, due to the cost of a survey with more than

10 questions

, due to the cost of a survey with more than

10 questions. This survey did however provide some insight into why our respondents began a barefoot running program. A recent survey study investigating the demographics of barefoot runners found the primary motivating factors for those who added barefoot or minimalist shod running to their training was prevention of future injury and performance enhancement. 9 Rothschild found fear of possible injury was the most prevalent perceived barrier in transitioning to Buparlisib molecular weight barefoot or minimalist shod running. However, consistent with our data, they also found that most of the respondents reported no adverse reactions or subsequent injuries after instituting barefoot or minimal running. 9 Similarly, a large number of runners in our study initially tried barefoot

running due to the promise of improved efficiency (60%) or an attempt to get past injury (53%). The runners in our survey ran barefoot on a variety of surfaces including streets, sidewalks, grass, and trails. It has been argued that the decrease in proprioception in cushioned running shoes modifies the body’s natural mechanism for attenuating impact forces, therefore increasing their magnitude.7 The body attempts to attenuate impact forces as failure to do so can result in micro trauma to soft tissue and bone.10 One way the body attempts to mitigate these forces is through adjusting leg stiffness. The body will adjust leg stiffness by altering muscular activity and Tenofovir in vivo joint angles across a variety of surfaces in order to minimize out stress and curtail injuries. Therefore, runners can experience similar impact forces on either hard or soft surfaces with no differences in impact loading whether they are barefoot or shod by appropriately adjusting their leg spring.7 and 11 Efficiency and performance enhancement with barefoot running is a controversial topic. It has been shown that heart rate, maximal oxygen consumption (VO2max), and relative perceived exertion are significantly

higher in the shod runner.12 This study also showed at 70% of VO2max pace, barefoot running is more economical than is running shod, both over ground and on a treadmill. Squadrone and Gallozzi8 found maximum oxygen uptake values to be 1.3% lower when running barefoot than when running in shoes. However, it was also shown that barefoot runners have higher step rates and higher metabolic rates than shod.8 Therefore, it is not clear if barefoot running is more economical metabolically than shod running. A majority of runners in this survey (55%) reported no or slight performance benefit secondary to barefoot running, and over 39% of the runners found moderate to significant improvements in their race times. However, only 6% of respondents claimed to have gotten slower after starting barefoot training. Barefoot running changes biomechanics by encouraging a shorter stride and increased step rate.

Of these, only full-scale IQ did (p = 0 02) Overall, the data sh

Of these, only full-scale IQ did (p = 0.02). Overall, the data showed a strong effect of large rare genic de novo CNVs on the presence or absence of an ASD diagnosis, but did not support either IQ or find more ASD severity as useful predictors for probands carrying these risk variants (Figure 3C). We did observe a trend toward more gene-rich de novo CNVs in females (Figure 2) and found females to be less vulnerable to the reduction in IQ associated with rare de novo CNVs. We next investigated whether individuals

with recurrent CNVs at 16p11.2 or 7q11.23 showed distinctive behavioral or cognitive profiles compared with probands who were not carrying rare de novo events. For each proband carrying a de novo CNV at 16p11.2 or 7q11.23, five other probands were selected as controls based on hierarchical matching criteria: first age, then sex, genetic distance, ascertainment site, and whether the sample was from a quartet or trio. Our primary Selleck Tofacitinib analysis focused on four variables: full-scale IQ, categorical diagnosis, severity of autism, and body mass index (BMI) (Table 2), with the latter motivated by multiple reports that 16p11.2 deletions contribute to obesity (Bijlsma et al., 2009 and Walters et al., 2010). We then pursued a broader exploratory

study of additional phenotypic variables, ten of which are presented in Table 2 with the remainder in Table S5. We found that probands carrying a 16p11.2 or 7q11.23 de novo CNV were indistinguishable from the larger group with regard to IQ, ASD severity, or categorical autism diagnosis (Table 2). However, we did find a relationship between body weight and 16p11.2 deletions and duplications. When we treated copy number as an ordinal variable (one, two, and three copies) and used the matched controls as the diploid sample, BMI diminished as 16p11.2 copy number increased (estimated β = −3.1kg/m2

for each extra copy, p = 0.02). The extensive phenotypic data available on the SSC sample constitute a great resource for fine-grained analyses of genotype-phenotype relationships. In the current study, the limiting factor with regard to recurrent de novo CNVs was the small sample size, even for 16p11.2 duplications and deletions. Nonetheless, we undertook to an exploratory analysis of a range of phenotypic features and found several that yielded significant p values. While none would survive correction for multiple comparisons, we report them here in the interest of generating hypotheses for future studies (Table 2 and Table S5). For example, individuals with 16p11.2 duplications had higher hyperactivity scores compared to matched control probands, while probands carrying 7q11.23 duplications showed significantly more behavioral problems (Aberrant Behavior Checklist total), but less severe social and communication impairment during ADOS administration.

The method of cumulative EPSC amplitudes (Figure 5E) revealed a s

The method of cumulative EPSC amplitudes (Figure 5E) revealed a significantly smaller pool size in Robo3 cKO mice (8.6 ± 3.5 nA; n = 5) as compared to control mice (25.7 ± 4.8 nA; n = 7; p = 0.012). Furthermore, there was a significant reduction of release probability during the first EPSC,

as estimated by dividing the first EPSC amplitude by the pool size estimate (Figures 5E1 and 5E2). In summary, direct pre- and postsynaptic recordings at ipsilateral calyx of Held synapses indicate a more variable Ca2+ current density, a smaller size of the fast-releasable vesicle pool (FRP), and selleck chemicals a significant reduction of the initial release probability in Robo3 cKO mice (Figure 5). These data show that processes of synapse maturation, Selleck Kinase Inhibitor Library including the acquisition of fast transmitter release properties characteristic for the calyx of Held, fail to take place in Robo3 cKO mice. The finding of functional deficits at the calyx of Held synapses in Robo3 cKO mice suggests that axon midline crossing conditions the functional maturation of commissural output synapses. Alternatively, Robo3 could have a so far unknown direct role in synapse formation and synapse maturation. In a first series of experiments, we addressed this possibility by studying the developmental expression of Robo3, to verify whether Robo3 is expressed at the time of synaptogenesis

(Figures 6A–6C). In situ hybridization showed Robo3 expression at E14 in the developing VCN, but transcript levels in the VCN were essentially absent at E18 and undetectable

at P10 (Figure 6A). Using an anti-human Robo3 antibody which stained crossing hindbrain axons at E12.5 (Figure 6B), we next attempted to localize Robo3 protein in developing calyces of Held early postnatally, at P1 and P3 (Figure 6C), and at P5 and P8 (see Figure S1 available online). Robo3 was undetectable in developing calyx of Held axons, despite occasional non-specific signals (Figure 6C, arrow); the latter persisted in Robo3 cKO mice (Figure S1). This data indicates that at the calyx of isothipendyl Held projection, Robo3 expression is developmentally downregulated before E18, similar as at other commissural projections (Marillat et al., 2004; Sabatier et al., 2004; Tamada et al., 2008). The absence of Robo3 argues against a direct role of this protein in synapse development. To address a possible direct role of Robo3 in synapse development with an independent approach, we used a conditional KO approach with an inducible Cre mouse line, the CAGGS::CreERTM mouse line ( Guo et al., 2002; Livet et al., 2007). CAGGSCreERTM/+, Robo3lox/lox mice were injected at P0 with tamoxifen (referred to as Robo3 cKOTMX-P0 mice), in order to inactivate the floxed Robo3 allele following axon midline crossing, but before calyx of Held formation and – maturation.

The aPFC may not encode only the value of an alternative

The aPFC may not encode only the value of an alternative

choice but also information about the diversity of alternative options available. Yoshida and Ishii (2006) trained their subjects to navigate through a virtual maze and then took fMRI scans while the subjects tried to work out where they had subsequently been placed within the maze. The BOLD signal in an aPFC region just anterior to that highlighted by Boorman et al. (2009) Doxorubicin manufacturer varied with the subjects’ uncertainty about their position in the maze and therefore with the range of alternative options that the subjects might reasonably choose as their next response. In other words, aPFC activity increases when one considers many alternative options as opposed to just a few. While aPFC may encode the number of possible alternative choices it is important to note that it does not code all options in the same way. The representation of one alternative, the one with the highest value, seems to have a special status. Boorman et al. (2011) tested subjects on a decision-making task with three choices. They found a positive correlation between aPFC BOLD signal and the value of the better alternative choice. There was, however, a negative relationship between aPFC BOLD signal

selleck and the value of both the chosen option and the worse alternative. It seems, therefore, that aPFC activity reflects the benefits of switching to the better alternative and the opportunity cost of switching away from both the current choice and the other worse alternative. Such a pattern of activity with just one alternative choice held in a pending state suggests that not all potential alternative choices are considered equally when we change our minds and pursue a different course of action. Behavioral evidence also suggests that we are not able to represent all alternative choices equally and that we switch more effectively to one alternative as opposed to another at any given time (Boorman

et al., 2011 and Charron and Koechlin, 2010). The same aPFC region has also been identified in a study of exploratory decision-making (Daw et al., 2006). Although it is obviously advantageous for organisms to choose the most valuable option they can identify it is essential that they also through explore alternative options; an organism that fails to explore alternatives will fail to identify choices that might be even higher in value, especially when the environment is changing. There is, therefore, a balance to be struck between exploiting choices of known value and exploring alternatives. Daw et al. (2006) found that vmPFC/mOFC activity was highest when exploitative choices of high-value options were being made but aPFC was more active when lower value, presumably exploratory, choices were made. The results reported by Boorman et al., 2009 and Boorman et al., 2011 suggest that the high-aPFC signal during exploration reported by Daw et al.

However, in 1999 human gliomas were analyzed

by Fassati e

However, in 1999 human gliomas were analyzed

by Fassati et al. for the infiltration of neutrophils using immunohistochemistry by staining sections for CD15-positive and myeloperoxidase-positive cells [43]. The authors observed a marked and significant correlation between tumor grade and the extent of the neutrophil infiltration. In the low grade tumors only 40–50% had significant infiltration, while in glioblastoma multiforme over 85% of the tumors had significant infiltration. When the circulating neutrophil count was scored against tumor neutrophil infiltration, a significant and remarkable positive correlation was observed between circulating neutrophil count and extent of neutrophil infiltration into the tumor. The highest numbers of circulating neutrophils were seen in the glioblastoma multiforme patients. Moreover, high-grade Tyrosine Kinase Inhibitor Library manufacturer glioblastoma multiforme were highly vascularized and contain

areas of necrosis. It is noteworthy that in these Romidepsin cell line tumors, neutrophils were detectable within capillaries in high numbers and were often observed in high numbers in areas of necrosis. More than 10 years later, Atai et al. confirmed that glioblastoma tissue was infiltrated with neutrophils and macrophages and demonstrated osteopontin is up-regulated and associated with neutrophil and macrophage infiltration [44]. Evaluating human head and neck squamous cell carcinoma (HNSCC) of the oral cavity, oropharynx, hypopharynx or larynx, Trellakis et al. published in 2011 observations regarding the role of neutrophils [45]. The authors observed most of T4 tumors

displayed medium or strong infiltration of CD66+ neutrophils, whereas smaller and less-invasive tumors exhibited a lower degree of neutrophil infiltration. The serum concentrations of CXCL8, CCL4 and CCL5 and the peripheral blood percentages of neutrophils and leukocytes as well as the NLR were significantly higher in HNSCC patients than in healthy controls. In multivariate analyses of patients with advanced disease, high CD66b+ neutrophil tumor infiltration was independently associated with poor survival. In 2011, An et al. published retrospective results of 363 consecutively, newly diagnosed, non-disseminated, and biopsy-proven nasopharyngeal carcinoma patients treated with standard curative radiotherapy with or without chemotherapy [46]. For patients with locoregionally advanced Mephenoxalone disease, high NLR (>3.73) was not only an independent prognostic factor for poor disease-specific survival, distant metastasis-free survival, and loco-regional recurrence-free survival, but was also a predictor of response to chemoradiotherapy. Thus, compared with radiation alone, chemoradiotherapy significantly improved disease-specific survival and loco-regional recurrence-free survival for patients with non-elevated NLR, but not for those with elevated NLR. This emphasizes high pre-treatment NLR as a strong prognostic factor for poor outcome of nasopharyngeal patients. He et al.

, 2008) The presence of polyQ-1C2-positive inclusions suggests t

, 2008). The presence of polyQ-1C2-positive inclusions suggests that two mechanisms might contribute to disease; a toxic RNA encoded by

one strand and a polyQ peptide encoded by the other ( Figure 1). In an attempt to clarify the molecular source of pathogenicity in HDL2, Yang and colleagues (as detailed in this issue of Neuron) engineered a series of transgenic mice expressing Sirolimus mw human JPH3 mutant alleles. Using bacterial artificial chromosomes (BACs), an approached pioneered by the Yang group for the study of HD, Wilburn et al. (2011) generated transgenic mice carrying 165 kb from the JPH3 locus with ∼120 CAG repeats. These mutant JPH3 BAC mice presented with several key features found in HDL2 patients. Among these are an age-dependent motor deficit, forebrain atrophy, and the presence of nuclear inclusions positive for ubiquitin and reactive with two polyQ antibodies, including

1C2. At a molecular level, Yang and colleagues provide evidence for a novel promoter that drives expression of a polyQ-encoding transcript Sorafenib concentration from the DNA strand in the antisense orientation to JPH3. Importantly, Wilburn et al. provide biochemical evidence that mutant BAC-JPH3 brains express insoluble polyQ peptides of a size range that would be expected to be encoded by the JPH3 CAG antisense strand of the BAC transgene. However, the nuclear TCL inclusions in the mutant BAC-JPH3

mice are also positive for RNA from the sense CUG strand. Given this finding, which of the two transcripts is the pathogenic species in the BAC-JPH3 mice? Does disease progression require functional expression of both transcripts? To address the extent to which the CAG-polyQ-encoding antisense transcript contributes to pathogenesis, Wilburn et al. developed a second version of an expanded CAG repeat BAC-JPH3 transgenic mouse, designated BAC-HDL2-STOP. In this mouse, exon 1 of the JPH3 transgene was replaced with a previously well-characterized transcription STOP sequence such that expression of the JPH3 CUG sense strand is selectively silenced while expression of the antisense CAG transcript remains intact. By behavioral and neurological measures, the BAC-HDL2-STOP mice expressing only the CAG antisense transcript develop motor deficits and degenerative pathology very similar to that seen in the original BAC-JPH3 mice expressing both transcripts. Although the extent to which the JPH3 sense CUG transcript contributes to disease was not assessed directly, these results provide strong evidence that the CAG antisense transcript is very pathogenic and a prominent contributor to disease progression in this mouse model of HDL2. In a final series of studies, Wilburn et al. provide evidence that, like other polyQ disorders (e.g.

The localization of Mib during asymmetric division is not known i

The localization of Mib during asymmetric division is not known in any experimental system. To address this question in the absence of a working Mib antibody, we used a GFP-tagged full-length Mib (Mib-GFP), which allows examination of the in vivo dynamics of the Mib protein. Multiple-tagged forms of Mib (including GST-, Myc-, and FLAG-tagged versions) have been previously shown to be functional (Itoh et al., 2003). Nevertheless,

we first verified whether the Mib-GFP reflected the endogenous Mib distribution. When transiently expressed in zebrafish embryos through either DNA electroporation or mRNA microinjection, Mib-GFP displayed a punctate pattern that is located in the cytosol near the membrane as well as adjacent to the nucleus (Figures 7A and 7B), in agreement with its previously reported localization and function in endosomes (Itoh et al., 2003 and Koo et al., 2005). In addition we performed double labeling with antibodies Fluorouracil against GFP and Dld at ∼24 hpf. Dld is expressed in the developing brain (Figure 3), albeit less prominently than Dla, for which a workable antibody was not available despite much failed effort with the previously published antibody (Tallafuss et al., 2009). This analysis showed that the Mib-GFP signal was colocalized with Dld (Figure 7C), although find more an exact colocalization was not expected

due to the transient nature of Mib-GFP expression and the presence of other Notch ligands in the brain. Together, these results suggest that Mib-GFP reflects the endogenous Mib distribution pattern. Next, we analyzed the Mib-GFP distribution in paired daughter cells. Coelectroporation of a red fluorescent lineage tracer together with the Mib-GFP construct at ∼22 hpf and analysis of paired daughters at ∼37 hpf showed that Mib-GFP was exclusively

detected in the apical daughter in 85% paired daughter cells analyzed (n = 26) (Figure 7D). This observed percentage is consistent with the idea that Mib asymmetry is likely present in both clone type 1 and 2 (as shown in Figure 1D). In addition the Mib asymmetry appeared to be stably maintained during INM (Figure S6). The unequal segregation of Mib-GFP into the apical daughter made us wonder whether it is dependent on the conserved intrinsic polarity regulator Par-3 because Par-3 has been found asymmetrically over localized to the apical domain of dividing neural progenitors in zebrafish (Alexandre et al., 2010 and von Trotha et al., 2006). We analyzed paired daughters in the embryos injected with a well-established morpholino antisense oligonucleotide targeting par-3 (referred to as the par-3 morphant) ( Alexandre et al., 2010 and Tawk et al., 2007). As expected, the par-3 morphants in our experiments displayed a loss of apicobasal cell polarity and suffered a mild defect in brain morphology at 37 hpf ( Figures S7A–S7I). In the par-3 morphant, Mib-GFP was detected in both daughter cells (91%, n = 23 pairs of daughter cells analyzed) ( Figures 7E and S6).

This indicates that in worms autophagy represents a pathway

This indicates that in worms autophagy represents a pathway

for endocytic lysosomal degradation of GABAARs ( Rowland et al., 2006). Third, GABARAP knockout mice show normal expression and punctate distribution of γ2-containing GABAARs ( O’Sullivan et al., 2005), possibly due to functional redundancy of GABARAP with GEC1 ( Mansuy-Schlick et al., 2006) and other GABARAP family members. Fourth, the function of GABARAP is complicated by its interactions with a very large number of other proteins. Among these the ER luminal Ca2+-dependent chaperone calreticulin stands out in that it binds GABARAP with exceptionally high affinity ( Mohrlüder et al., 2007). Compared to calreticulin the interaction GSK2656157 cost of GABARAP with γ2-derived peptides shows low affinity, suggesting that GABARAP might promote protein trafficking unspecifically along the secretory pathway ( Knight et al., 2002). Several GABARAP-interacting proteins contribute to GABAAR trafficking independently of GABARAP. The aforementioned NMDAR-induced and GABARAP-dependent Ferroptosis inhibitor increase in GABAAR clustering also depends on the synaptic PDZ domain-containing protein GRIP (Marsden et al., 2007), which interacts with GABARAP in vitro and in vivo (Kittler et al., 2004a). GRIP was first described as a trafficking factor of AMPARs (Dong

et al., 1997). It is present at both glutamatergic and GABAergic synapses, consistent with functions at both types of synapses (Dong et al., 1999, Charych et al., 2004a, Kittler et al., 2004a and Li et al., 2005). GABARAP further interacts with the phospholipase C-related catalytically inactive proteins 1 and 2 (PRIP1/2, PRIP1 was previously named p130; Kanematsu et al., 2002), a pair of GABAAR-associated adaptor proteins for phosphatases and kinases (Figure 3A)

(Kanematsu et al., 2002 and Uji et al., 2002). Likewise, GABARAP and its paralog GATE-16 (Sagiv et al., 2000) interact with NSF, an ATPase and chaperone of SNARE complexes that is critically important for regulated neurotransmitter release and also involved in trafficking of neurotransmitter receptors (Morgan and Burgoyne, 2004 and Zhao Adenosine et al., 2007). Both PRIP1/2 and NSF interact with GABAARs indirectly through GABARAP and directly via GABAAR β subunits (Figure 1C) (Kanematsu et al., 2002, Kittler et al., 2004a, Terunuma et al., 2004 and Goto et al., 2005). PRIP1/2 double knockout mice exhibit reduced expression and altered behavioral pharmacology of GABAARs, suggesting deficits in mainly γ2-containing GABAARs (Kanematsu et al., 2002, Kanematsu et al., 2006 and Mizokami et al., 2007). Brain extracts of these mice further show reduced association of GABAARs with GABARAP, indicating that PRIP facilitates indirect association of GABARAP with GABAARs (Mizokami et al., 2007). Moreover, PRIP and the γ2 subunit compete for binding to the same binding site on GABARAP (Kanematsu et al., 2002 and Uji et al., 2002).

For each event-related scan, the time course of the MR signal int

For each event-related scan, the time course of the MR signal intensity was first extracted by averaging the data from all the voxels within the predefined ROI. The average event-related time course was

then calculated for each type of trial, by selectively averaging to stimulus onset and using the average signal intensity during the fixation trials as a baseline to calculate percent signal change. Specifically, in each scan we averaged the signal intensity across the trials for each type of trial at each of 12 corresponding time points (s) starting from the stimulus onset. These event-related time courses of the signal intensities were then converted to time courses of percent signal change for each type of trials by subtracting the corresponding value for the fixation Alectinib chemical structure trials and then dividing by that value. Because M-sequences have the advantage that each type of trials was preceded and followed equally often by all types of trials, the overlapping BOLD responses due to the short interstimulus interval

are removed by this averaging procedure (Buracas and Boynton, 2002). The resulting time course for each type of trials was then averaged across scans for each subject and then across subjects. The V1 model with its original model parameters as in Li, 1999 and Li, 2002 was used to simulate V1 responses to the texture image of low luminance bars with a foreground region (i.e., 2 × 2 bars) like in our experiments. 3 Methyladenine The model mechanisms include (1), direct inputs to V1 neurons from each bar according to the classical receptive fields, and (2), interactions between V1 neurons by the intracortical connections implementing contextual influences (such as surround suppression) of the surround to the neural responses. At each grid location, the maximum response from all pyramidal model neurons was obtained. This maximum was averaged over all simulation

time steps within 50 ms (simulated by five membrane time constant of the model neurons). The saliency of each grid location is the Z-score of this maximum obtained as follows: take the difference between this maximum and the average of the maximums over all grid locations and then divide it by the standard deviation of all the maximums however ( Li, 1999). Saliency in the foreground region is the maximum of the Z-scores over the 4 × 4 bar region centered on the foreground region. The result for each orientation contrast (7.5°, 15°, 30°, and 90°) as plotted in Figure 2 was obtained by averaging the foreground region saliency from 24 simulations for 24 different background bar orientations evenly distributed between 0° and 180°. The saliency of the foreground region should be directly related to the strength of its attentional attraction (i.e., its cueing effect). We are grateful to Peter Dayan for reading the manuscript with helpful comments and Yan Song for help with dipole source localization.

The RT-PCR program consisted of 30 min at 50 °C and 15 min at 95 

The RT-PCR program consisted of 30 min at 50 °C and 15 min at 95 °C. A three-step cycling protocol was used as follows: 95 °C for 5 s, 58 °C for 15 s, and of 72 °C for 20 s for

45 cycles. In each PCR run a standard curve was included with a known virus concentration. Results of the PCR are expressed as TCID50-equivalents per swab or per gram of tissue. TCID50-equivalents are a relative measure and not necessarily represent live virus. Nasal swabs, oropharyngeal swabs, tissue homogenates and BALF were all tested in a virus isolation Fasudil in vitro with end-titration on MDCK-I-BD5 cells [15]. Samples were initially diluted with the same amount of GMEM/EMEM Modulators medium containing 1% bovine serum albumin and antibiotics (twofold dilution). This initial dilution was serially diluted tenfold in the same medium. The diluted samples (100 μl/well) were mixed with 150 μl of 2 × 105 MDCK-I-BD5 cells/ml and incubated CX-5461 concentration for 48 h at 37 °C and 5% CO2. The monolayers were subsequently washed with PBS, frozen at −20 °C and fixed with 4% cold (4 °C) paraformaldehyde for 10 min. After washing, viral NP-protein-containing cells were stained using HRPO-conjugated monoclonal antibody HB65 and 3-amino-9-ethyl-carbozole (AEC; Sigma–Aldrich,

The Netherlands) as a substrate for HRPO. Samples were tested in eightfold and titres were calculated according to the method of Spearman-Kärber [16]. and Virus titres are expressed as TCID50 per swab or per gram of tissue. The hemagglutination inhibition (HI) test was carried out as described before [17]. Before testing, samples were inactivated for 30 min at 56 °C. Subsequently

they were pre-treated with receptor destroying enzyme (RDE) and chicken red blood cells to remove non-specific agglutinins and hemagglutination inhibitors. Starting at an initial dilution of 1:10, sample were tested in two-fold dilution series. Samples were incubated for 60 min after adding antigen and another 45 min after adding chicken red blood cells and subsequently read. The antigens used in the test were the A/Netherlands/602/2009 (H1N1)v and, for swine influenza, the A/Swine/Best/96 (H1N1) [18] and A/Swine/Gent/7625/99 (H1N2) [19]. All were standardised at 4 hemagglutinating units per 25 μl. The virus neutralisation tests were performed on MDCK-I-BD5 cells [15]. Sera were heat inactivated for 30 min at 56 °C before testing. Twofold serial dilutions of the sera were made in GMEM/EMEM medium containing 1% bovine serum albumin and antibiotics in 96-well plates. The diluted sera (50 μl/well) were mixed with 100 TCID50 of the influenza viruses (50 μl) and incubated at 37 °C and 5% CO2 for 1 h. Thereafter 150 μl of 2 × 105 MDCK-I-BD5 cells/ml were added to each well. The plates were incubated at 37 °C and 5% CO2 for 48 h. The monolayers were washed with PBS, frozen at −20 °C and fixed with 4% cold (4 °C) paraformaldehyde for 10 min.