Local pH oscillations witness autocatalytic self-organization of biomorphic nanostructures.

Bottom-up self-assembly of simple molecular compounds is a prime pathway to complex materials with interesting structures and functions. Coupled reaction systems are known to spontaneously produce highly ordered patterns, so far observed in soft matter. Here we show that similar phenomena can occur during silica-carbonate crystallization, the emerging order being preserved. The resulting materials, called silica biomorphs, exhibit non-crystallographic curved morphologies and hierarchical textures, much reminiscent of structural principles found in natural biominerals. We have used a fluorescent chemosensor to probe local conditions during the growth of such self-organized nanostructures. We demonstrate that the pH oscillates in the local microenvironment near the growth front due to chemical coupling, which becomes manifest in the final mineralized architectures as intrinsic banding patterns with the same periodicity. A better understanding of dynamic autocatalytic crystallization processes in such simple model systems is key to the rational development of advanced materials and to unravel the mechanisms of biomineralization.

Distribution of IL28B Polymorphism in a Cohort of Italians and Immigrants with HCV Infection: Association with Viraemia, Stage of Fibrosis and Response to Treatment.

Aims of the study are to investigate, in a cohort of patients affected by HCV chronic hepatitis with genotypes 1 and 4, the prevalence of interleukin 28B (IL28B) genotypes, the possible association between IL28B polymorphism and severity of liver damage, the role of IL28B CC as a predictor of outcome. 365 patients with HCV infection were observed between 2013 and 2014. Demographic, virological, biochemical, and genetic characteristics of each patient were investigated. Liver fibrosis was assessed by transient elastometry. Mean age of the patients (72.9 % males, 27.1 % females) is 50 years. 91.5 % % of patients are Caucasian, 8.5 % African. In the patients with HCV1 and HCV4 a higher frequency of IL28B CT is observed with a prevalence of 52.1 and 61.8 % respectively. As regards ethnic group, African people have a prevalence of 35.5 % for CC, while Caucasians have a prevalence of 23.8 % for CC. In our cohort, IL28B polymorphism does not show significant differences among ethnic groups and in HCV1 and HCV4 genotypes. As described in literature, IL28B CC genotype is confirmed as predictor of sustained virological response in both Caucasians and Africans. A significant correlation between liver fibrosis and IL28B polymorphism emerges.

A fluorescent probe for ecstasy.

A nanostructure formed by the insertion in silica nanoparticles of a pyrene-derivatized cavitand, which is able to specifically recognize ecstasy in water, is presented. The absence of effects from interferents and an efficient electron transfer process occurring after complexation of ecstasy, makes this system an efficient fluorescent probe for this popular drug.

HBV whole-genome mutation profile in HIV-1/HBV coinfected patients in a long-term follow-up study.

PURPOSE: Human immunodeficiency virus (HIV-1)-infected patients frequently harbour hepatitis B and C viruses (HBV and HCV, respectively). Possible modifications of the natural history of hepatitis B may occur. The aim of this study was to characterise HBV diversity and evolutionary and mutational viral genome profiles in HIV-1/HBV coinfections. METHODS: HIV-1 and HBV markers determinations (Roche, FRG; Abbott, USA) and HBV genome-length retrospective analysis were performed in follow-up isolates from patients who were either stably HBsAg-negative with a low level of HBV DNA (occult hepatitis B infection, OBI) or HBsAg-positive with a high level of HBV DNA. Phylogenetic analysis (maximum likelihood method, MEGA5), statistical analysis and evolutionary rates calculation (d S/d N) were applied. RESULTS: Positive selection pressures in the PreS/S region and a significantly higher number of mutations in this region including the major hydrophilic region (MHR) and the "a" determinant were shown in HBsAg-negative (possibly OBI) compared to stably HBsAg-positive HIV-1/HBV subgenotypes D3/A2 coinfected patients. Mutants previously described in HIV-1/HBV coinfected patients were found. Known mutants Y100C, P127T and P120A associated to Y134H and S143T and new S mutants, which may potentially affect HBsAg expression and secretion and anti-HBs binding, were detected in baseline sera persisting up to the end of 9 years follow-up. Known mutations of BCP, Pre-C, C and X regions were also characterised. Natural mutants strictly known as being involved in diagnostic failure were not detected; however, numerous corresponding sites showed amino acid variations. CONCLUSIONS: Evolutionary and genotypic differences observed, particularly in the PreS/S region, between HBsAg-negative (OBI) and HBsAg-positive HIV-1/HBV coinfected patients, may contribute, in association with mutations of other genomic regions, to the HBsAg-negative phenotype.

Fractal dimension and mechanism of aggregation of apple juice particles.

Turbidity of freshly squeezed apple juice is produced by a polydisperse suspension of particles coming from the cellular tissue. After precipitation of coarse particles by gravity, only fine-colloidal particles remain in suspension. Aggregation of colloidal particles leads to the formation of fractal structures. The fractal dimension is a measure of the internal density of these aggregates and depends on their mechanism of aggregation. Digitized images of primary particles and aggregates of depectinized, diafiltered cloudy apple juice were obtained by scanning electron microscopy (SEM). Average radius of the primary particles was found to be a = 40 ± 11 nm. Maximum radius of the aggregates, R(L), ranged between 250 and 7750 nm. Fractal dimension of the aggregates was determined by analyzing SEM images with the variogram method, obtaining an average value of D(f) = 2.3 ± 0.1. This value is typical of aggregates formed by rapid flocculation or diffusion limited aggregation. Diafiltration process was found to reduce the average size and polydispersity of the aggregates, determined by photon correlation spectroscopy. Average gyration radius of the aggregates before juice diafiltration was found to be R(g) = 629 ± 87 nm. Average number of primary particles per aggregate was calculated to be N = 1174.

Virological and epitope evolution of HCV infection from acute hepatitis C to subsequent episodes of HCV-related acute liver cell necrosis.

AIM: To evaluate the virological and clinical events occurring during a 3-year follow-up in three patients who, after symptomatic acute hepatitis C (AHC), experienced subsequent episodes of HC virus (V)-related acute liver cell necrosis. PATIENTS AND METHODS: The three patients were investigated for viral variability in the core, E1/E2, and NS5b regions during different phases of infection, and a computer-assisted analysis of the variation of known predicted epitopes in the consensus sequence was performed. RESULTS: The first patient showed numerous genetic variations, which may be related to the maintenance of a chronic HCV infection state and to episodes of liver disease exacerbation. The second patient showed minimal viral variations associated with apparent resolution of the infection, but the same virus isolate, based on phylogenetic analysis, produced a second acute episode after the occult phase. The third patient, after the resolution of AHC, manifested a second episode of HCV infection by a different HCV sub-genotype. CONCLUSION: Episodes of HCV-related acute liver cell necrosis after AHC may be associated to different virological patterns, such as the establishment of a chronic HCV infection, a reactivation of an occult virus, or a reinfection by a different HCV genotype.

Epidemiological and virological characterization of a large community-wide outbreak of hepatitis A in southern Italy.

A large outbreak of hepatitis A virus (HAV) infection occurred in 2004 in Campania, a region of southern Italy, with 882 cases reported between 1 January and 1 August. The local public health authorities and the Italian National Institute of Health carried out investigations in order to characterize the agent, identify the source of infection and the route of transmission, and implement appropriate control measures. A web-based reporting system enhanced the flow of information between public health authorities, providing real-time epidemic curves and frequency distributions. The same 1B HAV genotype was found in 90% of sera from a subset of patients with acute disease, suggesting a local common source. A case-control study in the municipality with the highest attack rate showed that raw seafood consumption, in particular if illegally sold in water, was strongly associated with HAV illness. Samples of seafood systematically collected from retailers were found contaminated by HAV.

The rheology of colloidal and noncolloidal food dispersions.

Rheological data on a food together with data on its composition and structure or microstructure should lead to understanding the interrelationships between them. A number of foods are dispersions of solids in liquids, liquids in liquids, or gas in liquids. The dispersed particles may be colloidal in nature with dimensions < 10 mum, or larger noncolloidal particles (> 10 mum). For both colloidal and noncolloidal dispersions (either in dilute or concentrated regimes), several theoretical equations exist that provide insights into the role of key rheological parameters, such as particle volume fraction and size, interparticle forces, and fractal dimension on their viscosity, yield stress, and modulus. When theoretical models cannot be easily applied to foods with complex structures, structural analysis and structure-based models provide insight into the role of solids loading and interparticle bonding on rheological behavior. In this review, recent studies on colloidal and noncolloidal food dispersions in which theoretical models as well as structural analysis were employed are discussed.

Nosocomial transmission in simultaneous outbreaks of hepatitis C and B virus infections in a hemodialysis center.

Reported here are details of a simultaneous outbreak of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections that occurred in a hemodialysis centre in northern Italy, with three patients seroconverting for HBsAg and four patients seroconverting for HCV antibodies. Phylogenetic analysis of the E2 region of the isolates from HCV-seroconverted patients showed the sequences were grouped in the same distinct branch as in a chronically HCV-infected patient, suggesting that the chronically infected patient was the index case. For the patients with HBV infection, phylogenetic analysis showed strong clustering among the sequences of the three patients who seroconverted to HBsAg and no relatedness between them and the sequences of patients chronically infected with HBV. For one of the patients who seroconverted to HBsAg, the last test with negative results for HBV markers had been performed 18 months prior to HBsAg seroconversion. This patient may have been previously infected with HBV and is presumed to be the source of the outbreak. This report emphasizes the importance of using universal precaution measures and HBV vaccination to prevent the transmission of viral hepatitis among chronic hemodialysis patients.

Molecular epidemiology of hepatitis C virus genotype 4 isolates in Egypt and analysis of the variability of envelope proteins E1 and E2 in patients with chronic hepatitis.

We analyzed hepatitis C virus (HCV) genotype 4 isolates circulating in the Alexandria District (Egypt) in terms of genetic divergence and the presence of different subtypes. Hypervariable region 1 (HVR1) and the NH2 region of the E2 protein were characterized, and the heterogeneity of subtype 4a isolates was evaluated by analyzing epitope frequencies, immunoproteasome prediction, and possible glycosylation patterns. The heterogeneity of the nucleotide sequences was greater than that found in previous studies, which reported only subtype 4a. Subtype 4a was most common (78% of cases), yet four new subtypes were found, with subtype 4m representing 11% of the cases and the other three subtypes representing another 11%. Substantial heterogeneity was also found when the intrasubtype 4a sequences were analyzed. Differences in the probability of glycosylation and in the positions of the different sites were also observed. The analysis of the predicted cytotoxic-T-lymphocyte epitopes showed differences in both the potential proteosome cleavage and the prediction score. The Egyptian isolates in our study also showed high variability in terms of the HVR1 neutralization epitope. Five of these isolates showed amino acid substitutions never previously observed (a total of six positions). Four of these residues (in four different isolates) were in positions involved in anchoring to the E2 glycoprotein core and in maintaining the HVR1 conformation. The results of this study indicate that HCV genotype 4 in Egypt is extremely variable, not only in terms of sequence, but also in terms of functional and immunological determinants. These data should be taken into account in planning the development of vaccine trials in Egypt.

The multiphasic profile of free fatty acids during the intravenous glucose tolerance test is unresponsive to exogenous insulin.

As small increments in insulin concentration profoundly affect lipolysis, our goal was to describe the free fatty acid (FFA) profile during the frequently sampled intravenous glucose tolerance test (FSIGT) and determine if both endogenous and exogenous insulin influenced the FFA profile. Thirteen subjects had both a glucose-only (GO-FSIGT) and insulin-modified FSIGT (IM-FSIGT). Both protocols were of 6 hours duration. At baseline an intravenous glucose bolus (0.3 g/kg) was given. In the IM-FSIGT, insulin was infused from 20 to 25 minutes (4 mU/kg. min). Six additional subjects had both an IM-FSIGT and a normal saline study (NS-Study). For the NS-Study, normal saline solution was infused instead of glucose and insulin. Fasting glucose, insulin, FFA and epinephrine concentrations were similar for all tests. Endogenous insulin peaked at 4 +/- 1 minute in both FSIGTs. The mean calculated peak time of exogenous insulin in the IM-FSIGT was 26 +/- 1 minute. Glucose concentrations were lower and epinephrine concentrations higher in the IM-FSIGT versus GO-FSIGT. During the FSIGTs, the FFA time course revealed four distinct phases, which did not differ between protocols. In phase I (0 to 11 minutes), FFA levels remained near basal (491 +/- 183 micromol/L); in phase II (11 to 79 minutes), FFA levels declined achieving a nadir of 139 +/- 63 micromol/L; in phase III (79 to 188 minutes), FFA levels rose linearly and reattained basal levels; and in phase IV (188 to 360 minutes), FFA levels rose above basal and plateaued at 732 +/- 214 micromol/L (P <.001). In the NS-Study, FFA levels remained near baseline (388 +/- 118 mEq/L) until 180 minutes and then trended upward to 618 +/- 258 micromol/L at 360 minutes. FFA concentrations from 180 to 360 minutes did not differ in the IM-FSIGT versus NS-Study. As the 4 FFA phases did not differ between protocols, the insulin effect on FFA levels in the FSIGT can be attributed to endogenous insulin. But the similarity in FFA levels from 180 to 360 minutes in the IM-FSIGT and NS-Study suggests diurnal variation and not a dynamic related to insulin or the FSIGT protocol is responsible for the final suprabasal FFA plateau.

Molecular characterisation of SENV and TTV infections in hepatopathic liver-transplant patients. Brief Report.

The presence of SENV and TTV infections among 50 patients who had undergone liver transplantation was evaluated. UTR amplification showed that 46 (92%) sera were positive. ORF-1 amplification showed that 25 (50%) patients were positive for either SENV (51.3%), TTV (10.8%), or both (37.8%) all confirmed by sequencing and phylogenetic analysis. SENV-D and SENV-H were the most prevalent viruses. The phylogenetic analysis of isolates showed that whereas SENV-D and SENV-G viruses showed sequence stability and strain persistence, SENV-H had cleared or mutated. Biological differences seem to exist among different genotypes in terms of viral replication and their persistence.

Isolation and biological characterization of 3(2H)-isoflavene-resistant and -dependent poliovirus type 2 Sabin mutants.

Poliovirus type 2 Sabin mutants were selected for drug resistance and dependence by plating on HeLa cell monolayers in the presence of 3(2H)-isoflavene, a compound related to dichloroflavan, which prevents the shut-off of host translation and poliovirus RNA and protein synthesis. The drug-resistant mutants grew equally well in the presence and in the absence of the drug, while the drug-dependent mutants only grew in the presence of the compound. One dependent and one resistant mutant were characterized biologically in more detail. The resistant mutant did not exhibit thermolability. The mild thermolability exhibited by the dependent mutant was not affected by the addition of 3(2H)-isoflavene, indicating that the substance does not bind the poliovirus type 2 Sabin capsid. The translation of viral proteins and the shut-off of host protein translation during cell infection were not inhibited in either mutant. In the absence of the drug, the cleavage of the precursor VPO, a step in virus protein processing, was affected in the dependent mutant. The dependence of the mutant on the drug was due to the inability of 75S empty particles to reach maturation: our results strongly suggest that this phenomenon is strictly dependent on the reduction of RNA synthesis, confirming the existence of a dynamic equilibrium between RNA production and genome encapsidation during the poliovirus replication cycle.

Virological and molecular parameters of HIV-1 infection of human embryonic astrocytes.

Two different strains of HIV-1, the lymphotropic HIV-IIIB and the monocytotropic HIV-Ba-L, were able to infect tertiary cultures of astrocytes established from the human embryonic brain. The infection did not require contact with infected cells, as astrocytes were exposed to infectious cell-free supernatants. Except for an early transient peak of p24 consistently observed after infection with HIV-Ba-L, the infection of astrocytes appeared to be nonproductive. However, viral production was always observed when infected astrocytes were cocultured with permissive cells (CEM-SS or monocytes). To exclude the possibility that undetectable levels of virus are chronically produced by astrocytes, we exposed permissive cells to p24 negative supernatants taken from infected cultures. In such conditions permissive cells were never infected. Infection of astrocytes by HIV-1 was further supported by the finding that provirus persisted in these cells. Indeed, by a nested PCR, we detected HIV-1 DNA even one month after infection. Moreover, at the transcriptional level we observed expression of the multiply spliced RNA (tat and nef primers). Noteworthy, this pattern of HIV-1 expression did not change appreciably when astrocytes were pretreated and cultivated in the presence of IL-1 beta. Altogether, our data support the concept that astrocytes may play a role in the spread of HIV-1 infection within the brain and in the pathogenesis of neuro-AIDS.

Surveillance of suspected poliomyelitis in Albania, 1980-1995: suggestion of increased risk of vaccine associated poliomyelitis.

Surveillance of suspected poliomyelitis cases was conducted in Albania from 1980 through 1995. A total of 93 cases were reported, 11 of which were clinically defined as poliomyelitis cases according to WHO criteria. Poliovirus was isolated from six subjects who were defined as contact vaccine-associated cases. Characterization of isolates by both antigenic and molecular methods showed that, in all cases, the disease was associated with type 2 or 3 polioviruses of vaccine origin with retromutations known to be associated with loss of Sabin attenuated phenotype. Infection occurred despite the fact that all patients had records of previous immunization with oral polio vaccine (OPV), suggesting a failure of vaccination. Four of the five patients from which poliovirus could not be isolated were classified as possible recipient vaccine-associated poliomyelitis on the basis of serology data (presence of antibodies against all three polioviruses) and the temporal association between the latest dose of vaccine received and onset of paralysis. Virological investigation on healthy contacts of the poliomyelitic patients yielded the isolation of a further 12 Sabin-like polio revertant viruses, mostly type 2 and 3. A detailed study of the non-polio acute flaccid paralysis (AFP) cases and their healthy contacts revealed the presence of several enteroviruses, namely Echo, coxsackie and, in three cases type 2 or 3 Sabin-like polioviruses. Overall, these data suggest the absence of circulation of wild-type poliovirus in Albania from 1980 to 1995, before the recent outbreak of poliomyelitis in 1996, and emphasize the need for active surveillance of AFP and laboratory characterization of virus isolates to monitor vaccination efficacy.

Antigenic and molecular characterization of wild type 1 poliovirus causing outbreaks of poliomyelitis in Albania and neighboring countries in 1996.

Mass vaccination has led poliomyelitis to become a rare disease in a large part of the world, including Western Europe. However, in the past 20 years wild polioviruses imported from countries where polio is endemic have been responsible for outbreaks in otherwise polio-free European countries. We report on the characterization of poliovirus isolates from a large outbreak of poliomyelitis that occurred in Albania in 1996 and that also spread to the neighboring countries of Yugoslavia and Greece. The epidemics involved 145 subjects, mostly young adults, and caused persisting paralysis in 87 individuals and 16 deaths. The agent responsible for the outbreak was isolated from 74 patients and was identified as wild type 1 poliovirus by both immunological and molecular methods. Sequence analysis of the genome demonstrated the involvement of a single virus strain throughout the epidemics, and genotyping analysis showed 95% homology of the strain with a wild type 1 poliovirus strain isolated in Pakistan in 1995. Neutralization assays with both human sera and monoclonal antibodies were performed to analyze the antigenic structure of the epidemic strain, suggesting its peculiar antigenic characteristics. The presented data underline the current risks of outbreaks due to imported wild poliovirus and emphasize the need to improve vaccination efforts and also the need to implement surveillance in countries free of indigenous wild poliovirus.

Outbreak of paralytic poliomyelitis in Albania, 1996: high attack rate among adults and apparent interruption of transmission following nationwide mass vaccination.

After >10 years without detection of any cases of wild virus-associated poliomyelitis, a large outbreak of poliomyelitis occurred in Albania in 1996. A total of 138 paralytic cases occurred, of which 16 (12%) were fatal. The outbreak was due to wild poliovirus type 1, isolated from 69 cases. An attack rate of 10 per 100,000 population was observed among adults aged 19-25 years who were born during a time of declining wild poliovirus circulation and had been vaccinated with two doses of monovalent oral poliovirus vaccines (OPVs) that may have been exposed to ambient temperatures for prolonged periods. Control of the epidemic was achieved by two rounds of mass vaccination with trivalent oral poliovirus vaccine targeted to persons aged 0-50 years. This outbreak underscores the ongoing threat of importation of wild poliovirus into European countries, the importance of delivering potent vaccine through an adequate cold chain, and the effectiveness of national OPV mass vaccination campaigns for outbreak control.

Poliovirus Sabin type 1 neutralization epitopes recognized by immunoglobulin A monoclonal antibodies.

Immunity to poliomyelitis is largely dependent on humoral neutralizing antibodies, both after natural (wild virus or vaccine) infection and after inactivated poliovirus vaccine inoculation. Although the production of local secretory immunoglobulin A (IgA) antibody in the gut mucosa may play a major role in protection, most of information about the antigenic determinants involved in neutralization of polioviruses derives from studies conducted with humoral monoclonal antibodies (MAbs) generated from parenterally immunized mice. To investigate the specificity of the mucosal immune response to the virus, we have produced a library of IgA MAbs directed at Sabin type 1 poliovirus by oral immunization of mice with live virus in combination with cholera toxin. The epitopes recognized by 13 neutralizing MAbs were characterized by generating neutralization-escape virus mutants. Cross-neutralization analysis of viral mutants with MAbs allowed these epitopes to be divided into four groups of reactivity. To determine the epitope specificity of MAbs, virus variants were sequenced and the mutations responsible for resistance to the antibodies were located. Eight neutralizing MAbs were found to be directed at neutralization site N-AgIII in capsid protein VP3; four more MAbs recognized site N-AgII in VP1 or VP2. One IgA MAb selected a virus variant which presented a unique mutation at amino acid 138 in VP2, not previously described. This site appears to be partially related with site N-AgII and is located in a loop region facing the VP2 N-Ag-II loop around residue 164. Only 2 of 13 MAbs proved able to neutralize the wild-type Mahoney strain of poliovirus. The IgA antibodies studied were found to be produced in the dimeric form needed for recognition by the polyimmunoglobulin receptor mediating secretory antibody transport at the mucosal level.

Effect of chloro-, cyano-, and amidino-substituted flavanoids on enterovirus infection in vitro.

Synthetic flavans, isoflavans and isoflavenes substituted with chloro, cyano and amidino groups were tested for their in vitro activity against poliovirus type 2, Coxsackie virus B4, echovirus type 6 and enterovirus 71. Plaque-reduction assays showed that substituted 3-(2H)-isoflavenes, carrying a double bond in the oxygenated ring, possess antiviral activity higher than that of the corresponding isoflavans. The most effective compounds were 4'-chloro-6-cyanoflavan and 6-chloro-4'-cyanoflavan. Studies on the mechanism of action of these two compounds suggested an effect on the early stages of viral replication.

Mechanism of action of the antirhinovirus flavanoid 4',6-dicyanoflavan.

4',6-Dicyanoflavan (DCF), a new antirhinovirus compound, was shown to inhibit an early event of rhinovirus type 1B replication in HeLa cells. When DCF was present from the beginning of infection or was added no later than the first hour of infection, the compound completely prevented viral RNA and protein synthesis and the virus-induced shutoff of host translation. DCF had no adverse effect either on virus binding to the cell membrane or on virus penetration into cells, whereas it delayed the uncoating kinetics of neutral redencapsidated rhinovirus. DCF also prevented mild acid or thermal inactivation of virus infectivity, although it reversibly interacted with virions. These results suggest that the stabilizing effect of DCF on virion capsid conformation is responsible for uncoating inhibition.