Decoding of standard and non-standard visuomotor associations from parietal cortex.

OBJECTIVE: Neural signals can be decoded and used to move neural prostheses with the purpose of restoring motor function in patients with mobility impairments. Such patients typically have intact eye movement control and visual function, suggesting that cortical visuospatial signals could be used to guide external devices. Neurons in parietal cortex mediate sensory-motor transformations, encode the spatial coordinates for reaching goals, hand position and movements, and other spatial variables. We studied how spatial information is represented at the population level, and the possibility to decode not only the position of visual targets and the plans to reach them, but also conditional, non-spatial motor responses. APPROACH: The animals first fixated one of nine targets in 3D space and then, after the target changed color, either reached toward it, or performed a non-spatial motor response (lift hand from a button). Spiking activity of parietal neurons was recorded in monkeys during two tasks. We then decoded different task related parameters. MAIN RESULTS: We first show that a maximum-likelihood estimation (MLE) algorithm trained separately in each task transformed neural activity into accurate metric predictions of target location. Furthermore, by combining MLE with a Naïve Bayes classifier, we decoded the monkey's motor intention (reach or hand lift) and the different phases of the tasks. These results show that, although V6A encodes the spatial location of a target during a delay period, the signals they carry are updated around the movement execution in an intention/motor specific way. SIGNIFICANCE: These findings show the presence of multiple levels of information in parietal cortex that could be decoded and used in brain machine interfaces to control both goal-directed movements and more cognitive visuomotor associations.

Genome-wide association and epidemiological analyses reveal common genetic origins between uterine leiomyomata and endometriosis.

Uterine leiomyomata (UL) are the most common neoplasms of the female reproductive tract and primary cause for hysterectomy, leading to considerable morbidity and high economic burden. Here we conduct a GWAS meta-analysis in 35,474 cases and 267,505 female controls of European ancestry, identifying eight novel genome-wide significant (P < 5 × 10-8) loci, in addition to confirming 21 previously reported loci, including multiple independent signals at 10 loci. Phenotypic stratification of UL by heavy menstrual bleeding in 3409 cases and 199,171 female controls reveals genome-wide significant associations at three of the 29 UL loci: 5p15.33 (TERT), 5q35.2 (FGFR4) and 11q22.3 (ATM). Four loci identified in the meta-analysis are also associated with endometriosis risk; an epidemiological meta-analysis across 402,868 women suggests at least a doubling of risk for UL diagnosis among those with a history of endometriosis. These findings increase our understanding of genetic contribution and biology underlying UL development, and suggest overlapping genetic origins with endometriosis.

Plate failure by bending following tibial fracture stabilisation in 10 cats.

OBJECTIVE: To describe the clinical findings and management of tibial fractures in cats in which osteosynthesis failed due to plate bending. METHODS: Case records and radiographs of cat tibial fracture repairs from five referral centres were reviewed for signalment and to assess incidence of plate failure by bending. Cats that sustained plate bending following plate or plate-rod fixation were reviewed for fracture configuration, repair method, initial postoperative and postfailure tibial alignment, revision treatment and outcome. RESULTS: The incidence of plate bending in cat fractures managed with plate and plate-rod fixation in the four referral centres where the overall number could be established was 13% (8/60). In the 10 cats in which plates bent, initial fractures were generally oblique or spiral with mild comminution and located in the middle or distal third of the tibia. Mean time to implant failure was 24 days (range 2 to 56 days). Mean tibial valgus angle increased from 12·9° to 30·9° following bending of the plate. Short-term outcome following revision surgery using orthogonal plating or stacked medial plates was favourable with improvement in tibial valgus in all five fractures with follow-up data. CLINICAL SIGNIFICANCE: Plate bending following tibial fracture stabilisation in these 10 cats resulted in tibial valgus deformation. Consideration of plate and/or intramedullary rod selection and application should be given to avoid a plate strain environment that exceeds the yield point of the plate.

Exome sequencing-driven discovery of coding polymorphisms associated with common metabolic phenotypes.

AIMS/HYPOTHESIS: Human complex metabolic traits are in part regulated by genetic determinants. Here we applied exome sequencing to identify novel associations of coding polymorphisms at minor allele frequencies (MAFs) >1% with common metabolic phenotypes. METHODS: The study comprised three stages. We performed medium-depth (8×) whole exome sequencing in 1,000 cases with type 2 diabetes, BMI >27.5 kg/m(2) and hypertension and in 1,000 controls (stage 1). We selected 16,192 polymorphisms nominally associated (p < 0.05) with case-control status, from four selected annotation categories or from loci reported to associate with metabolic traits. These variants were genotyped in 15,989 Danes to search for association with 12 metabolic phenotypes (stage 2). In stage 3, polymorphisms showing potential associations were genotyped in a further 63,896 Europeans. RESULTS: Exome sequencing identified 70,182 polymorphisms with MAF >1%. In stage 2 we identified 51 potential associations with one or more of eight metabolic phenotypes covered by 45 unique polymorphisms. In meta-analyses of stage 2 and stage 3 results, we demonstrated robust associations for coding polymorphisms in CD300LG (fasting HDL-cholesterol: MAF 3.5%, p = 8.5 × 10(-14)), COBLL1 (type 2 diabetes: MAF 12.5%, OR 0.88, p = 1.2 × 10(-11)) and MACF1 (type 2 diabetes: MAF 23.4%, OR 1.10, p = 8.2 × 10(-10)). CONCLUSIONS/INTERPRETATION: We applied exome sequencing as a basis for finding genetic determinants of metabolic traits and show the existence of low-frequency and common coding polymorphisms with impact on common metabolic traits. Based on our study, coding polymorphisms with MAF above 1% do not seem to have particularly high effect sizes on the measured metabolic traits.

A role for coding functional variants in HNF4A in type 2 diabetes susceptibility.

AIMS/HYPOTHESIS: Rare mutations in the gene HNF4A, encoding the transcription factor hepatocyte nuclear factor 4α (HNF-4A), account for ~5% of cases of MODY and more frequent variants in this gene may be involved in multifactorial forms of diabetes. Two low-frequency, non-synonymous variants in HNF4A (V255M, minor allele frequency [MAF] ~0.1%; T130I, MAF ~3.0%)-known to influence downstream HNF-4A target gene expression-are of interest, but previous type 2 diabetes association reports were inconclusive. We aimed to evaluate the contribution of these variants to type 2 diabetes susceptibility through large-scale association analysis. METHODS: We genotyped both variants in at least 5,745 cases and 14,756 population controls from the UK and Denmark. We also undertook an expanded association analysis that included previously reported and novel genotype data obtained in Danish, Finnish, Canadian and Swedish samples. A meta-analysis incorporating all published association studies of the T130I variant was subsequently carried out in a maximum sample size of 14,279 cases and 26,835 controls. RESULTS: We found no association between V255M and type 2 diabetes in either the initial (p = 0.28) or the expanded analysis (p = 0.44). However, T130I demonstrated a modest association with type 2 diabetes in the UK and Danish samples (additive per allele OR 1.17 [95% CI 1.08-1.28]; p = 1.5 × 10⁻4), which was strengthened in the meta-analysis (OR 1.20 [95% CI 1.10-1.30]; p = 2.1 × 10⁻5). CONCLUSIONS/INTERPRETATION: Our data are consistent with T130I as a low-frequency variant influencing type 2 diabetes risk, but are not conclusive when judged against stringent standards for genome-wide significance. This study exemplifies the difficulties encountered in association testing of low-frequency variants.

High-density SNP association study and copy number variation analysis of the AUTS1 and AUTS5 loci implicate the IMMP2L-DOCK4 gene region in autism susceptibility.

Autism spectrum disorders are a group of highly heritable neurodevelopmental disorders with a complex genetic etiology. The International Molecular Genetic Study of Autism Consortium previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we performed a high-density association analysis in AUTS1 and AUTS5, testing more than 3000 single nucleotide polymorphisms (SNPs) in all known genes in each region, as well as SNPs in non-genic highly conserved sequences. SNP genotype data were also used to investigate copy number variation within these regions. The study sample consisted of 127 and 126 families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Further investigation of the strongest association results was conducted in an independent European family sample containing 390 affected individuals. Association and copy number variant analysis highlighted several genes that warrant further investigation, including IMMP2L and DOCK4 on chromosome 7. Evidence for the involvement of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family.

A note on the power to detect transmission distortion in parent-child trios via the transmission disequilibrium test.

Transmission distortion refers to deviation from the normal 50:50 transmission of alleles from parents to offspring. Identification of genomic regions which undergo distortion is necessary for the correct interpretation of linkage and association studies, since tests of linkage using affected relative pairs and family based tests of association will yield spurious results in the presence of transmission distortion. With the increasing availability of genome-wide high density SNP data (e.g. from the International HapMap project), identification of these loci is now a real possibility. Here we present an analytical formula which demonstrates that the power to detect transmission distortion is a simple function of the number of heterozygous parents in the sample and the level of distortion at the locus. Our results indicate that whilst it will be possible to identify loci undergoing major levels of distortion using tens or hundreds of trios, large sample sizes in the order of tens of thousands of trios will be necessary to detect minor levels of distortion with appreciable power. The corollary is that genome-wide searches are unlikely to identify loci where the level of distortion is small, although they may serve to identify interesting regions worthy of follow up.

Rotavirus NSP4 induces a novel vesicular compartment regulated by calcium and associated with viroplasms.

Rotavirus is a major cause of infantile viral gastroenteritis. Rotavirus nonstructural protein 4 (NSP4) has pleiotropic properties and functions in viral morphogenesis as well as pathogenesis. Recent reports show that the inhibition of NSP4 expression by small interfering RNAs leads to alteration of the production and distribution of other viral proteins and mRNA synthesis, suggesting that NSP4 also affects virus replication by unknown mechanisms. This report describes studies aimed at correlating the localization of intracellular NSP4 in cells with its functions. To be able to follow the localization of NSP4, we fused the C terminus of full-length NSP4 with the enhanced green fluorescent protein (EGFP) and expressed this fusion protein inducibly in a HEK 293-based cell line to avoid possible cytotoxicity. NSP4-EGFP was initially localized in the endoplasmic reticulum (ER) as documented by Endo H-sensitive glycosylation and colocalization with ER marker proteins. Only a small fraction of NSP4-EGFP colocalized with the ER-Golgi intermediate compartment (ERGIC) marker ERGIC-53. NSP4-EGFP did not enter the Golgi apparatus, in agreement with the Endo H sensitivity and a previous report that secretion of an NSP4 cleavage product generated in rotavirus-infected cells is not inhibited by brefeldin A. A significant population of expressed NSP4-EGFP was distributed in novel vesicular structures throughout the cytoplasm, not colocalizing with ER, ERGIC, Golgi, endosomal, or lysosomal markers, thus diverging from known biosynthetic pathways. The appearance of vesicular NSP4-EGFP was dependent on intracellular calcium levels, and vesicular NSP4-EGFP colocalized with the autophagosomal marker LC3. In rotavirus-infected cells, NSP4 colocalized with LC3 in cap-like structures associated with viroplasms, the site of nascent viral RNA replication, suggesting a possible new mechanism for the involvement of NSP4 in virus replication.

Little loss of information due to unknown phase for fine-scale linkage-disequilibrium mapping with single-nucleotide-polymorphism genotype data.

We present the results of a simulation study that indicate that true haplotypes at multiple, tightly linked loci often provide little extra information for linkage-disequilibrium fine mapping, compared with the information provided by corresponding genotypes, provided that an appropriate statistical analysis method is used. In contrast, a two-stage approach to analyzing genotype data, in which haplotypes are inferred and then analyzed as if they were true haplotypes, can lead to a substantial loss of information. The study uses our COLDMAP software for fine mapping, which implements a Markov chain-Monte Carlo algorithm that is based on the shattered coalescent model of genetic heterogeneity at a disease locus. We applied COLDMAP to 100 replicate data sets simulated under each of 18 disease models. Each data set consists of haplotype pairs (diplotypes) for 20 SNPs typed at equal 50-kb intervals in a 950-kb candidate region that includes a single disease locus located at random. The data sets were analyzed in three formats: (1). as true haplotypes; (2). as haplotypes inferred from genotypes using an expectation-maximization algorithm; and (3). as unphased genotypes. On average, true haplotypes gave a 6% gain in efficiency compared with the unphased genotypes, whereas inferring haplotypes from genotypes led to a 20% loss of efficiency, where efficiency is defined in terms of root mean integrated square error of the location of the disease locus. Furthermore, treating inferred haplotypes as if they were true haplotypes leads to considerable overconfidence in estimates, with nominal 50% credibility intervals achieving, on average, only 19% coverage. We conclude that (1). given appropriate statistical analyses, the costs of directly measuring haplotypes will rarely be justified by a gain in the efficiency of fine mapping and that (2). a two-stage approach of inferring haplotypes followed by a haplotype-based analysis can be very inefficient for fine mapping, compared with an analysis based directly on the genotypes.

DNA variation in a 13-Mb region including the F9 gene: inferring the genealogical history and causal role of a hemophilia B mutation (IVS 5+13 A-->G).

About 5.5% of all UK hemophilia B patients have the base substitution IVS 5+13 A-->G as the only change in their factor (F)IX gene (F9). This generates a novel donor splice site which fits the consensus better than the normal intron 5 donor splice. Use of the novel splice site should result in a missense mutation followed by the abnormal addition of four amino acids to the patients' FIX. In order to explain the prevalence of this mutation, its genealogical history is examined. Analysis of restriction fragment length polymorphism in the 21 reference UK individuals (from different families) with the above mutation showed identical haplotypes in 19 while two differed from the rest and from each other. In order to investigate the history of the mutation and to verify that it had occurred independently more than once, the sequence variation in 1.5-kb segments scattered over a 13-Mb region including F9 was examined in 18 patients and 15 controls. This variation was then analyzed with a recently developed Bayesian approach that reconstructs the genealogy of the gene investigated while providing evidence of independent mutations that contribute disconnected branches to the genealogical tree. The method also provides minimum estimates of the age of the mutation inherited by the members of coherent trees. This revealed that 17 or 18 mutant genes descend from a founder who probably lived 450 years ago, while one patient carries an independent mutation. The independent recurrence of the IVS5+13 A-->G mutation strongly supports the conclusion that it is the cause of these patients' mild hemophilia.

Cytoplasmic calcium measurement in rotavirus enterotoxin-enhanced green fluorescent protein (NSP4-EGFP) expressing cells loaded with Fura-2.

The green fluorescent protein (GFP) and its analogs are standard markers of protein expression and intracellular localization of proteins. The fluorescent properties of GFP complicate accurate measurement of intracellular calcium using calcium sensitive fluorophores, which show a great degree of spectral overlap with GFP, or their K(d) values are too high for accurate measurement of subtle changes in cytoplasmic calcium concentrations. Here we describe a simple modification of the standard microscope-based Fura-2 calcium-imaging technique which permits the quantitative measurement of intracellular calcium levels in cells expressing enhanced green fluorescent protein (EGFP) fusion proteins. Longpass emission filtering of the Fura-2 signal in cells expressing an EGFP fusion protein is sufficient to eliminate the EGFP-Fura-2 emission spectra overlap and allows quantitative calibration of intracellular calcium. To validate this technique, we investigated the ability of rotavirus enterotoxin NSP4-EGFP to elevate intracellular calcium levels in mammalian HEK 293 cells. We show here that inducible intracellular expression of NSP4-EGFP fusion protein elevates basal intracellular calcium more than two-fold by a phospholipase C (PLC) independent mechanism.

Chromosome-wide distribution of haplotype blocks and the role of recombination hot spots.

Recent studies of human populations suggest that the genome consists of chromosome segments that are ancestrally conserved ('haplotype blocks'; refs. 1-3) and have discrete boundaries defined by recombination hot spots. Using publicly available genetic markers, we have constructed a first-generation haplotype map of chromosome 19. As expected for this marker density, approximately one-third of the chromosome is encompassed within haplotype blocks. Evolutionary modeling of the data indicates that recombination hot spots are not required to explain most of the observed blocks, providing that marker ascertainment and the observed marker spacing are considered. In contrast, several long blocks are inconsistent with our evolutionary models, and different mechanisms could explain their origins.

Dietary pectin and calcium inhibit colonic proliferation in vivo by differing mechanisms.

Diet plays an important role in promoting and/or preventing colon cancer; however, the effects of specific nutrients remain uncertain because of the difficulties in correlating epidemiological and basic observations. Transmissible murine colonic hyperplasia (TMCH) induced by Citrobacter rodentium, causes significant hyperproliferation and hyperplasia in the mouse distal colon and increases the risk of subsequent neoplasia. We have recently shown that TMCH is associated with an increased abundance of cellular beta-catenin and its nuclear translocation coupled with up-regulation of its downstream targets, c-myc and cyclin D1. In this study, we examined the effects of two putatively protective nutrients, calcium and soluble fibre pectin, on molecular events linked to proliferation in the colonic epithelium during TMCH. Dietary intervention incorporating changes in calcium [high (1.0%) and low (0.1%)] and alterations in fibre content (6% pectin and fibre-free) were compared with the standard AIN-93 diet (0.5% calcium, 5% cellulose), followed by histomorphometry and immunochemical assessment of potential oncogenes. Dietary interventions did not alter the time course of Citrobacter infection. Both 1.0% calcium and 6% pectin diet inhibited increases in proliferation and crypt length typically seen in TMCH. Neither the low calcium nor fibre-free diets had significant effect. Pectin diet blocked increases in cellular beta-catenin, cyclin D1 and c-myc levels associated with TMCH by 70%, whereas neither high nor low calcium diet had significant effect on these molecules. Diets supplemented with either calcium or pectin therefore, exert anti-proliferative effects in mouse distal colon involving different molecular pathways. TMCH is thus a diet-sensitive model for examining the effect of specific nutrients on molecular characteristics of the pre-neoplastic colonic epithelium.

Unexpected benefit of sorbitol placebo in Mg intervention study of premenstrual symptoms: implications for choice of placebo in RCTs.

We carried out a randomized, double-blind, crossover study of 85 women, designed to investigate the dose-response of daily Mg supplementation on premenstrual symptoms. Each woman took one of four treatments: Mg (200, 350 or 500 mg/day) or sorbitol (placebo) for 2 months. This was followed by a washout of 1 month, and then each woman received one of the three remaining treatments for a further 2 months. Unexpectedly, sorbitol (1305 mg) reduced anxiety-related and total premenstrual symptoms after 2 months compared with Mg treatments (P<0.001 and P<0.001, respectively). We conclude that low-dose sorbitol reduces premenstrual symptoms beyond that expected of a placebo. After 2 months of treatment, sorbitol also reduced urinary Mg excretion compared to baseline (no intervention) and Mg treatments (P=0.005). A follow-up study on 17 healthy volunteers confirmed lack of effect on urinary Mg output of a similar sorbitol intervention regime compared with either baseline or cellulose placebo. It appears that sorbitol may influence Mg homeostasis in women suffering premenstrual symptoms, but not in healthy individuals. Implications for placebo choice in RCTs are discussed.

Fine-scale mapping of disease loci via shattered coalescent modeling of genealogies.

We present a Bayesian, Markov-chain Monte Carlo method for fine-scale linkage-disequilibrium gene mapping using high-density marker maps. The method explicitly models the genealogy underlying a sample of case chromosomes in the vicinity of a putative disease locus, in contrast with the assumption of a star-shaped tree made by many existing multipoint methods. Within this modeling framework, we can allow for missing marker information and for uncertainty about the true underlying genealogy and the makeup of ancestral marker haplotypes. A crucial advantage of our method is the incorporation of the shattered coalescent model for genealogies, allowing for multiple founding mutations at the disease locus and for sporadic cases of disease. Output from the method includes approximate posterior distributions of the location of the disease locus and population-marker haplotype proportions. In addition, output from the algorithm is used to construct a cladogram to represent genetic heterogeneity at the disease locus, highlighting clusters of case chromosomes sharing the same mutation. We present detailed simulations to provide evidence of improvements over existing methodology. Furthermore, inferences about the location of the disease locus are shown to remain robust to modeling assumptions.

Microbes and microbial toxins: paradigms for microbial-mucosal interactions. VIII. Pathological consequences of rotavirus infection and its enterotoxin.

Rotaviral infection in neonatal animals and young children leads to acute self-limiting diarrhea, but infected adults are mainly asymptomatic. Recently, significant in-roads have been made into our understanding of this disease: both viral infection and virally manufactured nonstructural protein (NSP)4 evoke intracellular Ca(2+) ([Ca(2+)]i) mobilization in native and transformed gastrointestinal epithelial cells. In neonatal mouse pup mucosa models, [Ca(2+)]i elevation leads to age-dependent halide ion movement across the plasma membrane, transepithelial Cl(-) secretion, and, unlike many microbial enterotoxins, initial cyclic nucleotide independence to secretory diarrhea. Similarities between rotavirus infection and NSP4 function suggest that NSP4 is responsible for these enterotoxigenic effects. NSP4-mediated [Ca(2+)]i mobilization may further facilitate diarrhea by signaling through other Ca(2+)-sensitive cellular processes (cation channels, ion and solute transporters) to potentiate fluid secretion while curtailing fluid absorption. Apart from these direct actions in the mucosa at the onset of diarrhea, innate host-mediated defense mechanisms, triggered by either or both viral replication and NSP4-induced [Ca (2+)]i mobilization, sustain the diarrheal response. This secondary component appears to involve the enteric nervous system and may be cyclic nucleotide dependent. Both phases of diarrhea occur in the absence of significant inflammation. Thus age-dependent rotaviral disease represents an excellent experimental paradigm for understanding a noninflammatory diarrhea.

Increased beta-catenin expression and nuclear translocation accompany cellular hyperproliferation in vivo.

Beta-catenin performs critical roles in development and cellular adhesion. More recently, an oncogenic role has been described. In colon cancer, decreased E-cadherin/beta-catenin association is causally linked to increased beta-catenin-regulated gene expression and increased cellular division. Whether the same pathway is active in native epithelia remains unknown. To address this question, we used the transmissible murine colonic hyperplasia model to measure changes in beta-catenin abundance, nuclear partitioning, target gene (c-myc and cyclin D1) expression, and subcellular distribution. Colonocyte hyperproliferation was associated with a 4.3 +/- 0.56 (SD)-fold increase in total cellular beta-catenin protein content, whereas modest changes in gamma-catenin and E-cadherin expression were recorded. The beta-catenin signal increased before changes in mucosal crypt length, a gross index of cellular proliferation/apoptosis. Beta-catenin detected in Triton X-100-soluble (cytosolic) cellular fractions was enriched 4.3 +/- 0.9 (SD)-fold, whereas a modest decrease of 0.9 +/- 0.09 (SD)-fold was recorded in Triton X-100-insoluble (cytoskeletal) fractions. After these changes, nuclear beta-catenin partitioning increased 2.4 +/- 0.4 (SD)-fold, accompanied by 2.5 +/- 0.4- and 4.0 +/- 0.8-fold (SD) increases in cellular c-myc and cyclin D1 levels, respectively. Thus, increased cellular cytosolic and nuclear beta-catenin levels were associated with increased beta-catenin target protein expression. Significant alterations in beta-catenin subcellular distribution were also recorded immunohistochemically. Apical/lateral junctional labeling was observed in normal crypts with increased lateral membrane staining within the upper regions. During transmissible murine colonic hyperplasia, these gradients were dissipated, and basilar plaques were formed within a subset of basal crypt cells. These findings predict that an oncogenic signaling mechanism related to non-E-cadherin-bound beta-catenin is active in hyperproliferating native colonocytes and is similar to that recorded during the early stages of colon carcinogenesis.

Family-based tests of association and/or linkage.

The large literature on family-based tests of association and/or linkage is reviewed, concentrating on the underlying principles and on recent methodological developments. We explain the distinction between testing for association and testing for linkage, and give our views on the circumstances in which each is the appropriate null hypothesis.

A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.

Previous studies have shown that the nonstructural glycoprotein NSP4 plays a role in rotavirus pathogenesis by functioning as an enterotoxin. One prediction of the mechanism of action of this enterotoxin was that it is secreted from virus-infected cells. In this study, the media of cultured (i) insect cells infected with a recombinant baculovirus expressing NSP4, (ii) monkey kidney (MA104) cells infected with the simian (SA11) or porcine attenuated (OSU-a) rotavirus, and (iii) human intestinal (HT29) cells infected with SA11 were examined to determine if NSP4 was detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western blotting, immunoprecipitation and N-terminal amino acid sequencing identified, in the early media from virus-infected cells, a secreted, cleavage product of NSP4 with an apparent molecular weight of 7,000 that represented amino acids 112 to 175 (NSP4 aa112-175). The secretion of NSP4 aa112-175 was not affected by treatment of cells with brefeldin A but was abolished by treatment with nocodazole and cytochalasin D, indicating that secretion of this protein occurs via a nonclassical, Golgi apparatus-independent mechanism that utilizes the microtubule and actin microfilament network. A partial gene fragment coding for NSP4 aa112-175 was cloned and expressed using the baculovirus-insect cell system. Purified NSP4 aa112-175 increased intracellular calcium mobilization in intestinal cells when added exogenously, and in insect cells when expressed endogenously, similarly to full-length NSP4. NSP4 aa112-175 caused diarrhea in neonatal mice, as did full-length NSP4. These results indicate that NSP4 aa112-175 is a functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.