Low-lying, Rydberg states of polycyclic aromatic hydrocarbons (PAHs) and cyclic alkanes.

TD-DFT calculations of low-lying, Rydberg states of a series of polycyclic hydrocarbons and cyclic alkanes are presented. Systematic variations in binding energies and photoelectron angular distributions for the first members of the s, p and d Rydberg series are predicted for increasing molecular complexity. Calculated binding energies are found to be in very good agreement with literature values where they exist for comparison. Experimental angle-resolved photoelectron spectroscopy results are presented for coronene, again showing very good agreement with theoretical predictions of binding energies and also for photoelectron angular distributions. The Dyson orbitals for the small "hollow" carbon structures, cubane, adamantane and dodecahedrane, are shown to have close similarities to atomic s, p and d orbitals, similar to the superatom molecular orbitals (SAMOs) reported for fullerenes, indicating that these low-lying, diffuse states are not restricted to π-conjugated molecules.

[Trusted person and living will: information and implementation defect]. / Personne de confiance et directives anticipées: défaut d'information et de mise en œuvre.

PURPOSE: The French law allows the persons of age to appoint a trusted person and to draft advance directives in case they are one day in a condition that prevents them from expressing their will regarding their health care. Our study objective was to assess patients' and relatives' knowledge and collecting their opinion regarding these means of expression of their will. METHODS: An anonymous survey by self-administered questionnaire was conducted in the admission offices of the University Hospital of Nancy in April 2011. The questions focused on trusted person and anticipated directives. RESULTS: We collected 367 answers, 61.8% of which were females. Average age of respondents was 48.7 years old (standard deviation: 15.6). Three fourths of respondents were informed of their possibility to appoint a trusted person and were able to establish the difference between a trusted person and a contact person. Respondents mainly chose their spouse (52%). They thought that the trusted person's opinion takes precedence over the family's or relatives' one (64.7%), given that this opinion is based on indications previously provided by the patient (74.8%). The majority of people surveyed were ignorant of the possibility to draft advance directives but were glad of it (57.5%). They would include herein their refusal of unreasonable obstinacy (75.8%), their wishes to withhold/withdraw of some treatments, to stop active treatments in case of high odds of chronic coma or vegetative state (52.8%) or their will to donate organ after death (50.6%). More than three fourths of the patients wished to include these informations on their health care card chip. CONCLUSION: Legal means of expression of the patient's wishes and are not systematically known by the population. The possibility to appoint a trusted person is much more known than that to draft advance directives. After the release in December 2012 of the Sicard report regarding the end of life in France, an important information campaign of the general public remains to be undertaken.

Mathematical treatment of transport data of bacterial transport systems to estimate limitation in diffusion through the outer membrane.

Bacterial transport systems are traditionally treated as enzymes exhibiting a saturable binding site giving rise to an apparent K(m)of transport, whereas the maximal rate of transport is regarded equivalent to the V(max)of enzymatic reactions. Thus, the Michaelis-Menten theory is usually applied in the analysis of transport data and K(m)and V(max)are derived from the treatment of data obtained from the rate of transport at varying substrate concentrations. Such an analysis tacitly assumes that the substrate recognition site of the transport system is freely accessible to substrate. However, this is not always the case. In systems endowed with high affinity in the micro M range or those recognizing large substrates or those exhibiting high V(max), the diffusion through the outer membrane may become rate determining, particularly at low external substrate concentrations. In such a situation the dependence of the overall rate of transport (from the medium into the cytoplasm) on the substrate concentration in the medium will no longer follow Michaelis-Menten kinetics. By analysing the deviation of transport data from the corresponding ideal Michaelis-Menten plot we developed a method that allows us to determine diffusion limitation through the outer membrane. The method allows us to find the correct K(m)of the transport system functioning at the inner membrane even under conditions of strong diffusion limitation through the outer membrane. The model was tested and validified with the Escherichia coli binding protein-dependent ABC transporter for maltose. The corresponding systems for sn -glycerol-3-phospate of Escherichia coli and the alpha -cyclodextrin transport of Klebsiella oxitoca were used as test systems.

Quantitative analysis of binding protein-mediated ABC transport systems.

We present a mathematical proof for the applicability of the Michaelis-Menten theory to the analysis of binding protein-dependent transport systems. Fitting the rate equation for transport of substrate to experimental data obtained with the Escherichia coli maltose system under conditions where the concentrations of the binding protein was varied, it was possible to determine without any further assumptions: (1) K1, the affinity constant of the binding protein towards its substrate; (2) K3, the affinity constant of the unloaded binding protein towards the membrane components; and (3) R, the concentration of the membrane component. This analysis allows determination of the alteration of KM and Vmax of the system at varying concentrations of binding protein.

The inhibition of maltose transport by the unliganded form of the maltose-binding protein of Escherichia coli: experimental findings and mathematical treatment.

Binding protein-dependent transport systems in Gram-negative enteric bacteria are multicomponent systems in which a soluble periplasmic binding protein of high substrate binding affinity establishes the major substrate recognition site. Usually, there are two integral membrane proteins which are thought to interact with the substrate loaded form of the binding protein to allow transport of substrate to occur. Transport is against the concentration gradient and needs energization by an ATP hydrolizing polypeptide. Overall transport is considered mainly unidirectional due to the high energy of ATP hydrolysis coupled to transport. In the study reported here, maltose transport in membrane vesicles in the presence of varying concentrations of unliganded maltose-binding protein but with constant amounts of maltose was measured. The conditions were chosen such that the concentration of maltose was always smaller than that of the binding protein and the initial concentration of the liganded binding protein was essentially constant. It was found that the initial rate of transport went through a maximum with increasing amounts of binding protein and declined thereafter. This finding strongly supports the conclusion that both the liganded and the unliganded forms of the binding protein interact with the membrane components of the transport system. The mathematical treatment of the experimental data allowed the ratio of the affinities for the membrane components of the substrate loaded and unloaded binding protein to be estimated. Published data on the binding protein-dependent transport of histidine in membrane vesicles of Salmonella typhimurium were also used. The data allowed the ratio of the binding affinity of the membrane components to the substrate-loaded and free binding protein to be determined. In addition, the KM of transport to the KD of binding protein was approximated.

The adaptive significance of social synchronisation of ultradian behaviour cycles: a computer model.

Daily behaviour patterns in a hunter-gatherer community of Colombian Indians show that individual activities are regulated by ultradian behaviour cycles of about 2 hr and that these cycles can be synchronised by social interaction. A computer model was developed which simulated an artificial community and generated dynamic portraits of locomotor activity and social aggregation similar to those of the observed community of Colombian Indians. Social phase-locking of ultradian behaviour cycles occurred, contributing to the safety of group members and their economy of effort in gathering and related activities. Social synchronisation of ultradian behaviour cycles may also have occurred in early hominid groups.

Mathematical treatment of the kinetics of binding protein dependent transport systems reveals that both the substrate loaded and unloaded binding proteins interact with the membrane components.

Binding-protein-dependent transport systems in Gram-negative bacteria are multicomponent systems in which a soluble periplasmic binding protein of high substrate binding affinity establishes the major substrate recognition site. Usually, there are two membrane proteins which are thought to interact with the substrate loaded form of the binding protein to allow transport of substrate to occur. Transport is against the concentration gradient and needs energization by an ATP hydrolyzing polypeptide. Overall transport is considered mainly unidirectional owing to the high energy of ATP hydrolysis coupled to transport. We dissected the overall transport process into three individual steps: (i) reversible binding of substrate to the binding protein; (ii) reversible binding of the binding protein to the membrane components forming the translocation complex; (iii) irreversible transport of substrate through the membrane and dissociation of the binding protein from the complex. Two models were considered. In the first, only the substrate-loaded binding protein interacts with the membrane components, while in the second model both the loaded and the unloaded form of the binding protein interact with the membrane components. The mathematical analysis of the second model revealed that the substrate concentration KM at half-maximal rate of transport approaches KD of the binding protein when the last step of transport becomes low and when the concentration of binding protein in the periplasm becomes large. This is usually observed in real systems. Under the same conditions, in model 1 KM approaches zero and is hence considerably smaller than KD. This has never been observed in any real system. In addition, the dependence of the overall rate of transport on the concentration of binding protein in the periplasm follows a sigmoidal curve only when model 2 is considered. The sigmoidal behavior becomes more pronounced when the substrate concentration is low and it is less pronounced when the last step in overall transport is low. This phenomenon has been observed with the Escherichia coli maltose transport system. Thus, at least for the maltose transport system, it seems likely that both the loaded and the unloaded forms of the binding protein interact with the membrane components. We propose that this should generally be considered in binding-protein-dependent transport systems.

Evaluation of a rapid, reliable, and inexpensive screening test for the serological diagnosis of human brucellosis.

In many laboratories the serological diagnosis of brucellosis is based on a tube agglutination test requiring incubation for two days and therefore causing delay in reporting of results. A slide agglutination test using an acidified, rose Bengal colored antigen that can be read after only 4 min was evaluated for its usefulness as a screening test for the presence of agglutinins in human sera. In sera of patients with culturally proven brucellosis or serological evidence for active infection the sensitivity of the slide agglutination test was 100%. Of 97 tube agglutination-negative sera none was positive in the slide test. From these results it was concluded that sera can be tested by the slide agglutination test which is quicker, easier to perform, and less expensive than the tube agglutination test. If positive, it is followed by a tube agglutination test which gives quantitative information and is necessary to follow the course of an infection.

Isolation and serotyping of porcine rotaviruses and antigenic comparison with other rotaviruses.

Seven rotavirus strains were isolated in cell cultures from the intestinal contents of piglets with diarrhea. MA104 cells with pancreatin in the cell culture medium was the host system of choice for virus isolation and replication. A cell culture immunofluorescence test in which MA104 cells were used in microtiter plates was very effective for detecting and assaying rotaviruses. A plaque reduction neutralization test, cross-protection studies in gnotobiotic pigs, and electrophoresis of rotaviral double-stranded RNA were used for comparing viruses. Three strains produced plaques on initial isolation attempts, replicated well in cell cultures, and were antigenically very similar. We suggest that these three strains be considered porcine rotavirus serotype 1, with The Ohio State University (OSU) strain serving as the prototype. The OSU strain was distinct from bovine, simian, canine, and human (Wa and M) rotaviruses by plaque reduction neutralization. Four strains did not produce plaques on initial isolation attempts, were difficult to adapt to cell cultures, and were related to each other but were distinct from the serotype 1 strains. We suggest that the Gottfried (G) strain be tentatively considered as a prototype for porcine rotavirus serotype 2. The G strain was antigenically closely related to canine and simian rotaviruses and less so to human M rotavirus (human rotavirus serotype 3). Canine, simian, and human M rotaviruses were closely related. All seven porcine rotavirus strains caused diarrhea in gnotobiotic pigs. Cell-cultured vaccines of the OSU and G strains caused only mild or no diarrhea in gnotobiotic pigs, and protection occurred when such pigs were challenged with homologous, bur not heterologous, virulent viruses. A survey indicated that 94% of 274 porcine serum samples and 100% of 75 herds were serologically positive to the porcine OSU rotavirus.

Immune response of pregnant cows to bovine rotavirus immunization.

Fifteen pregnant Holstein cows were freely assigned to 3 experimental groups (5 cows in each group). Cows in group I were inoculated IM and intramammarily (IMm) with Ohio Agricultural Research and Development Center (OARDC) tissue culture-propagated modified-live Nebraska calf diarrhea bovine rotavirus with added adjuvant (OARDC vaccine-immunized cows). Group II cows were given IM injections of a commercial modified-live rotavirus-coronavirus vaccine (commercial vaccine-immunized cows), and the remaining 5 cows were noninoculated controls (group III). Rotavirus antibody in colostrum and milk was mainly associated with immunoglobulin (Ig)G1, and less so with IgG2, IgA, and IgM, as analyzed by the enzyme-linked immunosorbent assay (ELISA), using monospecific anti-bovine IgG1, IgG2, IgM, and IgA sera. In serum, the rotavirus antibody was distributed almost equally between IgG1 and IgG2. The same relationships appeared in both immunized and nonvaccinated cows. All OARDC vaccine-injected cows had virus-neutralization (VN) and ELISA IgG1 rotavirus antibody titers in serum and mammary secretions at significantly increased levels (at least 100-fold; P less than 0.05) compared with the titers in groups II (commercial vaccine-immunized cows) and III (controls). Serum, colostrum, and milk antibody titers from these latter 2 groups did not differ statistically. The ELISA IgG2, IgA, and IgM rotavirus antibody titers also were significantly greater in mammary secretions from OARDC vaccine-immunized cows than in groups II and III cows. There was a high correlation between ELISA IgG1 and VN rotavirus antibody titers for all samples tested (r = 0.97, P less than 0.001), but ELISA IgG1 antibody titers were consistently higher than VN titers. The ELISA IgG1 and VN antibody titers of milk samples collected from cows 30 days after parturition were higher from the OARDC vaccine-immunized cows (ELISA IgG1, geometric mean titer (GMT) = 3,511; VN GMT = 1,689) than were titers from the group II cows (ELISA IgG1 GMT = 39; VN GMT = 33) or group III cows (ELISA IgG1 GMT = 21; VN GMT = 19). These results indicate that IM plus IMm immunization of pregnant cows, using modified-live bovine rotavirus with added adjuvant, may significantly enhance serum, colostrum, and milk rotavirus antibody titers, whereas IM vaccinal inoculation of pregnant cows with a commercial modified-live rotavirus-coronavirus vaccine may not.

Passive immunity to transmissible gastroenteritis virus: intramammary viral inoculation of sows.

Sows were injected intramammarily with live-attenuated TGE virus, an enteric coronavirus--one sow during pregnancy and three sows during lactation. All sows were TGE antibody seronegative prior to inoculation except for one naturally infected sow inoculated during lactation. The animal injected during pregnancy had primarily IgG TGE antibodies in milk from all glands. By contrast, sows injected during lactation had IgA and IgM initially, and later IgA and IgG TGE antibodies in milk from injected and noninjected glands. The seropositive sow had elevated IgA TGE antibody titers in milk after IMm injection. Both seronegative sows inoculated intramammarily during lactation shed TGE virus in milk from injected glands, and their nursing piglets developed mild diarrhea and shed virus in their feces at three to nine DPE of the sows. Milk from IMm injected glands generally had higher TGE antibody titers than milk from noninjected glands. These results suggest that TGE virus replicates in lactating mammary gland tissue, thereby stimulating IgA immunocytes, leading to secretion of IgA antibodies in milk. Whether the intramammary route presents a natural route of enteric virus exposure in lactating animals (by way of infected nursing piglets), leading to IgA-antibody secretion in milk, requires further investigation.

Porcine pararotavirus: detection, differentiation from rotavirus, and pathogenesis in gnotobiotic pigs.

Some characteristics of a newly recognized porcine enteric virus are described. Tentatively, the virus was referred to as porcine pararotavirus (PaRV) because it resembled rotaviruses in respect to size, morphology, and tropism for villous enterocytes of the small intestine. However, it was antigenically distinct from porcine, human, and bovine rotaviruses and reoviruses 1, 2, and 3, and the electrophoretic migration pattern of PaRV double-stranded RNA was distinct from the electrophoretic migration patterns of the rotaviral and reoviral genomes. By passage in gnotobiotic pigs, PaRV was isolated from two suckling diarrheic pigs originating from two herds. After oral exposure of gnotobiotic pigs, villous enterocytes of the small intestines became infected as judged by immunofluorescence, resulting in villous atrophy and diarrhea. Mortality was high when gnotobiotic pigs less than 5 days old were infected. The C strain of this virus was serially passed 10 times in gnotobiotic pigs, and electron microscopy, immunofluorescence, and serological tests indicated no extraneous agents. The virus was serially passed five times in cell cultures which contained pancreatin in the medium, but replication was negligible or absent, as the number of immunofluorescent cells decreased with each passage. Since rotaviral infections are frequently diagnosed by direct electron microscopy of fecal specimens, the presence of other morphologically similar viruses, such as PaRV, should be considered. The use of immune electron microscopy is suggested as a means of helping recognize this situation.

Rapid, simple method of preparing rotaviral double-stranded ribonucleic acid for analysis by polyacrylamide gel electrophoresis.

A procedure for extracting rotaviral double-stranded ribonucleic acid (RNA) directly from fecal and intestinal specimens collected from calves and pigs is described. This procedure provides a rapid, simple, reproducible method of obtaining rotaviral double-stranded RNA preparations suitable for electrophoretic analysis in polyacrylamide-agarose composite gels. The rotaviral genome electrophoretic migration pattern produced by double-stranded RNA extracted directly from a specimen by this procedure was qualitatively identical to the electrophoretic migration pattern obtained with double-stranded RNA extracted from purified rotavirus derived from the same specimen. Direct extraction of specimens containing porcine rotavirus-like virus by this procedure gave preparations that had electrophoretic migration patterns similar, but not identical, to the characteristic electrophoretic migration pattern of the rotaviral genome. Sufficient rotaviral double-stranded RNA could be extracted from 6 ml of fecal or intestinal specimen by this procedure to permit 15 or more electrophoretic assays.

Rotavirus-like, calicivirus-like, and 23-nm virus-like particles associated with diarrhea in young pigs.

Virus particles morphologically similar to caliciviruses and rotaviruses were detected by electron microscopy (EM) in the intestinal contents of a 27-day-old diarrheic nursing pig. A third small spherical 23-nm virus-like particle was also observed. Calicivirus-like particles averaged 33 nm in diameter. Similar to rotaviruses, rotavirus-like particles were present as single-capsid 55-nm forms or double-capsid 70-nm particles. Most gnotobiotic pigs orally exposed to samples containing these three viruses developed diarrhea and villous atrophy of the small intestine, and all shed the three viruses in their intestinal contents. Attempts to propagate these viruses in cell culture were unsuccessful. The antigenic relationship of the rotavirus-like particles to known rotaviruses was explored by immune EM and immunofluorescent staining. By these techniques, the rotavirus-like particles did not cross-react with antisera to porcine, bovine, or human rotaviruses or to reovirus type 3. Antisera from gnotobiotic pigs exposed to all three viruses had enzyme-linked immunosorbent assay and virus neutralization titers of <4 against porcine rotavirus. Previous infection of gnotobiotic pigs with the mixture containing rotavirus-like particles failed to protect them against a subsequent challenge with porcine rotavirus. The antigenic relationship of the calicivirus-like particles to known caliciviruses was investigated by immune EM and virus neutralization. By these tests, the calicivirus-like particles did not react with antisera against feline calicivirus strain 255 or M-8. In a study conducted at Plum Island Animal Disease Center, antiserum against the three combined agents did not specifically neutralize any serotype of swine vesicular exanthema virus.

Human rotavirus type 2: cultivation in vitro.

A strain of type 2 human rotavirus (Wa) was grown to relatively high titer through 14 passages in primary cultures of African green monkey kidney (AGMK) cells. This passage series was initiated with virus that had been passaged 11 times serially in newborn gnotobiotic piglets. In contrast, virus present in the stool of patient Wa as well as virus from the first, second, or third passage in piglets could not be propagated successfully in African green monkey kidney cells. Prior to each passage in cell culture, the virus was treated with trypsin and the inoculated cultures were centrifuged at low speed. Cultivation of a type 2 human rotavirus should aid attempts to characterize this virus and to develop a means of immunoprophylaxis for a serious diarrheal disease of human infants.

Porcine rotaviral infection of cell culture: effects of certain enzymes.

Porcine rotaviral infectivity for continuous porcine kidney (PK-15) cells was enhanced by incorporation of pancreatic endopeptidases into the cell culture maintenance medium. Marked enhancement of infectivity was induced by trypsin, whereas elestase and alpha-chymotrypsin enhanced infectivity to a lesser extent. Bacterial protease also induced some enhancement of porcine rotaviral infectivity. A synergistic enhancement of porcine rotaviral infectivity was noticed with trypsin and alpha-chymotrypsin combined. Porcine rotaviral infectivity was not affected by incorporation of alpha-amylase, alkaline phosphatase, beta-galactosidase, carboxypeptidase-A, deoxyribonuclease, enterokinase, lipase, or ribonuclease into the maintenance medium.

Concurrent porcine rotaviral and transmissible gastroenteritis viral infections in a three-day-old conventional pig.

A 3-day-old suckling pig with diarrhea was necropsied, and immunofluorescent microscopic examination of the small intestinal mucosa, together with immune electron microscopic examination of the large intestinal contents, provided a presumptive diagnosis of a concurrent infection with transmissible gastroenteritis (TGE) virus and porcine rotavirus. Immunofluorescent microscopic, immune electron microscopic, and serologic data obtained from gnotobiotic pigs experimentally inoculated with the large intestinal contents of the suckling pig confirmed this diagnosis. Two gnotobiotic pigs, convalescent from previous TGE viral infections, became infected with porcine rotavirus only. However, another gnotobiotic pig, convalescent from a previous porcine rotaviral infection, became infected with TGE virus only, following inoculation with the large intestinal contents of the suckling pig.

Rotaviral diarrhea in pigs: brief review.

Rotavirus is a name given to a group of viruses that have similar characteristics and are generally capable of causing diarrhea in the young. Infection of pigs with porcine rotavirus is common and widespread and can result in diarrhea, especially in 1- to 4-week-old pigs. This virus is frequently associated with a diarrheal syndrome popularity known as "white scours," "milk scours," or "3-week-old scours." Pigs less than 1 week old are infrequently infected, presumably because of adequate passive immunity. The infection resembles enzootic transmissible gastroenteritis. Diagnosis can be made by immunofluorescent staining of mucosal scrappings from the small intestines.