African swine fever (ASF), have been introduced into the Russian Federation from Transcaucasia countries, has spread widely across the territory of the southern region of Russia since 2008. In this work we present an analysis of the spatial and temporal spread of the disease, determine risk factors by means of GIS tools and model the dynamics of the epidemic process both within infected premises (farms) and at the between-farm level to estimate the basic reproduction ratio R(0). The analysis allowed us to make a conclusion about the anthropogenic nature of the risk factors for disease spread. The major significant risk factors identified were: density of the road network, density of domestic swine population and density of water bodies in the study area. The basic reproduction ratio was estimated to range from 2 to 3 at the between-farm level and from 8 to 11 within the infected farms. These initial studies of the ASF epidemic provide information on which to based control and prevention programs.

Ribonucleases (RNases) have therapeutic potential against cancer and viral diseases and have been reported to inhibit replication of the human immunodeficiency virus type 1 (HIV-1) in chronically infected cell lines. The ribonuclease eosinophil-derived neurotoxin (EDN) is responsible for the anti-HIV-1 activity of a soluble factor produced in response to human alloantigens (ASF). Four recombinant RNases (EDN; a four amino acid extension of the N-terminus EDN, -4EDN; RNase A; and angiogenin) were tested for inhibition of HIV-1 replication in PHA blasts. All RNases showed anti-HIV-1 activity, irrespective of whether the RNases were added before, during, or 2 h after infection. Polyclonal antibodies against the four RNases blocked the antiviral activity. ASF inhibited HIV-1 replication in vitro if added up to 4 h after infection. We demonstrated that allostimulation induced EDN, RNase A, and angiogenin mRNA expression in peripheral blood mononuclear cells (PBMCs), although only EDN protein was detected. We identified monocytes and dendritic cells, but not macrophages or T cells, as EDN-producing cells. These findings raise the possibilities that multiple naturally occurring RNases may contribute to protection against HIV-1 infection and could be considered for utilization in HIV-1 therapy.

African swine fever (ASF) is a viral hemorrhagic disease with different clinical and lesional changes depending of virulence of strains/isolates and immunological status of pigs. In acute and subacute forms of ASF, severe vascular changes are present, with hemorrhages in different organs (mainly melena, epistaxis, erythema, renal petechiaes and diffuse hemorrhages in lymph nodes), pulmonary edema, disseminate intravascular coagulation and thrombocytopenia. Lymphopenia and monocytopenia are developed during acute and subacute ASF. Lymphopenia is associated with lymphoid depletion in primary and secondary lymphoid organs, which is caused by apoptosis. All these lesions are not related to viral replication in endothelial cells or lymphocytes. Monocytes-macrophages show viral replication and cytophatic effect, including hemadsorption. The more significant changes in these cells are increased number and secretory activation (increased levels of proinflammatory cytokines) in targets organs. Proinflammatory activation is the initial cause of clinical and lesional pictures in ASF, including fever and changes in levels of acute phase proteins. Levels of IFN-β and -γ are increased from initial phase of acute ASF. Anti-inflammatory response, represented by increased level of IL-10, is observed also, although in the final phase of acute ASF only.

The cognitive neurosciences combine behavioral experiments with acquiring physiological data from different modalities, such as electroencephalography, magnetoencephalography, transcranial magnetic stimulation, and functional magnetic resonance imaging, all of which require excellent timing. A simple framework is proposed in which uni- and multimodal experiments can be conducted with minimal adjustments when one switches between modalities. The framework allows the beginner to quickly become productive and the expert to be flexible and not constrained by the tool by building on existing software such as MATLAB and the Psychophysics Toolbox, which already are serving a large community. The framework allows running standard experiments but also supports and facilitates exciting new possibilities for real-time neuroimaging and state-dependent stimulation.

There is presently no vaccine to combat African swine fever (ASF), a viral hemorrhagic fever of domestic pigs that causes up to 100% morbidity and mortality in naive, commercial pig populations. In its endemic setting, ASF virus cycles between asymptomatic warthogs and soft ticks, with persistence in exotic locations being ascribed to the almost global distribution of susceptible soft tick and suid hosts. An understanding of the role played by diverse hosts in the epidemiology of this multi-host disease is crucial for effective disease control. Unlike the intensively studied Ornithodoros tick vector, the role of many wild suids remains obscure, despite growing recognition for suid-exclusive virus cycling, without the agency of the argasid tick, at some localities. Because the four wild suid genera, Phacochoerus, Potamochoerus, Hylochoerus, and Sus differ from each other in taxonomy, distribution, ecology, reservoir host potential, virus shedding, ASF symptomology, and domestic-pig contact potential, their role in disease epidemiology is also varied. This first consolidated summary of ASF epidemiology in relation to wild suids summarizes current knowledge and identifies information gaps and future research priorities crucial for formulating effective disease control strategies.

Postoperative C5 palsy is a common complication after cervical spine decompression surgery. However, the incidence, prognosis, and etiology of C5 palsy after anterior decompression with spinal fusion (ASF) have not yet been fully established. In the present study, we analyzed the clinical and radiological characteristics of patients who developed C5 palsy after ASF for cervical degenerative diseases. The cases of 199 consecutive patients who underwent ASF were analyzed to clarify the incidence of postoperative C5 palsy. We also evaluated the onset and prognosis of C5 palsy. The presence of high signal changes (HSCs) in the spinal cord was analyzed using T2-weighted magnetic resonance images. C5 palsy occurred in 17 patients (8.5%), and in 15 of them, the palsy developed after ASF of 3 or more levels. Among ten patients who had a manual muscle test (MMT) grade ≤2 at the onset, five patients showed incomplete or no recovery. Sixteen of the 17 C5 palsy patients presented neck and shoulder pain prior to the onset of muscle weakness. In the ten patients with a MMT grade ≤2 at the onset, nine patients showed HSCs at the C3-C4 and C4-C5 levels. The present findings demonstrate that, in most patients with severe C5 palsy after ASF, pre-existing asymptomatic damage of the anterior horn cells at C3-C4 and C4-C5 levels may participate in the development of motor weakness in combination with the nerve root lesions that occur subsequent to ASF. Thus, when patients with spinal cord lesions at C3-C4 and C4-C5 levels undergo multilevel ASF, we should be alert to the possible occurrence of postoperative C5 palsy.

Phosphorylation of proteins by kinases is the most commonly studied class of posttranslational modification, yet its structural consequences are not well understood. The human SR (serine-arginine) protein ASF/SF2 relies on the processive phosphorylation of the serine residues of eight consecutive arginine-serine (RS) dipeptide repeats at the C terminus by SRPK1 before it can be transported into the nucleus. This SR protein plays critical roles in spliceosome assembly, pre-mRNA splicing, and mRNA export, and the phosphorylation process of the RS repeats has been extensively studied experimentally. However, knowledge of the conformational changes associated with the phosphorylation of this simple sequence and how it triggers the importation of the SR protein is lacking. Here, we have carried out extensive molecular dynamics simulations to show that phosphorylation of the eight RS repeats significantly alters the peptide's conformation and leads to the formation of very stable structures that are likely to be involved in the recognition, binding, and transport of the SR protein. Specifically, we found an unusual symmetry-broken phase of conformations of the repetitive and quasi-symmetric phosphorylated peptide sequence. One of the main characteristics of these conformations is the exposed phosphate groups on the periphery, which possibly could serve as the recognition platform for the transport protein transportin-SR2.

Heat shock induces the transcriptional activation of large heterochromatic regions of the human genome composed of arrays of satellite III DNA repeats. A number of RNA-processing factors, among them splicing factor SF2/ASF, associate with these transcription factors giving rise to nuclear stress bodies (nSBs). Here, we show that the recruitment of SF2/ASF to these structures is mediated by its second RNA recognition motif. Amino acid substitutions in the first alpha-helix of this domain, but not in the beta-strand regions, abrogate the association with nSBs. The same mutations drastically affect the in vivo activity of SF2/ASF in the alternative splicing of adenoviral E1A transcripts. Sequence analysis identifies four putative high-affinity binding sites for SF2/ASF in the transcribed strand of the satellite III DNA. We have verified by gel mobility shift assays that the second RNA-binding domain of SF2/ASF binds at least one of these sites. Our analysis suggests that the recruitment of SF2/ASF to nSBs is mediated by a direct interaction with satellite III transcripts and points to the second RNA-binding domain of the protein as the major determinant of this interaction.

The purpose of this paper is to provide characteristics of the spread of African Swine Fever (ASF) in Poland from February to August, 2014. The samples from dead wild boar and domestic pigs were submitted to the National Veterinary Research Institute, National Reference Laboratory for ASF in Pulawy, Poland, for testing by PCR and ELISA methods. In the studied period, fourteen cases of ASF in wild boar and two outbreaks in backyard pigs were confirmed. In addition to the results of laboratory tests performed in 2014, the article describes the ASF surveillance programme in wild boar and pigs in Poland carried out in 2011-2013. The spread of ASF in Poland is compared with the epidemiological situation in Lithuania, Latvia, Belarus and the Russian Federation.

African swine fever (ASF) is an acute and often fatal disease affecting domestic pigs and wild boar, with severe economic consequences for affected countries. ASF is endemic in sub-Saharan Africa and the island of Sardinia, Italy. Since 2007, the virus emerged in the republic of Georgia, and since then spread throughout the Caucasus region and Russia. Outbreaks have also been reported in Belarus, Ukraine, Lithuania, Latvia, Estonia, Romania, Moldova, Czech Republic, and Poland, threatening neighboring West European countries. The causative agent, the African swine fever virus (ASFV), is a large, enveloped, double-stranded DNA virus that enters the cell by macropinocytosis and a clathrin-dependent mechanism. African Swine Fever Virus is able to interfere with various cellular signaling pathways resulting in immunomodulation, thus making the development of an efficacious vaccine very challenging. Inactivated preparations of African Swine Fever Virus do not confer protection, and the role of antibodies in protection remains unclear. The use of live-attenuated vaccines, although rendering suitable levels of protection, presents difficulties due to safety and side effects in the vaccinated animals. Several African Swine Fever Virus proteins have been reported to induce neutralizing antibodies in immunized pigs, and vaccination strategies based on DNA vaccines and recombinant proteins have also been explored, however, without being very successful. The complexity of the virus particle and the ability of the virus to modulate host immune responses are most likely the reason for this failure. Furthermore, no permanent cell lines able to sustain productive virus infection by both virulent and naturally attenuated African Swine Fever Virus strains exist so far, thus impairing basic research and the commercial production of attenuated vaccine candidates.

Triplication of chromosome 21 in Down syndrome (DS) results in overexpression of the minibrain kinase/dual-specificity tyrosine phosphorylated and regulated kinase 1A gene (DYRK1A). DYRK1A phosphorylates cytoplasmic tau protein and appears in intraneuronal neurofibrillary tangles (NFTs). We have previously shown significantly more DYRK1A-positive NFTs in DS brains than in sporadic Alzheimer disease (AD) brains. This study demonstrates a gene dosage-proportional increase in the level of DYRK1A in DS in the cytoplasm and the cell nucleus, and enhanced cytoplasmic and nuclear immunoreactivity of DYRK1A in DS. The results suggest that overexpressed DYRK1A may alter both phosphorylation of tau and alternative splicing factor (ASF). Two-dimensional electrophoresis revealed modification of ASF phosphorylation in DS/AD and AD in comparison to controls. Altered phosphorylation of ASF by overexpressed nuclear DYRK1A may contribute to the alternative splicing of the tau gene and an increase by 2.68 × of the 3R/4R ratio in DS/AD, and a several-fold increase in the number of 3R tau-positive NFTs in DS/AD subjects compared with that in sporadic AD subjects. These data support the hypothesis that phosphorylation of ASF by overexpressed DYRK1A may contribute to alternative splicing of exon 10, increased expression of 3R tau, and early onset of neurofibrillary degeneration in DS.

Alternative splicing of RNA increases the coding potential of the genome and allows for additional regulatory control over gene expression. The full extent of alternative splicing remains to be defined but is likely to significantly expand the size of the human transcriptome. There are several examples of mammalian viruses regulating viral splicing or inhibiting cellular splicing in order to facilitate viral replication. Here, we describe a viral protein that induces alternative splicing of a cellular RNA transcript. Epstein-Barr virus (EBV) SM protein is a viral protein essential for replication that enhances EBV gene expression by enhancing RNA stability and export. SM also increases cellular STAT1 expression, a central mediator of interferon signal transduction, but disproportionately increases the abundance of the STAT1beta splicing isoform, which can act as a dominant-negative suppressor of STAT1alpha. SM induces splicing of STAT1 at a novel 5' splice site, resulting in a STAT1 mRNA incapable of producing STAT1alpha. SM-induced alternative splicing is dependent on the presence of an RNA sequence to which SM binds directly and which can confer SM-dependent splicing on heterologous RNA. The cellular splicing factor ASF/SF2 also binds to this region and inhibits SM-RNA binding and SM-induced alternative splicing. These results suggest that viruses may regulate cellular gene expression at the level of alternative mRNA splicing in order to facilitate virus replication or persistence in vivo.

The factors involved in regulating alternative splicing of the human adenylyl cyclase stimulatory G-protein G alpha(s) in different cell types remain undefined. We have designed a G alpha(s) minigene that retains the signals required for G alpha(s) alternative splicing in vivo. Employing transient transfection of human myometrial smooth muscle cells and HeLa cells, as well as in vitro splicing assays, we have provided evidence that the antagonistic splicing factors SF2/ASF and hnRNPA1 act as potent regulators of G alpha(s) isoform expression in these cells. Both SF2/ASF and hnRNPA1 control the selection of competing 5'-splice sites and respectively promote inclusion or skipping of the small cassette-type exon 3 of G alpha(s) transcripts, resulting in the generation of G alpha(s)-long and G alpha(s)-short mRNA isoforms. We have also provided evidence that SF2/ASF and hnRNPA1 play a role in 3'-splice site selection involving the use of a non-canonical TG 3'-splice site preceding exon 4. Using a score-matrix analysis to identify putative exonic enhancer sequences (ESEs), we found multiple high score ESE motifs for SF2/ASF, SC35, and SRp40 in exon 3 of G alpha(s). These results suggest that tissue-specific expression of SF2/ASF and hnRNPA1 governs the expression of alternative isoforms of G alpha(s) in these different cells types.

Thymic stromal lymphopoietin (TSLP) is constitutively expressed in the intestine and is known to regulate inflammation in models of colitis. We show that steady-state TSLP expression requires intestinal bacteria and has an important role in limiting the expansion of colonic T helper type 17 (Th17) cells. Inappropriate expansion of the colonic Th17 cells occurred in response to an entirely benign intestinal microbiota, as determined following the colonization of germ-free C57BL/6 or TSLPR(-/-) mice with the altered Schaedler flora (ASF). TSLP-TSLPR (TSLP receptor) interactions also promoted the expansion of colonic Helios(-)Foxp3(+) regulatory T cells, necessary for the control of inappropriate Th17 responses following ASF bacterial colonization. In summary, these data reveal an important role for TSLP-TSLPR signaling in promoting steady-state mutualistic T-cell responses following intestinal bacterial colonization.

BACKGROUND

SR family and SR-related proteins assemble on exonic splicing enhancer (ESE) sequences to promote both constitutive and regulated splicing. The SRm160 splicing coactivator, an SR-related nuclear matrix protein of 160 kDa, is important for the splicing of specific constitutive and ESE-dependent pre-mRNAs.

RESULTS

In the present study, we show that SRm160 is required to promote pre-mRNA splicing mediated by a large population of functional ESE sequences within a randomized 18 nucleotide sequence. This suggests that it functions as a general coactivator by interacting with different SR family/SR-related proteins bound to different ESE sequences. Consistent with this, several SR family and SR-related proteins coimmunoprecipitated specifically with SRm160 in the presence of low salt. We used RNA interference (RNAi) in Caenorhabditis elegans to determine whether interactions between CeSRm160 and different CeSR family proteins are important in a whole-organism context. Previously we showed that RNAi of CeSRm160 and individual CeSR family genes other than CeSF2/ASF results in no obvious phenotype, which is indicative of gene redundancy. In the present study, we demonstrate that RNAi of CeSRm160 in combination with any CeSR family gene results in the production of unfertilized oocytes by the injected mother.

CONCLUSIONS

The observation that simultaneous suppression of CeSRm160 and individual CeSR family proteins results in a distinct phenotype is indicative of critical functional interactions between these factors. Our results provide biochemical and genetic evidence indicating that interactions between SRm160 and multiple SR family proteins are important for both optimal splicing activity and for proper development.

Parasitic nematodes of the genus Strongyloides are remarkable for their ability to switch between alternative free-living developmental pathways in response to changing internal environmental conditions. After exiting the host, soil-dwelling larval stages may develop either to infectivity via 2 microbiverous stages (homogonic development) or to free-living adulthood via 4 microbiverous larval stages (heterogonic development). The progeny of these adults then give rise to the infective stage. In the latter case, free-living existence is extended in time and the number of infective larvae is greatly amplified. Anterior chemosensory neurons (amphidial neurons) are thought to respond to environmental cues and via signal transduction pathways control the direction of larval development. We now demonstrate by laser microbeam ablation that 2 classes of amphidial neurons (ASF and ASI), acting together, control the direction of free-living larval development. Larvae in which the neurons were killed developed to infectivity via the homogonic route rather than to adulthood via the otherwise predominant heterogonic route. These neurons are probable homologues of neurons ADF (=ASF) and ASI in Caenorhabditis elegans, suggesting the control of development at the cellular level is conserved among divergent taxa of nematodes. These observations also have important implications for the evolution of nematode parasitism and the design of new prophylactic measures against parasitic nematodes of medical and veterinary medical importance.

Most studies on the structure of DNA in telomeres have been dedicated to the double-stranded region or the guanosine-rich strand and consequently little is known about the factors that may bind to the telomere cytosine-rich (C-rich) strand. This led us to investigate whether proteins exist that can recognise C-rich sequences. We have isolated several nuclear factors from human cell extracts that specifically bind the C-rich strand of vertebrate telomeres [namely a d(CCCTAA)(n)repeat] with high affinity and bind double-stranded telomeric DNA with a 100xreduced affinity. A biochemical assay allowed us to characterise four proteins of apparent molecular weights 66-64, 45 and 35 kDa, respectively. To identify these polypeptides we screened alambdagt11-based cDNA expression library, obtained from human HeLa cells using a radiolabelled telomeric oligonucleotide as a probe. Two clones were purified and sequenced: the first corresponded to the hnRNP K protein and the second to the ASF/SF2 splicing factor. Confirmation of the screening results was obtained with recombinant proteins, both of which bind to the human telomeric C-rich strand in vitro.

The expression profile of a panel of RNA-binding proteins (heterogeneous ribonucleoprotein (hnRNP) A1, hnRNP C1/C2, hnRNP H, hnRNP I, ASF/SF2, SR proteins, HuR and U2AF(65)) and markers of differentiation, proliferation and neoplasia (cytokeratin (CK) 13, CK-14, proliferating cell nuclear antigen (PCNA), Syndecan-1 and p16INK4a) were analyzed in 50 formalin fixed paraffin embedded cervical tissues using immunohistochemistry. The samples included histologically normal cervical epithelium, human papillomavirus (HPV) induced low-grade and high-grade pre-malignant lesions and cervical cancers. All samples were tested for HPV DNA using nested PCR. Forty-nine of the 50 tissue samples tested positive for HPV, 27 tissue samples (54%) were HPV-16 positive and 4 samples (8%) were HPV-18 positive. The immunohistochemistry results detected different expression levels of the various proteins in basal epithelial cells in histologically normal epithelium followed by an increase in expression in the intermediate layers, whereas the superficial layers remained negative for all tested RNA-binding proteins. Expression of all RNA-binding proteins increased in neoplastic lesions and highest expression was detected in cervical cancers. p16INK4a had a stronger association with high-grade lesions when compared with the RNA-binding proteins. The expression profile of the RNA-binding proteins is similar to PCNA expression in histologically normal epithelium as well as in lesions (low-grade and high-grade) and cervical cancers. As PCNA expression has been suggested to mimic HPV E6/E7 expression in cervical epithelium, the results suggest the RNA-binding protein analyzed here regulate HPV early gene expression directly and late gene expression indirectly.

BACKGROUND

Clear cell renal cell carcinoma (ccRCC) is the most common type of renal cancer. One of the processes disturbed in this cancer type is alternative splicing, although phenomena underlying these disturbances remain unknown. Alternative splicing consists of selective removal of introns and joining of residual exons of the primary transcript, to produce mRNA molecules of different sequence. Splicing aberrations may lead to tumoral transformation due to synthesis of impaired splice variants with oncogenic potential. In this paper we hypothesized that disturbed alternative splicing in ccRCC may result from improper expression of splicing factors, mediators of splicing reactions.

RESULTS

Using real-time PCR and Western-blot analysis we analyzed expression of seven splicing factors belonging to SR proteins family (SF2/ASF, SC35, SRp20, SRp75, SRp40, SRp55 and 9G8), and one non-SR factor, hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) in 38 pairs of tumor-control ccRCC samples. Moreover, we analyzed splicing patterns of five genes involved in carcinogenesis and partially regulated by analyzed splicing factors: RON, CEACAM1, Rac1, Caspase-9, and GLI1.

CONCLUSIONS

We found that the mRNA expression of splicing factors was disturbed in tumors when compared to paired controls, similarly as levels of SF2/ASF and hnRNP A1 proteins. The correlation coefficients between expression levels of specific splicing factors were increased in tumor samples. Moreover, alternative splicing of five analyzed genes was also disturbed in ccRCC samples and splicing pattern of two of them, Caspase-9 and CEACAM1 correlated with expression of SF2/ASF in tumors. We conclude that disturbed expression of splicing factors in ccRCC may possibly lead to impaired alternative splicing of genes regulating tumor growth and this way contribute to the process of carcinogenesis.

METHODS

Prospective, single center, nonblinded radiographic analysis of anterior and posterior adult spinal deformity fusions performed with bone morphogenetic protein (rhBMP-2).

OBJECTIVE

To determine the ability of rhBMP-2 to achieve multilevel spinal fusion in the deformity patient.

BACKGROUND

No previous study has evaluated rhBMP-2 for multilevel adult spinal deformity fusion with 2-year results. We postulated fusion could be achieved without distant autogenous graft harvest.

METHODS

Prospective analysis was performed for 98 patients (308 levels; mean age, 51.4 years) who underwent multilevel anterior or posterior spinal fusion (PSF) with minimum 2-year follow-up (average, 2.6 years). Group 1 (10 mg/level) contained 47 patients (109 levels; 2.33 levels/patient) who underwent anterior spinal fusion (ASF): BMP on an absorbable collagen sponge (ACS) with a titanium mesh cage. Group 2 (20 mg/level) included 43 patients (156 levels; 3.63 levels/patient) with PSF: BMP on an ACS with local bone graft (LBG) and bulking agent [tricalcium phosphate/hydroxyapatite (TCP-HA)]. Group 3 (40 mg/level) contained 8 patients (43 levels; 5.38 levels/patient) with PSF: rhBMP-2 and TCP-HA with no autologous bone. Confounding negative factors were present in the study population: medical comorbidities (26%), tobacco use (17%), revision surgery (34%), previous laminectomy (51%), and preoperative pseudarthrosis (27%). Postoperative films (AP, lateral, oblique) were evaluated by independent observers. Average fusion grade was based on a published scale.

RESULTS

Overall fusion rate was 95%. (group 1 91%, group 2 97%, group 3 100%). No confounding factor demonstrated a detrimental statistical significance to fusion.

CONCLUSIONS

In multilevel ASF, BMP (10 mg/level) generates fusion without autogenous bone. In multilevel PSF, BMP (20 mg/level) with LBG and TCP-HA produced fusion. BMP (40 mg/level) and TCP-HA without LBG achieved fusion. In multilevel spinal fusion, rhBMP-2 eliminated the necessity for iliac crest bone graft and yielded an excellent fusion rate.

Mitochondria are key organelles for ATP production and apoptosis. Therefore, impairment of mitochondria can modulate or accelerate cancer progression. p32, originally identified as a pre-mRNA splicing factor SF2/ASF-associated protein, is localized predominantly in the mitochondrial matrix and involved in mitochondria respiration. Recently, p32 was implicated in apoptosis and resultantly cancer progression. However, little is known about the expression and function of p32 in human tumors including prostate cancer. Here, we investigated the expression of p32 in 148 prostate carcinoma tissues by immunohistochemistry and found a positive correlation of p32 expression to clinicopathological parameters including follow-up data. p32 is highly expressed in prostate tumor samples and its expression is significantly associated with the Gleason score, pathological stage and relapse. For localized cancers, high p32 is a strong and independent predictor of clinical recurrence in multivariate analysis (P=0.01). In addition, p32 is overexpressed in the prostate cancer cell lines examined. The selective knockdown of p32 by RNA interference inhibits the growth of prostate cancer cell lines but not of a non-cancerous cell line. The p32 RNA interference decreases cyclin D1, increases p21 expression and causes a G1/S cell cycle arrest in prostate cancer cells. These data suggest that p32 is critical for prostate cancer cell proliferation and may be a novel marker of clinical progression in prostate cancer.

African swine fever virus (ASFV) causes a contagious and often lethal disease of feral and domestic swine. Experimental vaccines derived from naturally occurring, genetically modified, or cell culture-adapted ASFV have been evaluated, but no commercial vaccine is available to control African swine fever (ASF). We report here the genotypic and phenotypic analysis of viruses obtained at different passages during the process of adaptation of a virulent ASFV field isolate from the Republic of Georgia (ASFV-G) to grow in cultured cell lines. ASFV-G was successively passaged 110 times in Vero cells. Viruses obtained at passages 30, 60, 80, and 110 were evaluated in vitro for the ability to replicate in Vero cells and primary swine macrophages cultures and in vivo for assessing virulence in swine. Replication of ASFV-G in Vero cells increased with successive passages, corresponding to a decreased replication in primary swine macrophages cultures. In vivo, progressive loss of virus virulence was observed with increased passages in Vero cells, and complete attenuation of ASFV-G was observed at passage 110. Infection of swine with the fully attenuated virus did not confer protection against challenge with virulent parental ASFV-G. Full-length sequence analysis of each of these viruses revealed significant deletions that gradually accumulated in specific areas at the right and left variable ends of the genome. Mutations that result in amino acid substitutions and frameshift mutations were also observed, though in a rather limited number of genes. The potential importance of these genetic changes in virus adaptation/attenuation is discussed.

OBJECTIVE

The main problem in controlling ASF is the lack of vaccines. Attempts to produce vaccines by adaptation of ASFV to cultured cell lines have been made. These attempts led to the production of attenuated viruses that conferred only homologous protection. Specifics regarding adaptation of these isolates to cell cultures have been insufficiently described. Details like the numbers of passages required to obtain attenuated viruses, genetic modifications introduced into the virus genomes along passages, and the extent of attenuation and induced protective efficacy are not readily available. In this study, we assessed the changes that lead to decreased growth in swine macrophages and to attenuation in swine. Loss of virulence, probably associated with limited replication in vivo, may lead to the lack of protective immunity in swine observed after challenge. This report provides valuable information that can be used to further the understanding of ASFV gene function, virus attenuation, and protection against infection.

African swine fever (ASF) virus was introduced in Latvia in June 2014. Thirty-two outbreaks in domestic pigs and 217 cases in wild boar were notified in 2014. Twenty-eight outbreaks (87.5%) were primary outbreaks. The contagiosity within pig herds was low. Failure to use simple biosecurity measures to reduce the chance of virus introduction, for example by inadvertent feeding of locally produced virus contaminated fodder were the main causes for the outbreaks in backyard holdings. The infection in wild boar survived locally in two different areas with a low prevalence and a slow spread. The persistence of the infection in wild boar within an area was most probably linked to wild boar scavenging the carcasses of infected wild boar.

OBJECTIVE

The aim of this observer-blind, controlled, three-cell cross-over study was to evaluate the influence of an amine fluoride/stannous fluoride (Meridol, 250 ppm; ASF) and a chlorhexidine mouthrinse (CHX; Chlorhexamed forte, 0.2%) compared with water on in situ biofilm growth.

METHODS

After a professional toothcleaning seven volunteers had to wear a special acrylic appliance, in which six specimens each were inserted to allow the build-up of intra-oral biofilms. The volunteers had to rinse twice daily for 1 min. with 10 ml of the allocated mouthrinse. After 48 h of wearing, the specimens with the adhering biofilms were removed from the splints and stained with two fluorescent dyes, which selectively stain vital bacteria green and dead bacteria red. Under the confocal laser scanning microscope biofilm thickness (BT) was evaluated. To examine bacterial vitality (BV%) the biofilms were scanned (1 microm sections) and digital images were made. An image analysis program was used to calculate the mean BV as well as the BV of the single sections. After a wash-out period of 14 days a new test cycle was started.

RESULTS

The use of CHX and ASF resulted in a BT of 8.4+/-4.4 mum and 15.7+/-9.9 compared with 76.7+/-29.4 mum using water. The mean vitality (in %) was reduced from 66.1+/-20.4 to 23.3+/-11.6 and 23.9+/-12.4 using CHX and ASF, respectively. Both active solutions reduced BT and BV significantly compared with water (p<0.001). Differences between the two active solutions were not significant (p>0.05).

CONCLUSIONS

Both mouthrinses showed antibacterial and plaque-reducing properties against the in situ biofilm. The study design enables the examination of an undisturbed oral biofilm and for the first time shows the influence of antibacterial components applied under clinical conditions regarding biofilm formation.