting. Concanamycin A was added to 0.5 µM to assess the dependence of 45Ca uptake by vacuolar vesicles. For MβCD treatment, vacuolar vesicles were incubated with MβCD or MβCD preloaded with cholesterol  for 30 min at 4°C with gentle shaking. Vesicles were spun down and suspended in buffer C with proteinase inhibitors  for V-ATPase function assays and SDS-PAGE Vacuolar vesicles were prepared as described previously except that 10% Ficoll, instead of 8% Ficoll, was used in the second ultracentrifugation step to facilitate purification of vesicles from 490–540+ pumping rates were calculated from the absorbance change in the first 60 seconds. ATPase activity was assessed by an enzyme-coupled assay monitoring depletion of NADH through oxidation at 340 nm. ATPase activity was calculated based on the absorbance decrease between three and six minutes after initiating the reaction. Concanamycin A was added at a final concentration of 0.1 µM to assess the specificity of V-ATPase.ATP-dependent proton pumping was assayed by monitoring change of ΔATEF1 promoter in plasmid pZR4.1. Early log phase cells harboring plasmid pZR4.1 were grown for 6 hours to mid log phase (OD ∼1) in SC minus leucine medium with or without fluconazole. Cells were collected by centrifugation and suspended in SC to OD 3 in a 96-well plate, with 200 µl culture in each well. Amiodarone (stock 300 µM in H2O) was injected to give the specified final concentration. Fluorescence emission at 520 nm was measured in triplicate with excitation at 485 nm and 410 nm in a Fluostar Optima plate reader. Cytosolic pH was calculated based on the ratio of emission at 520 nm excited at 485 nm and 410 nm against a calibration curve covering pH from 4 to 8 established as described previously Cytosolic pH was measured using pHluorin, a pH-sensitive GFP Vacuolar pH was measured with BCECF AM, a pH-sensitive fluorophore that accumulates in the yeast vacuole erg24Δ cells as described previously 8 cells were pelleted by centrifugation and washed twice with distilled water. The washed cell pellet was resuspended in distilled water and placed on ice for 2–3 h. Just prior to use, the cells were centrifuged and resuspended in 3 ml of water. Once a stable pH base line was established, glucose was added to 2% to initiate proton efflux.Proton efflux was assayed in wild-type and C. albicans (SC5314) were diluted to OD 0.025 and grown in YPD with or without fluconazole (1 µg/ml) for 5 hours. FM4-64 was added to 5 µM and the cultures were grown for another 30 min. Cells were washed twice in YPD, suspended in YPD with or without fluconazole, and shaken for 20 min. Quinacrine was then added to 100 µg/ml and the cultures were shaken for another 5 min. The cells were collected and washed twice in YPD and examined by fluorescence microscopy immediately.Log phase cells of All animal experimentation was conducted following the United States Public Health Service guidelines for housing and care of laboratory animals and performed in accordance with Institutional regulations after pertinent review and approval by the Institutional Animal Care and Use Committee of the University of Medicine and Dentistry of New Jersey.C. albicans (ATCC 36082) was induced by injection of 5.0×105 blastoconidia  in 0.1 ml of sterile saline via the lateral tail vein of female BALB/c mice. Therapy was initiated 3 hours after challenge. Mice were treated with vehicle, Fluconazole  (0.1 to 60 mg/kg of body weight/dose), Amiodarone  (5.0 or 25 mg/kg of body weight/dose) or Fluconazole (0.1 to 60 mg/kg of body weight/dose) + Amiodarone (5.0 or 25 mg/kg of body weight/dose) administered intraperitoneally once a day for a total of 4 days. On day 4 post-infection, kidneys from euthanized mice  were aseptically removed, weighed, and homogenized in sterile saline using an IKA Works ULTRA TURRAX Tube Disperser Workstation . Serial dilutions of homogenized kidneys were plated onto YPD agar plates containing chloramphenicol (70 µg/ml) and ampicillin (50µg/ml) to eliminate bacterial cross-contamination. Culture plates were incubated at 30°C for 48 h, after which the CFU were counted and the number of CFU per gram of tissue was calculated. The method was sensitive for detection of ≥10 CFU/g. The culture-negative plates were counted as having 0 CFU/g.Female BALB/c mice   were used throughout the experiments. The mice were housed in micro-isolator cages with five animals per group and had access to food and water ad libitum. All animal experiments were conducted in the PHRI-UMDNJ Research Animal Facility. Disseminated infection with C. albicans proliferation with treatment group was assessed using multiple linear regression of log-transformed CFU counts. Preliminary model fit analysis was conducted to confirm that a parsimonious model which included dose-response slopes for AMD and FLZ, and a term for synergistic effect of dual drug treatment fit the data as well as the saturated model, where each of the nine treatment and dosage combinations were independent predictors (likelihood ratio test p = 0.47). The regression coefficients for drug dose are interpreted as slopes of the dose response curves. For the synergistic term, the exponentiated coefficient is the ratio of geometric means of the CFU for any given dose of each drug when the other drug is present versus when the other drug is absent. The significance of the association of CFU with predictors was assessed using Wald t-statistics for the regression coefficients.The association of Figure S1upc2-1 cells treated with fluconazole and ergosterol. Total sterol from control WYP361 (upc2-1) cells (A), cells treated with fluconazole (100 µg/ml) for 6 hours (B), and those treated with fluconazole (100 µg/ml) and ergosterol (50 µM) together for 6 hours (C) was extracted and analyzed by gas chromatography. Cholesterol was added to the samples to normalize extraction efficiency. Ergosterol and lanosterol were identified with standards. A major sterol derivative, likely 14-methyl 3,6-diol, is marked with an asterisk. Analysis was done in triplicates for each treatment and a representative chromatogram is shown. (D) Quantification of ergosterol, lanosterol and the major sterol derivative. Averages of relative amounts and standard deviations were calculated from triplicate samples and plotted.Sterol profiles of (0.86 MB TIF)Click here for additional data file.


The purpose of the study was to compare radiographic and histopathologic findings with regard to number and extent of calcified discs in the dachshund.The intervertebral discs of 20 dachshunds were subjected to a radiographic and histopathologic examination. The dogs were selected randomly from clinical cases euthanased for reasons unrelated to research at the Norwegian School of Veterinary Science. Lateral radiographs were taken of the vertebral columns after removing them from the carcasses. The histopathologic examination included 5 μm thick sections in the transverse plane, stained with hematoxylin-eosin and von Kossa. Radiographs and histological sections were evaluated independently.A total of 148 (28.5%) calcified discs were identified at the radiographic and 230 (45.7%) at the histopathologic examination. Of 92 discs found to be calcified by histopathology, but not by radiography, the degree of calcification was evaluated as 'slight' in 84 (91.3%). All the intervertebral discs (n = 138) that were found to be calcified by radiography were also found to be calcified by histopathology.A sensitivity of 0.6 and specificity of 1.0 for radiography was calculated when using histopathology as the gold standard. Intervertebral disc disease (IDD) is the most common cause of neurologic dysfunction in the dog . The disIn dachshunds and other chondrodystrophoid breeds, IDD is mainly due to extrusion of nuclear material from the disc into the vertebral canal. Hansen  classifiThe occurrence of calcified discs is found to differ between dachshunds of the same age, size and coat variety . FurtherLow-field magnetic resonance (MR) imaging is found to be very sensitive in the evaluation of disc degeneration in dogs . HoweverDifferent degrees of calcification are found in degenerated chondrodystrophoid discs ,12. DiscThe vertebral columns were obtained from 20 dachshunds that were euthanased for reasons unrelated to research at the Department of Companion Animal Clinical Sciences, Norwegian School of Veterinary Science. The case material, including size and coat varieties, sex, age and reason for euthanasia is presented in Table The vertebral columns were separated from the skull, ribs and pelvis by dearticulation. The tail, including the coccygeal vertebrae, was also removed. The spines were freed of muscle, loose connective tissue and ligaments.Within 24 hours of death, conventional lateral radiographs were taken of the cervical, thoracic, lumbar and sacral vertebral columns . The radAt least four exposures were taken of each dog covering the vertebral column from the first cervical C1) to the first sacral (S1) vertebra. The total number of calcified discs and their location in the vertebral column were recorded according to earlier described methods ,14. Thre to the fSLIGHT:- one or more calcified bodies with diameter less than 2 mm, or- calcification of part of the transitional zone between the nucleus pulposus and the annulus fibrosus, or- indistinct calcification of a larger part of the nucleus pulposusMODERATE:and calcification of part of the transitional zone between the nucleus pulposus and the annulus fibrosus, or- calcified bodies with diameter less than 2 mm - distinct calcification of a larger part of the nucleus pulposus, or- indistinct calcification of the entire nucleus pulposusSEVERE:- distinct calcification of the entire nucleus pulposusIf there was doubt about calcification or the degree of calcification, additional radiographs were taken of the vertebral section of current interest.extremitas cranialis et caudalis) and removed.Immediately after the radiographic examination, specimens for histological examination were collected by removing each intervertebral disc between the second cervical (C2) and first sacral (S1) vertebra in all of the 20 vertebral columns. With tissue forceps, a scalpel handle and no. 11 blade the discs were carefully separated from the adjacent vertebral body end plates (All specimens (520 discs) were fixed in 4% phosphate-buffered formaldehyde, pH 7.2. Subsequently the discs were dehydrated in ethanol, equilibrated in xylene and embedded in paraffin. Decalcifications of the specimens were not performed. The discs were sectioned transversely at about 5 μm and stained with hematoxilin-eosin and von Kossa, a method for demonstration of calcium . During Three degrees of calcification of the individual discs were noted, and for each category, all findings might appear either separately or in combination with other lesions. The degrees of calcification were defined as follows:SLIGHT Figure :- single or multiple foci of calcification occupying less than half of the area of the nucleus pulposus- single or multiple small foci of calcification within the annulus fibrosusMODERATE Figure :- single or multiple foci of calcification occupying more than half, but not all, of the area of the nucleus pulposus- single or multiple medium size foci of calcification within the annulus fibrosus- thin calcified ring, continuous or discontinuous, either surrounding the nucleus pulposus or localised within the annulus fibrosusSEVERE Figure :- widespread or total calcification of the nucleus pulposus- single or multiple large foci of calcification within the annulus fibrosus- broad calcified ring, continuous or discontinuous, either surrounding the nucleus pulposus or localised within the annulus fibrosusIf two or more sections of the same disc were read to have different degrees of calcification, the section with the most severe degree of calcification determined the evaluation of that disc.Radiographs and histological sections were evaluated independently.At the radiographic examination, calcification was found in 148 (28.5%) of the 520 discs. Of all affected discs, a severe degree of calcification was found in 15, a moderate degree in 43 and a slight degree in 90 discs. Calcified discs were identified in 18 (90.0%) of the dogs and the number of calcified discs in each affected dog ranged from one (dog no. 19) to 20 (dog no. 15).At the histopathologic examination, calcification was found in 230 (45.7%) of the 503 discs. Of all affected discs, a severe degree of calcification was found in 30, a moderate degree in 45 and a slight degree in 155 discs. Calcified discs were identified in all the dogs and the number of calcified discs in each dog ranged from one (dog no. 19) to 24 (dog no. 17).The individual number and location of calcified discs together with the degree of calcification of each affected disc, identified both by radiography and histopathology, are presented in Additional file Comparative results by radiographic and histopathologic examination concerning 503 discs are presented in Table In 95 discs the degree of calcification was found to be equal in histopathology and radiography. In 125 discs the degree of calcification was higher in histopathology and in 10 it was higher in radiography.The present study shows that a significantly higher number (45.7% versus 27.4%) of calcified discs are identified by a histopathologic examination compared to a radiographic examination of intervertebral discs in dachshunds. This finding verifies the hypothesis presented at the beginning of the present paper.At the radiographic examination, a total of 148 calcified discs were identified in 18 dogs. This makes a mean of 8.2 calcified discs in each affected individual. Both the percentage number of dogs with calcified discs (90.0%) and the mean number of such discs in each affected dog are higher than values for corresponding parameters found in previous radiographic studies ,7,12. ThIn dogs no. 2 and 3, no calcified discs were found at the radiographic examination, but at the histopathologic examination a slight degree of calcification was found in five and 15 discs, respectively. This finding shows that a total absence of calcified discs visible on radiographs of a dachshund is no guarantee for the dog not having extensive calcification at histopathologic examination.Of the 92 discs that were found to be calcified only at the histopathologic examination, 84 (91.3%) were noted to have a 'slight' degree, eight a 'moderate' degree and none a 'severe' degree of calcification. This agrees with the general understanding that an ordinary radiographic examination of the vertebral column in dogs is not very sensitive at detecting minor calcifications of intervertebral discs.caput costae and os ilium) was avoided on the radiographs. Also motion blurring was not a problem with the examination of dead specimens. These factors contributed to high technical quality of the radiographs. Therefore, the sensitivity of the radiographic examination in the present study is likely higher than could be expected in similar studies on live dogs. As a consequence, the presented results concerning total number of calcified discs and degree of calcification of individual discs, cannot directly be compared to results from previous radiographic studies [The radiographic examination in the present study was done on vertebral columns separated from the skull, ribs and pelvis and freed of soft tissue. This meant that superimposition of tissue adjacent to the discs  declare that they have no competing interests.ØS conceived of the study, carried out the radiographic examination and is the main author of the paper. ØK carried out the histopathologic examination. Both authors read and approved the final manuscript.1 slight. 2 moderate. 3 severe. Other note: -: intervertebral disc not available for a final histopathologic examination.Table 3. Degree of calcification, evaluated by a radiographic and a histopathologic examination in 26 intervertebral discs from each of 20 dachshunds. Degrees of calcification evaluated by radiography: light grey: slight. dark grey: moderate. black: severe. Degrees of calcification evaluated by histopathology: Click here for file


Ayurveda, the traditional medical system of India, is understudied in western contexts. Using data gathered from an Ayurvedic treatment program, this study examined the role of psychosocial factors in the process of behavior change and the salutogenic process. This observational study examined associations with participation in the 5-day Ayurvedic cleansing retreat program, Panchakarma. Quality of life, psychosocial, and behavior change measurements were measured longitudinally on 20 female participants. Measurements were taken before the start of the program, immediately after the program, and 3 months postprogram. The program did not significantly improve quality of life. Significant improvements were found in self-efficacy towards using Ayurveda to improve health and reported positive health behaviors. In addition, perceived social support and depression showed significant improvements 3 months postprogram after the subjects had returned to their home context. As a program of behavior change, our preliminary results suggest that the complex intervention Panchakarma may be effective in assisting one's expected and reported adherence to new and healthier behavior patterns.


Although all these antibodies sensitize trypomastigotes and prepare them to activate the complement (C) system, as measured by consumption of total C, C4, B and C3, only the polyclonal antiserum or its IgG, IgM and Fabμ fragments were able to induce trypanosome lysis by the alternative C pathway.A Mouse polyclonal antiserum against Trypanosoma cruzi or its IgG and IgM fractions and five monoclonal antibodies (two IgM, two IgG


Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor superfamily, is amplified and over-expressed in various cancers. The objective of the present study was to investigate the concentration of DcR3 in sera of hepatocellular carcinoma (HCC) patients and its clinical significance.Serum concentrations of DcR3 were measured by enzyme-linked immunosorbent assay (ELISA) in 67 patients with HCC, 8 with liver cirrhosis, 17 with cholecystitis, and in 28 healthy individuals. Immunohistochemistry was employed to access protein expression of DcR3 in the corresponding HCC tissues.P < 0.01). Moreover, serum concentrations of DcR3 in HCC patients were associated with TNM stage, para-cirrhosis, capsular infiltration, and metastasis or recurrence of disease (P < 0.05). There was a positive correlation between the serum concentration of DcR3 and protein expression in HCC tissues .Serum concentrations of DcR3 in patients with HCC or cirrhosis were significantly higher than in healthy individuals (The high serum concentration of DcR3 might play a certain role in pathogenesis, progress, and metastasis of HCC. Moreover, DcR3 might serve as a valuable molecular indicator in early diagnosis and contribute to predicting the clinical outcome in HCC patients. Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer deaths in the world with an increasing incidence . Althougn = 67), cirrhosis (n = 8), or cholecystitis (n = 17) and also from healthy controls (n = 28). Clinical information was obtained from medical records. The age of the HCC patients ranged from 29 to 74 years, with a mean of 48 years. Among the 67 patients, 58 were male and 9 female. HBV infection status was based on hepatitis B surface antigen (HBsAg). Fifty-one patients were HBsAg-positive, and ten were found to be anti-HCV-positive. There were six anti-HCV-positive cases that were co-infected with HBV, and six cases were both HBsAg-negative and anti-HCV-negative. The histopathologic diagnoses were made according to the World Health Organization international histological classification of HCC: well differentiated (n = 16), moderately differentiated (n = 37), or poorly differentiated (n = 14). According to the clinical tumor–node–metastasis (TNM) standard, their clinical stages were stage I (n = 26), stage II (n = 11), stage III (n = 19), and stage IV (n = 11). All 67 HCC patients were followed up for 20 months by measurements of tumor markers (AFP) and ultrasound (US), as well as helical computed tomographic (CT) scan. Forty cases had metastasis or recurrence of disease, 27 cases did not. Forty-four cases had para-cirrhosis. Sixty-four samples of HCC tissues were collected from hepatectomies. All 64 cases were initial hepatectomies in order to avoid the secondary changes of healing post biopsy. All the 67 HCC patients had never received any radiation therapy or chemotherapy. Diagnosis and classification were based on histology or cytology by the same pathologist. Among eight cirrhotic samples, six were male, two female. The range of age was between 28 and 60 years, with a mean of 44 years. In the cholecystitis group, nine were male, eight female. The age ranged from 25 to 79 years, with a mean of 55 years. After formation of a clot, sera were collected and centrifuged at 1,000 rpm for 20 min. The supernatant was carefully aspirated and stored at −80°C until analysis. The study protocol was approved by Guangxi Medical University Ethics Committee. Written informed consent to use the samples for research was obtained from the patients and clinicians.Serum samples were collected at the First Affiliated Hospital, Guangxi Medical University, China, between September 2006 and September 2007 from patients with HCC  was used according to the manufacturer's protocol. Briefly, 100 μL of each sample was incubated in duplicate in microplates coated with anti-DcR3 monoclonal antibody for 2 h. Following incubation, biotin-conjugate anti-DcR3 monoclonal antibody was added and incubated as a primary antibody for 2 h. After the microplate had been washed, streptavidin-horseradish peroxidase (HRP) was added and incubated for 1 h. After washing off any unbound streptavidin-HRP, tetramethylbenzidine was added as a substrate, and the absorbance was measured at 450 nm after 20 min in a microplate reader . The absorbance of each sample was plotted against a standard curve produced by serial dilutions of recombinant human DcR3-Fc in duplicate. The concentrations of DcR3 in sera were calculated by logarithmic analysis. All samples with an absorbance of less than zero according to the standard curve were discarded from the final analysis. A concentration higher than 122.22 pg/mL was set as the positive level according to the DcR3 average level in the normal control group in our study.The procedure employed has previously been described . Sectiont test and the Mann–Whitney test were used for comparisons between the two groups with SPSS 17.0 software for Windows . Correlations were calculated by Pearson chi-square test. In all cases, a P value of less than 0.05 was considered statistically significant.All data from ELISA were expressed as mean ± standard deviation (SD). For normally distributed data, the two-tailed Student's P < 0.01). Likewise, in the cirrhosis patients the DcR3 serum concentration was higher than that in the healthy controls . The rate of DcR3 protein expression in HCC tissues was 61% (39/64), which was lower than the corresponding rate of DcR3 serum-positive (higher than 122.22 pg/mL) patients . The sensitivity of combined detection of AFP and DcR3 increased to 93% (detailed data not shown).Human DcR3 is a soluble receptor that is mostly expressed in many different classes of tumors. DcR3 is short of the transmembrane domain of conventional Tumor Necrosis Factor Receptors (TNFRs) and is thus believed to be a secreted protein . In the It is known that HCC can result from pre-cancerous liver disease, for instance cirrhosis. However, the molecular mechanisms in such a process remain unclear. The high serum concentrations of DcR3 in the cirrhosis patients and in the para-cirrhosis HCC patients in the present study prompted us to formulate a hypothesis concerning the potential role of DcR3 in hepatocarcinogenesis. However, further studies have to be carried out to prove the hypothesis and investigate the value and mechanism of DcR3 in liver cirrhosis and tumorigenesis of HCC.P < 0.05), revealing an obvious relation between DcR3 and HCC malignant characteristics. As a rule, the capsular infiltration and metastasis reflect tumor growth and tumor invasive capability.To our knowledge, this is the first study investigating a possible relationship between serum concentrations and clinicopathological parameters of patients in HCC. Another interesting observation in the present study was that the serum concentration of DcR3 directly correlated with para-cirrhosis, capsular infiltration, and metastasis or recurrence of disease  was higher than the corresponding tissue DcR3 protein expression rate (39%). This was due to 14 cases of negative expression of DcR3 protein in patients, who were all that serum-positive. The variation between serum and tissue DcR3 levels might result from a difference in the sensitivity of the detecting assays (ELISA versus immunohistochemistry). Thus, using ELISA to estimate serum DcR3 concentrations seems more advantageous in clinical application.Taken together, our study demonstrates that the measurements of serum DcR3 concentrations might become of value for clinical early diagnosis, prognosis prediction, and curative effects in HCC.