African swine fever (ASF), an acute, viral hemorrhagic disease in domestic swine with mortality rates approaching 100%, is arguably the most significant emerging disease threat for the swine industry worldwide. Devastating ASF outbreaks and continuing epidemic in the Caucasus region and Russia (2007-to date) highlight significance of this disease threat. There is no vaccine for ASF, thus leaving animal slaughter the only effective disease control option. It is clear, however, that vaccination is possible since protection against reinfection with the homologous strain of African swine fever virus (ASFV) has been clearly demonstrated. Vaccine development has been hindered by large gaps in knowledge concerning ASFV infection and immunity, the extent of ASFV strain variation in nature and the identification of viral proteins (protective antigens) responsible for inducing protective immune responses in the pig. This review focuses on the challenges surrounding ASF vaccine design and development, with an emphasis on existing knowledge gaps.

Age-appropriate complementary feeding practices are far from optimal among low- and middle-income countries with available data. The evidence on the association between feeding practices and linear growth is mixed. We sought to systematically examine the association between two indictors of dietary quality-dietary diversity and animal source food (ASF) consumption (WHO, 2008)-and stunting (length-for-age z-score) employing existing data from 39 Demographic and Health Surveys. Data on 74,548 children aged 6-23 months were pooled and multiple logistic regression models, adjusting for child, maternal, and household characteristics, employed to assess the association between dietary quality and stunting. Stratified models by child age and by World Bank country-income classifications (World Bank, 2015) were also applied. Children aged 6-23 months consuming zero food groups in the previous day had a 1.345 higher odds of being stunted when compared to the reference group (≥5 food groups); those who did not consume any ASF in the previous day had a 1.436 higher odds of being stunted compared to children consuming all three types of ASF (egg, meat, and dairy). We estimated that 2,629 cases of stunting would have been averted (12.6% of those stunted) among the population studied if all children had consumed five or more food groups. Outcomes by country-income groupings showed larger associations of diet diversity and ASF consumption for upper- and lower-middle income countries compared to low-income countries. In summary, dietary diversity and ASF consumption were associated with stunting, with associations varying by stratified groups.

The gastrointestinal microbiota influences immune function throughout the body. The gut-lung axis refers to the concept that alterations of gut commensal microorganisms can have a distant effect on immune function in the lung. Overgrowth of intestinal Candida albicans has been previously observed to exacerbate allergic airways disease in mice, but whether subtler changes in intestinal fungal microbiota can affect allergic airways disease is less clear. In this study we have investigated the effects of the population expansion of commensal fungus Wallemia mellicola without overgrowth of the total fungal community. Wallemia spp. are commonly found as a minor component of the commensal gastrointestinal mycobiota in both humans and mice. Mice with an unaltered gut microbiota community resist population expansion when gavaged with W. mellicola; however, transient antibiotic depletion of gut microbiota creates a window of opportunity for expansion of W. mellicola following delivery of live spores to the gastrointestinal tract. This phenomenon is not universal as other commensal fungi (Aspergillus amstelodami, Epicoccum nigrum) do not expand when delivered to mice with antibiotic-depleted microbiota. Mice with Wallemia-expanded gut mycobiota experienced altered pulmonary immune responses to inhaled aeroallergens. Specifically, after induction of allergic airways disease with intratracheal house dust mite (HDM) antigen, mice demonstrated enhanced eosinophilic airway infiltration, airway hyperresponsiveness (AHR) to methacholine challenge, goblet cell hyperplasia, elevated bronchoalveolar lavage IL-5, and enhanced serum HDM IgG1. This phenomenon occurred with no detectable Wallemia in the lung. Targeted amplicon sequencing analysis of the gastrointestinal mycobiota revealed that expansion of W. mellicola in the gut was associated with additional alterations of bacterial and fungal commensal communities. We therefore colonized fungus-free Altered Schaedler Flora (ASF) mice with W. mellicola. ASF mice colonized with W. mellicola experienced enhanced severity of allergic airways disease compared to fungus-free control ASF mice without changes in bacterial community composition.

Because of the clinical and pathologic similarity to common endemic diseases, introduction of CSFV or ASFV strains of moderate to low virulence represents the greatest risk to North American swine herds. Producers, veterinarians, and diagnosticians should increase their awareness of these devastating diseases and request specific diagnostic testing whenever they are suspected. Production practices that improve biosecurity will reduce the risk of introduction of CSF and ASF and limit the spread if an incursion occurs. Additional resources. The following Web sites contain excellent color photographs that will assist producers and practitioners in identifying clinical signs and gross lesions associated with CSFV and ASFV: http://www.vet.uga.edu/vpp/gray_book/FAD and http://www.pighealth.com. The latter Web site and the OIE Web site (http://www.oie.int) offer updated information on current worldwide epizootics of ASF and CSF and other swine diseases. Details of biosecurity procedures can be found at http://www.agebb.missouri.edu; see publication G2340.

African swine fever (ASF) is a highly lethal hemorrhagic disease of swine caused by African swine fever virus (ASFV), a unique and genetically complex virus. The disease continues to be a huge burden to the pig industry in Africa, Europe and recently in Asia, especially China. The purpose of this review is to recapitulate the current scenarios and evolving trends in ASF vaccine development. The unavailability of an applicable ASF vaccine is partly due to the complex nature of the virus, which encodes various proteins associated with immune evasion. Moreover, the incomplete understanding of immune protection determinants of ASFV hampers the rational vaccine design. Developing an effective ASF vaccine continues to be a challenging task due to many undefined features of ASFV immunobiology. Recent attempts on DNA and live attenuated ASF vaccines have been reported with promising efficacy, and especially live attenuated vaccines have been proved to provide complete homologous protection. Single-cycle viral vaccines have been developed for various diseases such as Rift Valley fever, bluetongue and the rational extension of these strategies could be helpful for developing single-cycle ASF vaccines. Therefore, live attenuated vaccines in short-term and single-cycle vaccines in long-term would be the next-generation of ASF vaccines.

African swine fever virus (ASFV) is the sole member of the family Asfarviridae, and the only known DNA arbovirus. Since its identification in Kenya in 1921, ASFV has remained endemic in Africa, maintained in a sylvatic cycle between Ornithodoros soft ticks and warthogs (Phacochoerus africanus) which do not develop clinical disease with ASFV infection. However, ASFV causes a devastating and economically significant disease of domestic (Sus scrofa domesticus) and feral (Sus scrofa ferus) swine. There is no ASFV vaccine available, and current control measures consist of strict animal quarantine and culling procedures. The virus is highly stable and easily spreads by infected swine, contaminated pork products and fomites, or via transmission by the Ornithodoros vector. Competent Ornithodoros argasid soft tick vectors are known to exist not only in Africa, but also in parts of Europe and the Americas. Once ASFV is established in the argasid soft tick vector, eradication can be difficult due to the long lifespan of Ornithodoros ticks and their proclivity to inhabit the burrows of warthogs or pens and shelters of domestic pigs. Establishment of endemic ASFV infections in wild boar populations further complicates the control of ASF. Between the late 1950s and early 1980s, ASFV emerged in Europe, Russia and South America, but was mostly eradicated by the mid-1990s. In 2007, a highly virulent genotype II ASFV strain emerged in the Caucasus region and subsequently spread into the Russian Federation and Europe, where it has continued to circulate and spread. Most recently, ASFV emerged in China and has now spread to several neighboring countries in Southeast Asia. The high morbidity and mortality associated with ASFV, the lack of an efficacious vaccine, and the complex makeup of the ASFV virion and genome as well as its lifecycle, make this pathogen a serious threat to the global swine industry and national economies. Topics covered by this review include factors important for ASFV infection, replication, maintenance, and transmission, with attention to the role of the argasid tick vector and the sylvatic transmission cycle, current and future control strategies for ASF, and knowledge gaps regarding the virus itself, its vector and host species.

Keywords: African swine fever virus; DNA arbovirus; domestic swine; soft tick; transmission; virus replication; wild boar.

African swine fever (ASF) is a viral hemorrhagic disease with exceptionally high lethality in domestic pigs and Eurasian wild boar. Over the last decade, ASF has emerged in several European and Asian countries and has now an unprecedented distribution. Against this background, the presented review focuses on current knowledge and advances in ASF virology, clinical disease upon infection with recent strains, epidemiology, diagnosis, and control. This review highlights knowledge gaps and controversial opinions related to ASF.

Keywords: African swine fever; Clinics; Control; Domestic pigs; Epidemiology; Vaccine; Wild boar.

Three discrete regions of the African swine fever virus (ASFV) were analysed in the genomes of a wide range of isolates collected from wild and domestic pigs in Sardinia, over a 31-year period (1978-2009). The analysis was conducted by genotyping based on sequence data from three single copy ASF genes. The E183L gene encoding the structural protein p54 and part of the gene encoding the p72 protein were used to delineate genotypes, before intra-genotypic resolution of viral relationships by analysis of tetramer amino acid repeats within the hypervariable central variable region (CVR) of the B602L gene. The data revealed that these isolates did not show significant variation in their p72 and p54 sequence when compared between different isolates showing a remarkable genetic stability of these genome regions. In particular, the phylogeny revealed that all the Sardinian isolates belong to the same largest and most homogeneous p72 genotype I together with viruses from Europe, South America, the Caribbean and West Africa, and p54 genotype Ia which comprises viruses from Europe and America. The analysis of B602L gene revealed a minor difference in the number of tetramer repeats, placing the Sardinian isolates into two clusters, accordingly to their temporal distribution, namely sub-group III and sub-group X, this latter showing a deletion of 12 tetramer repeats located in the centre of the array. The genetic variation of this fragment suggests that one sub-group could be derived from the other supporting the hypothesis of a single introduction of ASFV in Sardinia.

Proteolipid protein (PLP) and DM20 are generated by alternative splicing of exon 3B of PLP1 transcript in differentiating oligodendrocytes. We investigated the role of exonic splicing enhancers (ESE) in the selection of PLP 5' donor site, focusing on putative ASF/SF2, and SC35 binding motifs in exon 3B on the basis of mutations that cause disease in humans. Mutations in a putative ASF/SF2 binding motif (nucleotides 406-412) reduced PLP 5' donor site selection, whereas a mutation in a putative SC35 binding motif (nucleotides 382-389) had no effect. UV crosslinking and immunoprecipitation (IP) assays using an antibody to ASF/SF2 showed that the ASF/SF2 protein specifically binds to the ESE (nucleotides 406-412). The single nucleotide mutations that reduced PLP splice site selection greatly diminished ASF/SF2 protein binding to this motif. We next tested the effect of overexpressed ASF/SF2 on PLP 5'splice selection in differentiating oligodendrocytes. ASF/SF2 positively regulates PLP splice site selection in a concentration-dependent manner. Disruption of the putative ASF/SF2 binding site in exon 3B reduced the positive effect of ASF/SF2 on PLP splicing. We conclude that an ESE in exon3B regulates PLP 5' donor site selection and that ASF/SF2 protein participates in the regulation of PLP alternative splicing in oligodendrocytes.

Owing to frequent reports of suspected outbreaks and the presence of reservoir hosts and vectors (warthogs, bushpigs and O. moubata ticks), African swine fever (ASF) is believed to be an endemic disease in Uganda. There have, however, been very few studies carried out to confirm its existence in Uganda. This study was carried out to describe the prevalence of ASF based on pathologic lesions and analysis of serum samples from slaughtered pigs during a suspected outbreak in the Mubende district of Uganda. The study was based on visits to 22 slaughterhouses where individual pigs were randomly selected for a detailed ante-mortem and post-mortem inspections. Sera were also collected for laboratory analysis. A total of 997 pigs (53.7% male and 46.3% female) were examined for lesions suggestive of ASF and sero-positivity of sera for ASF antibodies. The sera were tested using enzyme-linked immunosorbent assay (ELISA) and positive samples were further confirmed with an immunoblot assay. The results showed that 3.8% (38/997) of the pigs examined had clinical signs and post-mortem lesions suggestive of ASF. Two of 997 (0.2%) sera analysed were positive for ASF antibodies. Of the sub-counties investigated, Bagezza (12%) and Kiyuni (11%) had the highest prevalence of lesions suggestive of ASF based on ante- and post-mortem examination results, while Mubende town council (1.7%) had the lowest. This study found a low number of pigs (3.8%) with lesions suggestive of ASF at slaughter and an even lower number of pigs (0.2%) that were seropositive at slaughter, however a significantly higher number of pigs were slaughtered during the outbreak as a strategy for farmers to avoid losses associated with mortality.

African swine fever (ASF) is a notifiable viral pig disease with high mortality and serious socio-economic consequences. Since ASF emerged in Georgia in 2007 the disease has spread to several neighbouring countries and cases have been detected in areas bordering the European Union (EU). It is uncertain how fast the virus would be able to spread within the unrestricted European trading area if it were introduced into the EU. This project therefore aimed to develop a model for the spread of ASF within and between the 27 Member States (MS) of the EU during the high risk period (HRP) and to identify MS during that period would most likely contribute to ASF spread ("super-spreaders") or MS that would most likely receive cases from other MS ("super-receivers"). A stochastic spatio-temporal state-transition model using simulated individual farm records was developed to assess silent ASF virus spread during different predefined HRPs of 10-60 days duration. Infection was seeded into farms of different pig production types in each of the 27 MS. Direct pig-to-pig transmission and indirect transmission routes (pig transport lorries and professional contacts) were considered the main pathways during the early stages of an epidemic. The model was parameterised using data collated from EUROSTAT, TRACES, a questionnaire sent to MS, and the scientific literature. Model outputs showed that virus circulation was generally limited to 1-2 infected premises per outbreak (95% IQR: 1-4; maximum: 10) with large breeder farms as index case resulting in most infected premises. Seven MS caused between-MS spread due to intra-Community trade during the first 10 days after seeding infection. For a HRP of 60 days from virus introduction, movements of infected pigs will originate at least once from 16 MS, with 6 MS spreading ASF in more than 10% of iterations. Two thirds of all intra-Community spread was linked to six trade links only. Denmark, the Netherlands, Lithuania and Latvia were identified as "super-spreaders"; Germany and Poland as "super-receivers". In the sensitivity analysis, the total number of premises per country involved in intra-Community trade was found to be a key determinant for the between-MS spread dynamic and needs to be further investigated. It was concluded that spread during the HRP is likely to be limited, especially if the HRP is short. This emphasises the importance of having good disease awareness in all MS for early disease detection.

As shown in animal studies, 5HT1B/D agonists can inhibit activity in the trigeminal nucleus caudalis, which may be advantageous for their antimigraine effect. To demonstrate a possible central nervous system (CNS) action of these compounds in man we studied their effect on the intensity dependence of the cortical auditory evoked potentials (IDAPs), thought to be inversely related to central serotonergic transmission. An amplitude/stimulus intensity function (ASF) slope was computed in healthy volunteers and migraine patients between attacks before and 2 h after oral 311C90 (zolmitriptan "Zomig") 10 mg (n=14), 311C90 5 mg (n=7), sumatriptan 100 mg (n=14), dexfenfluramine 15 mg (n=4), lorazepam 1.25 mg (n=4) and placebo (n=14). 311C90 10 mg and, to a lesser degree, 5 mg significantly increased the mean ASF slope (p=0.007 and 0.05 vs placebo). There was a significant positive correlation between plasma levels of 311C90 and ASF slope changes. Sumatriptan and lorazepam had little effect, but dexfenfluramine produced a significant ASF slope decrease. 311C90 is able to modify a CNS activity that is modulated by serotonin, i.e. the IDAP. This effect is probably the consequence of its superior lipophilicity compared to sumatriptan and of activation of prejunctional 5HT1B/D autoreceptors, which lowers central serotonin release and thus the preactivation level of sensory cortices.

Following recruitment to solid tissues, peripheral blood monocytes express a number of proinflammatory molecules including TF, a trigger of coagulation that also promotes cell-cell interactions and tissue remodeling. Monocytes express two forms of TF: flTF, a highly coagulant transmembrane form, and asTF, a highly proangiogenic, soluble TF form. Biosynthesis of the two TF forms occurs via alternative processing of exon 5 during pre-mRNA splicing. Its inclusion results in flTF mRNA and its exclusion, asTF mRNA. We developed a splicing reporter system recently and determined that two spliceosomal constituents, SR proteins ASF/SF2 and SRp55, play a pivotal role in exon 5 inclusion. In this report, we show for the first time that two other SR proteins expressed in human monocytes, SRp40 and SC35, antagonize ASF/SF2 and SRp55 by competing for binding to certain sites in exon 5, thereby promoting TF exon 5 exclusion, an event unique to asTF biosynthesis. We also show that the intron preceding TF exon 5 possesses characteristics rarely found in U2 introns. Our findings indicate that modulation of TF pre-mRNA splicing can be accomplished via modification of SR proteins' activity, facilitating development of novel therapeutic strategies to modulate the "TF profile" of monocytes/macrophages.

METHODS

Prospective, single-cohort study.

OBJECTIVE

To determine the immediate change in pulmonary function test (PFT) in children following scoliosis surgery.

BACKGROUND

The number of pediatric scoliosis surgeries is increasing each year because of recent advances in spinal instrumentation, surgical techniques, and improved perioperative monitoring. Pulmonary function decreases immediately following scoliosis surgery, but the extent of this decrease is not well documented in pediatric patients. To use preoperative PFTs to assess the risk of postoperative complications, knowledge of the postoperative decline in PFT is necessary.

METHODS

We measured preoperative and daily postoperative PFT in 24 children who had scoliosis surgery (age 12.7 +/- 2.7 [SD] years) from January 2002 to June 2003. There were 10 male and 14 female patients. Two (8%) patients had congenital scoliosis, 11 (46%) had idiopathic scoliosis, 9 (38%) had scoliosis due to a neuromuscular disease, and 2 (8%) had kyphoscoliosis. Fifteen (62%) patients had posterior spinal fusion (PSF), 5 (21%) had anterior spinal fusion (ASF), and 4 (17%) had both ASF and PSF performed. PFT parameters (forced expiratory volume in 1 second [FEV1], forced vital capacity [FVC], FEV1/FVC, and forced expiratory rate between 25% and 75% of FVC [FEF25%-75%]) were measured before surgery and daily after surgery by bedside spirometry until hospital discharge.

RESULTS

PFT declined up to 60% after surgery. The PFT nadir is at 3 days. PFT values remained significantly decreased at 1 week, with values at about half of preoperative baseline. No patient required postoperative mechanical ventilation>> or =3 days. There was no statistical significance between the degree of decline in PFT with etiology of either the scoliosis or the type of surgery performed.

CONCLUSIONS

Our study found that patients are still at risk for postoperative complications as long as 1 week postoperatively and that PFTs do not return to near baseline until 1 to 2 months after surgery. The postoperative decrease in PFT should be considered during preoperative prediction of postoperative risk.

Cajal bodies (CBs) are subnuclear structures involved in RNA metabolism. Here we show that, following infection of HeLa cells by adenovirus type 5 (Ad5), CBs fragment and form ordered structures, which we have termed "rosettes". Formation of CB rosettes was prevented by inhibition of viral DNA synthesis and preceded expression of the L4-33K protein. CB rosettes localised to the periphery of E2A-72K-containing replication centers and to the edges of ASF/SF2 and hnRNP A1 ring structures that demarcate sites of viral transcription and splicing. At later times of infection, CB rosettes were undetectable. Furthermore, knock-down of p80-coilin (the major structural protein of CBs) by RNA interference reduced the yield of infectious Ad5 and expression of the late proteins IIIa (from L1), hexon (from L3) and fiber (from L5), whereas the E2A-72K protein was unaffected. We conclude that CBs have an important role in the expression of adenovirus major late gene products.

Cap formation is the first step of pre-mRNA processing in eukaryotic cells. Immediately after transcription initiation, capping enzyme (CE) is recruited to RNA polymerase II (Pol II) by the phosphorylated carboxyl-terminal domain of the Pol II largest subunit (CTD), allowing cotranscriptional capping of the nascent pre-mRNA. Recent studies have indicated that CE affects transcription elongation and have suggested a checkpoint model in which cotranscriptional capping is a necessary step for the early phase of transcription. To investigate further the role of the CTD in linking transcription and processing, we generated a fusion protein of the mouse CTD with T7 RNA polymerase (CTD-T7 RNAP). Unexpectedly, in vitro transcription assays with CTD-T7 RNAP showed that CE promotes formation of DNA.RNA hybrids or R loops. Significantly, phosphorylation of the CTD was required for CE-dependent R-loop formation (RLF), consistent with a critical role for the CTD in CE recruitment to the transcription complex. The guanylyltransferase domain was necessary and sufficient for RLF, but catalytic activity was not required. In vitro assays with appropriate synthetic substrates indicate that CE can promote RLF independent of transcription. ASF/SF2, a splicing factor known to prevent RLF, and GTP, which affects CE conformation, antagonized CE-dependent RLF. Our findings suggest that CE can play a direct role in transcription by modulating displacement of nascent RNA during transcription.

Single genes are often subject to alternative splicing, which generates alternative mature mRNAs. This phenomenon is widespread in animals, and observed in over 90% of human genes. Recent data suggest it may also be common in Apicomplexa. These parasites have small genomes, and economy of DNA is evolutionarily favoured in this phylum. We investigated the mechanism of alternative splicing in Toxoplasma gondii, and have identified and localized TgSR3, a homologue of ASF/SF2 (alternative-splicing factor/splicing factor 2, a serine-arginine-rich, or SR protein) to a subnuclear compartment. In addition, we conditionally overexpressed this protein, which was deleterious to growth. qRT-PCR was used to confirm perturbation of splicing in a known alternatively-spliced gene. We performed high-throughput RNA-seq to determine the extent of splicing modulated by this protein. Current RNA-seq algorithms are poorly suited to compact parasite genomes, and hence we complemented existing tools by writing a new program, GeneGuillotine, that addresses this deficiency by segregating overlapping reads into distinct genes. In order to identify the extent of alternative splicing, we released another program, JunctionJuror, that detects changes in intron junctions. Using this program, we identified about 2000 genes that were constitutively alternatively spliced in T. gondii. Overexpressing the splice regulator TgSR3 perturbed alternative splicing in over 1000 genes.

African swine fever (ASF) virus has been reported to infect cells of the monocyte family, probably macrophage-like cells, but there is variation in the apparent susceptibility of these cells. We have demonstrated that the phenotype and activity of porcine monocytic cells varies between different isolations and also upon culture. The variation during culture is dependent upon the phenotype of the cells at the time of isolation. As for the susceptibility of porcine monocytes/macrophages to infection by ASF virus, it was seen that this could be related to the variation in cell phenotype and activity. The susceptibility was determined by the relative density of particular subpopulations of cells present. Whilst inflammatory macrophages did not have an apparent role to play, phagocytic activity was influential. Furthermore, the expression of CD44 and the DH59 myeloid cell marker was important, whereas the relevance of MHC Class II expression was variable. Overall, it was concluded that susceptibility to infection required that a culture be dominated by CD44-positive cells which were non-inflammatory, of low phagocytic activity, and characterizable as being of the myeloid (DH59-positive) lineage.

Digital tomosynthesis is an imaging technique to produce a tomographic image from a series of angular digital images in a manner similar to conventional focal plane tomography. Unlike film focal plane tomography, the acquisition of the data in a C-arm geometry causes the image receptor to be positioned at various angles to the reconstruction tomogram. The digital nature of the data allows for input images to be combined into the desired plane with the flexibility of generating tomograms of many separate planes from a single set of input data. Angular datasets were obtained of a low contrast detectability (LCD) phantom and cadaver breast utilizing a Lorad stereotactic biopsy unit with a coupled source and digital detector in a C-arm configuration. Datasets of 9 and 41 low-dose projections were collected over a 30 degrees angular range. Tomographic images were reconstructed using a Backprojection (BP) algorithm, an Iterative Subtraction (IS) algorithm that allows the partial subtraction of out-of-focus planes, and an Algebraic Reconstruction (AR) algorithm. These were compared with single view digital radiographs. The methods' effectiveness at enhancing visibility of an obscured LCD phantom was quantified in terms of the Signal to Noise Ratio (SNR), and Signal to Background Ratio (SBR), all normalized to the metric value for the single projection image. The methods' effectiveness at removing ghosting artifacts in a cadaver breast was quantified in terms of the Artifact Spread Function (ASF). The technology proved effective at partially removing out of focus structures and enhancing SNR and SBR. The normalized SNR was highest at 4.85 for the obscured LCD phantom, using nine projections and IS algorithm. The normalized SBR was highest at 23.2 for the obscured LCD phantom, using 41 projections and an AR algorithm. The highest normalized metric values occurred with the obscured phantom. This supports the assertion that the greatest value of tomosynthesis is in imaging fibroglandular breasts. The ASF performance was best with the AR technique and nine projections.

The expression of parA, an auxin-regulated gene expressed during the culture of tobacco (Nicotiana tabacum L.) mesophyll protoplasts, is induced by cadmium. To identify the cadmium-responsive element, we examined the parA promoter using the GUS reporter gene. Cadmium responsiveness was retained in a 5' deletion of the parA promoter to -78 bp, but it was nullified by further deletion to -49bp, which implies that the region -49 to -78 bp contained a cadmium-responsive element. This region contains a sequence similar to as-1, an enhancer sequence from the cauliflower mosaic virus 35S RNA promoter that binds the nuclear factor ASF-1. We named the sequence in the parA promoter pas. Gel-shift assays revealed that pas and as-1 compete for the same DNA-binding nuclear protein(s). Since pentamers of either pas and as-1 were able to confer cadmium responsiveness on a minimal promoter but mutant as-1 was not, we propose that pas and as-1 are involved in cadmium-responsive gene expression. Neither pas nor as-1 conferred responsiveness to copper. The specificity of this response, involving the function of as-1-related elements including pas, is discussed.

In March 1978 an outbreak of African swine fever (ASF) occurred in Malta. The disease spread rapidly and by April 13, ASF had been found on 304 premises involving 25,100 pigs. A census carried out on April 15/16 showed that there were at least 1440 premises containing 70,700 pigs on the island. A slaughter policy was implemented and depopulation of known infected premises started on April 15. Pigs which appeared normal on these premises were stored in freezers for subsequent processing for human consumption and by the end of June more than 4500 carcases were in cold store. The most consistent clinical signs were fever, anorexia and reluctance to move. Haemorrhagic lymph nodes and petechial haemorrhages in the kidneys were the predominant macroscopic lesions. A serum survey, using the immunoelectroosmophoresis technique, was carried out on 2409 sera from 200 farms collected at the Government abattoir during a four-week period. Of these sera, 308 (12.8 per cent) from 65 (32.5 per cent) of the farms contained antibodies to ASF virus. By August the original pig population had been reduced to one-third and a second census taken on August 15/16 showed that a total of 501 owners and 13,975 pigs remained. The decision was taken to slaughter all the remaining pigs and by the end of January 1979 there were no pigs in Malta. The outbreak cost an estimated 5 million pounds and provided the first occasion when any country had slaughtered all members of a species of domestic animal in order to eliminate a disease.

The cell nucleus is highly organized with chromosomes occupying discrete, partially overlapping territories, and proteins that localize to specific nuclear compartments. This spatial organization of the nucleus is considered to be dynamic in response to environmental and cellular conditions to support changes in transcriptional programs. Chromatin, however, is relatively immobile when analyzed in living cells and shows a constrained Brownian type of movement. A possible explanation for this relative immobility is that chromatin interacts with a nuclear matrix structure and/or with nuclear compartments. Here, we explore the use of photoactivatable GFP fused to histone H4 as a potential tool to analyze the mobility of chromatin at various nuclear compartments. Selective photoactivation of photoactivatable-GFP at defined nuclear regions was achieved by two-photon excitation with 820 nm light. Nuclear speckles, which are considered storage sites of splicing factors, were visualized by coexpression of a fluorescent protein fused to splicing factor SF2/ASF. The results reveal a constrained chromatin motion, which is not affected by transcriptional inhibition, and suggests an intimate interaction of chromatin with speckles.

We report a new mechanism of aberrant pre-mRNA splicing resulting in constitutive activation of a mis-spliced oncoprotein (KIT) leading to malignancy (gastrointestinal stromal tumor) in contrast to loss of function of mis-spliced proteins resulting in diverse human diseases in the literature. The mechanisms of three consecutive molecular events, deletion of noncoding and coding regions encompassing the 3' authentic splice site, creation of a novel intra-exonic pre-mRNA 3' splice acceptor site leading to in-frame loss of 27 nucleotides (nine amino acids; Lys550-Lys558), and the mechanism of constitutive activation of the mis-spliced KIT are elucidated. Loss of a peptide in a critical location unleashed the protein from autoinhibition (as evidenced by three-dimensional structural analysis), causing KIT to become constitutively activated and resulting in the GIST phenotype. We also demonstrated that only one of the following two exonic splicing enhancers is sufficient for inclusion of the KIT exon 11 in vivo: AACCCATGT (nucleotides 2-10 from the 5' end, which are recognized by SC35, SRp55, and SF2/ASF) or GGTTGTTGAGG (nucleotides 27-37 from the 5' end, which are recognized by SC35 and SRp55), suggestive of exonic enhancer redundancy.

Motivated by strong correlations between plasma levels of triglycerides (TG) and adiposity traits, we conducted a series of bivariate genome-wide linkage analyses of TG with body mass index (BMI), total fat mass (FAT), percentage of body fat (FATPC), and abdominal subcutaneous fat (ASF). Maximum lod scores of 3.3, 3.0, 2.2 and 2.4, respectively, were found on chromosome 19q13. This linkage region includes the APOE gene, a predictor of variation in lipid-lipoprotein levels, and the hormone-sensitive lipase (LIPE) gene, a key enzyme in the mobilization of fatty acids from triglyceride stores. In addition, the adiposity measures together with the APOE marker showed significant association with TG levels (p = 0.02 to p = 0.03). In summary, these results suggest that one or more QTLs in the 19q13 region jointly influence TG levels and adiposity. Polymorphisms in the APOE gene, and possibly LIPE gene, appear to be strong candidates for the source of this pleiotropic QTL.