Theoretical investigations on electronic structure and optoelectronic properties of vinyl fused monomeric and oligomeric benzimidazole derivatives using DFT and TDDFT techniques.

CONTEXT: The present work encompasses the theoretical investigation of 14 benzimidazole-based (seven vinyl fused monomeric benzimidazole (VFMBI) and seven vinyl fused oligomeric benzimidazole (VFOBI)) derivatives using density functional theory (DFT) and time-dependent density functional theory (TD-DFT) techniques. The effects of electron donor and acceptor groups on the electronic structure such as HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) energies, HOMO-LUMO energy gap, ionization potentials (IPs), electron affinities (EAs), internal reorganization energies of holes and electrons (λh/e), and excited state properties have been explored in the present work. In addition, natural bond orbital (NBO) analysis of these compounds has been investigated to reveal the typical stabilization interactions in these molecules. Hence, the aim of the present work is to explore the electronic structures and optoelectronic properties of the title molecules on the basis of the DFT quantum chemical calculations and to make an idea on the parameters influencing the optoelectronic efficiency toward a better understanding of the structure-property relationships. Moreover, the calculated results reveal the suitable optoelectronic properties of benzimidazole oligomer derivatives using theoretical techniques. Of the investigated molecules, 4_MABIMCY and 4_MABIOCY show potential optoelectronic properties and can be used as a potential charge transport material due to their narrow band gap, high hyperpolarizability, low ionization potential, and high electron affinity. The larger λab and λem values favor the system to be used as a potential optoelectronic material with better optical properties. METHODS: All quantum chemical calculations were carried out using Gaussian09 theoretical chemistry code. Ground state calculations were made using the B3LYP/6-31+G(d,p) method. All excited state calculations had been computed using TDB3P86/6-311++(d,p). The initial structure for excited state calculations was optimized using the AM1 semi-empirical method.

Structural and optical characterization of the local environment of Er3+ ions in PbO-ZnO tellurite glasses.

Erbium activated PbO-ZnO tellurite glasses ((70TeO(2)-(30-x)ZnO-xPbO)(0.99)-(Er(2)O(3))(0.01) (TZPE), (x = 5, 10, 15, 20)) were prepared by a melt quenching process and studied by optical absorption, luminescence, Raman and x-ray absorption spectroscopy measurements as a function of the PbO/ZnO ratio. The glass structure, as monitored by Raman scattering, shows important changes with the PbO/ZnO ratio, attributed to a glass former action of PbO. The local environment of Er(3+) ions, as measured by extended x-ray absorption spectroscopy, does not appreciably change as regards the first oxygen shell. However, the intensity of the optical transitions is quite sensitive to the PbO/ZnO ratio, indicating a progressive increase of the site symmetry with the PbO content. The emission probability and radiative lifetime of several excited states of Er(3+) ions were calculated using Judd-Ofelt analysis.

Fecal occult blood testing in a noncompliant inner city minority population: increased compliance and adherence to screening procedures without loss of test sensitivity using stool obtained at the time of in-office rectal examination.

OBJECTIVES: Fecal occult blood screening is cost-effective, is easily administered to large groups of patients, and reduces mortality associated with colorectal cancer. Within our predominant African American and Latino inner city clinic populations, compliance with common screening procedures is suboptimal. A procedure with increased compliance is needed to adequately screen this population at high risk for colorectal cancer. The objective of this study was to compare the results of the 3-day at-home hemoccult test for occult blood to those of a hemoccult test performed from stool obtained at rectal examination in the office. METHODS: A total of 350 consecutive patients referred to the GI clinic of University Hospital or Jersey City Medical Center for colorectal cancer screening had both the 3-day at-home hemoccult test and an in-office hemoccult examination performed, followed by either sigmoidoscopy (for negative results) or by colonoscopy (for positive results). RESULTS: Patients were noncompliant with dietary restrictions, 3-day card return, follow-up appointments, and endoscopy with conventional screening methods. Decisions based on the in-office examination with direct scheduling of endoscopy significantly improved compliance with follow-up. There was no statistical difference between the two detection methods, suggesting that the in-office examination was the more effective screening test. CONCLUSIONS: Endoscopy based on an in-office hemoccult examination is an acceptable alternative to using the 3-day at-home stool collection to govern endoscopic choices. In a noncompliant inner city population, use of the in-office examination increased compliance with follow up, potentially allowing more patients exposure to screening.

Borrelia burgdorferi proteins whose expression is similarly affected by culture temperature and pH.

Previously, we had demonstrated the upregulation in the expression of several proteins, including the lipoproteins OspC and P35, of Borrelia burgdorferi in the stationary growth phase. Since the expression of OspC is also known to be affected by culture temperature and pH, we examined the effects of both variables on the expression of the remaining stationary-phase-upregulated proteins. Our study revealed that the expression of each of the remaining stationary-phase-upregulated proteins, P35 included, was also influenced by culture temperature; these proteins were selectively expressed at 34 degrees C but not at 24 degrees C. Significantly, the expression of a majority of these proteins was also affected by culture pH, since they were abundantly expressed at pH 7.0 (resembling the tick midgut pH of 6.8 during feeding) but only sparsely at pH 8.0 (a condition closer to that of the unfed tick midgut pH of 7.4). We propose that this group of B. burgdorferi proteins, which in culture is selectively expressed under conditions of 34 degrees C and pH 7.0, may be induced in the tick midgut during the feeding event. Furthermore, the differential and coordinate expression of these proteins under different environmental conditions suggests that the encoding genes may be coregulated.

Transcriptional regulation in spirochetes.

Spirochetes belong to a widely diverse family of bacteria. Several species in this family can cause a variety of illnesses including syphilis and Lyme disease. Despite the fact that the complete genome sequence of two species, Borrelia burgdorferi and Treponema pallidum, have been deciphered, much remains to be understood about spirochetal gene regulation. In this review we focus on the environmental transitions that spirochetes undergo during their life cycles and the mechanisms of transcriptional regulation that might possibly mediate spirochetal adaptations to such changes.

An immunodominant conserved region within the variable domain of VlsE, the variable surface antigen of Borrelia burgdorferi.

Antigenic variation is an effective strategy evolved by pathogenic microbes to avoid immune destruction. Variable Ags such as the variable major protein of Borrelia hermsii, the variant surface glycoprotein of African trypanosomes, and the pilin of Neisseria gonorrhoeae include an immunodominant variable domain and one or more invariable domains that are not antigenic. Short, nonantigenic, invariable regions also may be present within the variable domain. VlsE (variable major protein-like sequence, expressed), the variable surface Ag of Borrelia burgdorferi, the Lyme disease spirochete, also contains both variable and invariable domains. In addition, interspersed within the VlsE variable domain there are six invariable regions (IR1-6) that together amount to half of this portion's primary structure. We show here that these IRs are conserved among strains and genospecies of the B. burgdorferi sensu lato complex. Surprisingly, unlike the invariable regions of variable major protein, variant surface glycoprotein, and pilin, which are not antigenic in natural infections, the most conserved of the IRs, IR6, is immunodominant in Lyme disease patients and in monkeys infected with B. burgdorferi. IR6 is exposed on the surface of VlsE, as assessed by immunoprecipitation experiments, but is inaccessible to Ab on the spirochete's outer membrane, as demonstrated by immunofluorescence and in vitro killing assays. VlsE thus significantly departs from the antigenic variation paradigm, whereby immunodominance is only manifest in variable portions. We submit that IR6 may act as a decoy epitope(s) and contribute to divert the Ab response from other, perhaps protective regions of VlsE.

Differential expression of Borrelia burgdorferi proteins during growth in vitro.

In an earlier paper we described the transcriptionally regulated differential levels of expression of two lipoproteins of Borrelia burgdorferi, P35 and P7.5, during growth of the spirochetes in culture from logarithmic phase to stationary phase (K. J. Indest, R. Ramamoorthy, M. Solé, R. D. Gilmore, B. J. B. Johnson, and M. T. Philipp, Infect. Immun. 65:1165-1171, 1997). Here we further assess this phenomenon by investigating whether the expression of other antigens of B. burgdorferi, including some well-characterized ones, are also regulated in a growth-phase-dependent manner in vitro. These studies revealed 13 additional antigens, including OspC, BmpD, and GroEL, that were upregulated 2- to 66-fold and a 28-kDa protein that was downregulated 2- to 10-fold, during the interval between the logarithmic- and stationary-growth phases. Unlike with these in vitro-regulated proteins, the levels of expression of OspA, OspB, P72, flagellin, and BmpA remained unchanged throughout growth of the spirochetes in culture. Furthermore, ospAB, bmpAB, groEL, and fla all exhibited similar mRNA profiles, which is consistent with the constitutive expression of these genes. By contrast, the mRNA and protein profiles of ospC and bmpD indicated regulated expression of these genes. While bmpD exhibited a spike in mRNA expression in early stationary phase, ospC maintained a relatively higher level of mRNA throughout culture. These findings demonstrate that there are additional genes besides P7.5 and P35 whose regulated expression can be investigated in vitro and which may thus serve as models to facilitate the study of regulatory mechanisms in an organism that cycles between an arthropod and a vertebrate host.

Borrelia burgdorferi escape mutants that survive in the presence of antiserum to the OspA vaccine are killed when complement is also present.

As an initial attempt to investigate the possible role of outer surface protein A (OspA) escape mutants of Borrelia burgdorferi in decreasing the efficacy of the OspA vaccine, mutants of the HB19 strain of B. burgdorferi sensu stricto were selected in vitro from an uncloned, low-passage-number isolate. The antiserum used for selection was obtained from rhesus monkeys that had been given a vaccine of the same formulation and dose, and by the same route of administration, as that given to humans in several trials. All of the mutants selected in liquid medium and subsequently cloned twice in solid medium expressed a single abundant protein of 28 to 34 kDa instead of both OspA and OspB. Depending on the mutant, this protein reacted strongly, weakly, or not detectably with the anti-OspA antibody used for selection. Analysis of the ospAB locus of each of four representatives from these three groups of mutants by PCR with oligonucleotide primers that hybridize to flanking regions of the ospAB operon, and of the corresponding phenotype with monoclonal antibodies that bind to the amino or carboxyl terminus of the OspA or OspB polypeptide, indicated that in all cases a deletion within the operon had occurred. Spirochetes from the four mutant strains chosen for further analysis could be killed in antibody-dependent, complement-mediated killing assays with the selecting anti-OspA antibody, despite their resistance to killing with this antibody in the absence of complement. Complement-mediated killing occurred at an antibody concentration higher than that required to kill wild-type spirochetes. If anti-OspA antibody acts only within the tick, where complement is probably ineffective due to tick-derived decomplementing factors, then OspA escape mutants, if infectious, could seriously diminish the efficacy of OspA vaccines. On the other hand, if the killing of B. burgdorferi with anti-OspA antibody also takes place within the human host, then our results indicate that chimeric/deletion escape mutants will be killed as well.

Cell-density-dependent expression of Borrelia burgdorferi lipoproteins in vitro.

Previously, we had identified non-OspA-OspB surface proteins of Borrelia burgdorferi that are targeted by the antibody-dependent complement-mediated killing mechanism. Here we demonstrate by Western blotting that one of these proteins, P35, is upregulated at the onset of stationary phase in vitro. Northern analysis revealed that the upregulation of P35 is at the level of transcription. In addition, the expression of an open reading frame (ORF) located downstream of the p35 gene was found to be regulated in the same fashion as that of P35. This ORF encodes a 7.5-kDa lipoprotein. The transcriptional start sites for both of these genes were determined, to aid in the identification of the putative promoter regions. Additional sequencing of the 5' flanking region of the p35 gene revealed a region of dyad symmetry 52 bp upstream of the transcription start site. Southern analysis demonstrated that the expression of these genes was not due to a cell-density-dependent rearrangement in the genome of B. burgdorferi. These findings provide an in vitro model for studying mechanisms of gene regulation in B. burgdorferi.

5' sequences essential for trans-splicing of msp (gp63) RNAs in Leishmania chagasi.

The 5' sequences essential for spliced leader (SL) addition to RNA encoding the abundant Leishmania surface protease gp63 were determined. A DNA segment found upstream of all Leishmania chagasi gp63 genes was cloned in front of a luciferase gene, and luciferase activity was measured in transiently transfected L. chagasi promastigotes. Two hundred and twenty bp of the upstream region was needed for optimal luciferase activity. Deletions and point mutations were placed in this segment to determine which nucleotides participate in the splicing events. A region containing 87% pyrimidines located between 31 and 69 bp upstream of the splice acceptor dinucleotide and containing three potential branch point A residues was highly beneficial for reported gene activity. In contrast, a 30-nt pyrimidine-rich sequence immediately upstream of the splice acceptor site did not by itself enhance luciferase activity. Luciferase activity was associated with the presence of trans-spliced luciferase mRNA. The results suggest that sequences 31-69 bp upstream of gp63 genes enhance gene expression through provision of signals for efficient trans-splicing.

Molecular characterization, genomic arrangement, and expression of bmpD, a new member of the bmp class of genes encoding membrane proteins of Borrelia burgdorferi.

An expression library made with Borrelia burgdorferi DNA in the vector lambda ZapII was screened with serum from a monkey infected with the Lyme disease agent. This serum killed B. burgdorferi in vitro by an antibody-dependent, complement-mediated mechanism and contained antibodies to at least seven spirochetal antigens, none of which were the major outer surface proteins OspA or OspB. Among several positive clones, a clone containing the B. burgdorferi bmpA gene encoding the immunodominant antigen P39 was obtained. Chromosome walking and DNA sequence analysis permitted the identification of two additional upstream genes homologous to the bmpA gene and its related companion, bmpB. The first of these was the recently characterized bmpC gene, and adjacent to it was the fourth and new member of this class, which has been designated bmpD. The gene product encoded by bmpD is 34l residues long, contains a signal sequence with a potential signal peptidase II cleavage site, and has 26% identity with TmpC of Treponema pallidum. Southern blotting confirmed the tandem arrangement of all four bmp genes in the chromosome of B. burgdorferi JD1. However, Northern (RNA) blotting revealed that bmpD is expressed as a monocistronic transcript, which indicates that it is not part of an operon at the bmp locus. The bmpD gene was found to be conserved in representative members of the three species of the B. burgdorferi sensu lato complex, suggesting that it serves an important biological function in the spirochete.

Intergenic regions between tandem gp63 genes influence the differential expression of gp63 RNAs in Leishmania chagasi promastigotes.

The major surface protease, gp63, of Leishmania chagasi is encoded by 18 or more tandem msp genes that can be grouped into three classes on the basis of their unique 3'-untranslated sequences (3'-UTRs) and their differential expression. RNAs from the mspLs occur predominantly during the logarithmic phase of promastigote growth in vitro, RNAs from the mspSs are present mainly in stationary phase, and RNAs from mspCs occur throughout growth in culture. All three classes of gp63 genes are constitutively transcribed during all growth phases, indicating that their expression is post-transcriptionally regulated. Chimeric plasmids containing the three different 3'-UTRs and downstream intergenic regions (IRs) fused downstream of the beta-galactosidase (beta-gal) coding region were transfected into L. chagasi, and their effects on beta-gal RNA processing and enzymatic activity were examined. The presence of the 3'-UTRs by themselves had no substantive effect on beta-gal expression. However, the 3'-UTR from a mspS plus its IR resulted in about 20-fold more beta-gal activity and RNA in stationary phase relative to logarithmic phase cells. In contrast, the 3'-UTRs plus IRs of mspL and mspC had either no or little effect, respectively, on beta-gal expression. Thus, differential expression of the mspLs and mspSs is post-transcriptionally controlled by different mechanisms.

Sequence diversity and organization of the msp gene family encoding gp63 of Leishmania chagasi.

During in vitro growth Leishmania chagasi promastigotes differentially express 3 classes of RNAs encoding the major surface protease (MSP) gp63 that can be distinguished by their unique 3' untranslated regions. Here we show that the three classes (logarithmic-specific, stationary-specific and constitutively expressed) are encoded by a family of at least 4 tandem stationary genes (mspS2, mspS1, mspS3 and mspS5) followed by twelve or more logarithmic genes (mspL genes), one constitutive gene (mspC) and a final stationary gene (mspS4). Some of the stationary genes can be distinguished from each other by groups of nucleotide differences within the coding regions that result in localized amino acid differences. Northern blots confirm that RNAs from the individual stationary genes are present in stationary, but not logarithmic, phase promastigotes. Western blots using sera directed against synthetic peptides indicate that correspondingly heterogeneous gp63 proteins are expressed in L. chagasi promastigotes. A 200-bp region upstream of all three gp63 gene classes is conserved except for a variable number of 6-bp repeats. Downstream of the gp63 coding regions are highly conserved, class-specific sequences that include the 3' untranslated regions and extend past the polyadenylation site for 65 bp (mspL), 345 bp (mspC) or 2.8 kb (mspS). These sequence features flanking the msp coding regions are likely important in the growth phase-specific expression of the three gp63 RNA classes.

The effect of ongoing protein synthesis on the steady state levels of Gp63 RNAs in Leishmania chagasi.

G63, the major surface glycoprotein of Leishmania chagasi promastigotes, increases 11-fold in amount as promastigotes grow from logarithmic to stationary phase. Transcripts from three different classes of gp63 genes are differentially expressed during this development (Ramamoorthy, R., Donelson, J. E., Paetz, K. E., Maybodi, M., Roberts, S. P., and Wilson, M. E. (1992) J. Biol. Chem. 267, 1888-1895). We studied the effect of protein synthesis inhibitors on gp63 mRNAs. The steady state level of log class gp63 RNA, expressed primarily in logarithmic phase promastigotes, increased 16.5-fold after incubation in cycloheximide. A similar increase in log gp63 RNAs was caused by inhibitors that block different steps in translation. In contrast, the levels of stationary class gp63 RNA, expressed in stationary phase parasites, and constitutive class gp63 RNA, expressed throughout promastigote growth, increased only 2.3- and 1.5-fold, respectively. The latter was not statistically significant. Nuclear run-on assays showed that the cycloheximide effect was not due to an increased rate of transcription. However, the t1/2 of log RNAs was prolonged 6.5-fold after incubation in cycloheximide, in contrast to a 1.7-fold increase in the t1/2 of ATPase RNA, suggesting that cycloheximide specifically stabilizes log gp63 mRNAs. Thus, a highly labile negative regulatory protein, such as an RNase, may specifically target log gp63 RNAs for degradation.

Three distinct RNAs for the surface protease gp63 are differentially expressed during development of Leishmania donovani chagasi promastigotes to an infectious form.

Leishmania sp. protozoa contain an abundant surface protease (gp63) that is important for the virulence of the parasite. We found that the average amount of gp63 expressed by Leishmania donovani chagasi promastigotes increases 6-11-fold as they develop from a less infectious form in logarithmic phase to a highly infectious form during stationary phase of cultivation in vitro. The predominant gp63 RNA switches from a 2.7 to a 3.0 kilobase (kb) RNA during the transition from log to stationary phase. Sequence analysis of gp63 cDNAs reveals that three different classes of gp63 RNAs, containing unique 3'-untranslated regions (3' UTRs), are expressed during growth to stationary phase. The predominant 2.7-(log) and 3.0-kb (stationary) class gp63 RNAs possess nearly identical coding regions, but they diverge in their 3' UTRs. A third class, consisting of 3.1- and 2.6-kb (constitutive) gp63 RNAs, is expressed at low levels throughout cultivation. This latter class encodes a gp63 with an additional 41 amino acids at its C terminus, replacing a potential signal for attachment of a glycolipid membrane anchor with a sequence that could be a transmembrane region. These findings are consistent with the regulated expression of different gp63 genes, resulting in different amounts of gp63 protein, during the promastigote's in vitro development to an infectious form.

Transcript hairpin structures are not required for RNA polymerase pausing in the gene encoding the E. coli RNase P RNA, M1 RNA.

Strong pauses at nucleotides +118 and +121 relative to the transcriptional start occur during in vitro transcription of the E. coli rnpB gene encoding the catalytic M1 RNA subunit of Ribonuclease P. These pauses are immediately downstream of 2 phylogenetically conserved stem-loop structures in the RNA. In the present work, single-base changes which disrupted Watson-Crick base-pairing in the hairpins were introduced into rnpB. Transcription studies in vitro with these modified templates revealed that none of the nucleotide changes predicted to increase or decrease the stability of the first hairpin significantly affected the pause half-lives. A mutation which disrupted the second hairpin increased the pause half-life 2-fold. The data suggest that the upstream stem and loop structures in the transcript are not involved in the pausing event.

Postauricular cerebellar encephalocoele secondary to chronic suppurative otitis media and mastoid surgery.

Cerebellar herniation into the mastoid through the posterior aspect of the temporal bone as a result of chronic suppurative otitis media and mastoid surgery is a rare event. A case is reported in which such a hernia presented subcutaneously behind the pinna; its repair is discussed.

Carbohydrate metabolic studies during twelve months of treatment with a low-dose combination oral contraceptive.

Carbohydrate metabolism was evaluated in twenty healthy women volunteers using a low-dose combination oral contraceptive (OC) containing 30 micrograms of ethinyl estradiol and 500 micrograms of dl-norgestrel by measurement of serum glucose and insulin levels during 3-hour oral glucose tolerance test (OGTT) before and after 3, 6 and 12 months of medication. There were no significant differences in body weight or blood pressure between pretreatment and posttreatment. Fasting serum glucose levels were slightly reduced, though not significantly, during all periods of treatment. But serum glucose levels were increased at 1, 2 and 3 hours in association with high insulin responses during OGTT in all periods of OC therapy, indicating mild to moderate insulin resistance. These data suggest that the low-dose combination OC used in the study exerts alterations on carbohydrate metabolism in women during one year of OC use.

PIP: Carbohydrate metabolism was evaluated in 20 health women volunteers using a low-dose combination oral contraceptive (OC) containing 30 mcg ethinyl estradiol +500 mcg of dl-norgestrel by measurement of serum glucose and insulin levels during a 3-hour oral glucose tolerant test (OGTT). The test was conducted before and after 3, 6, and 12 months of medication. There were no significant differences in body weight or blood pressure between pretreatment and posttreatment levels. Fasting serum glucose levels were slightly reduced, although not significantly, during all periods of treatment. But serum glucose levels were increased at 1, 2, and 3 hours in association with high insulin responses during OGTT in all periods of OC therapy, indicating mild to moderate insulin resistance. These data suggest that the low-dose combination OC used in the study exerts alterations on carbohydrate metabolism in women during 1 year of OC use.

Sites of initiation and pausing in the Escherichia coli rnpB (M1 RNA) transcript.

DNA sequences affecting the transcription of the Escherichia coli rnpB transcript encoding the catalytic M1 RNA subunit of RNase P have been analyzed. Previous work (Motamedi, H., Lee, Y., and Schmidt, F.J.) (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 3959-3963) identified S1 nuclease protection products corresponding to transcripts originating upstream of the M1 RNA gene. Sequence analysis of the upstream region of rnpB identified three regions homologous to the E. coli consensus promoter sequence. In the present work, analysis of in vitro transcription products by S1 nuclease mapping indicated that all three promoter homologies were capable of directing transcription. The nearest promoter, P-1, was approximately 100 times more active than either of the upstream homologies P-2 and vivo experiments, wherein the three promoter homologies preceding rnpB were cloned into the galactokinase (GalK) expression vector pKO100. The promoter homology nearest to the M1 RNA gene directed the synthesis of GalK above background. The upstream promoter homologies did not direct the synthesis of GalK at a level greater than 1% of transcription from P-1. Deletion of the upstream homologies did not affect transcription from P-1. It was concluded that P-1 is responsible for essentially all M1 RNA transcription in vivo. Single-round transcription experiments in vitro detected strong NusA-independent transcriptional pausing at nucleotides +118 and +121 of the rnpB transcript, with a half-life of 27 s when concentrations of NTPs were near the average Km for elongation. Pausing at these points was eliminated by substitution of ITP for GTP in the transcription mixture. This suggests that pausing is dependent on transcript secondary structure. The position of pausing corresponds to that of a dual stem and loop structure of M1 RNA which has recently been proposed on the basis of phylogenetic sequence analysis.