Ordered Mesoporous Electrodes for Sensing Applications.

Electrochemical sensors have become increasingly relevant in fields such as medicine, environmental monitoring, and industrial process control. Selectivity, specificity, sensitivity, signal reproducibility, and robustness are among the most important challenges for their development, especially when the target compound is present in low concentrations or in complex analytical matrices. In this context, electrode modification with Mesoporous Thin Films (MTFs) has aroused great interest in the past years. MTFs present high surface area, uniform pore distribution, and tunable pore size. Furthermore, they offer a wide variety of electrochemical signal modulation possibilities through molecular sieving, electrostatic or steric exclusion, and preconcentration effects which are due to mesopore confinement and surface functionalization. In order to fully exploit these advantages, it is central to develop reproducible routes for sensitive, selective, and robust MTF-modified electrodes. In addition, it is necessary to understand the complex mass and charge transport processes that take place through the film (particularly in the mesopores, pore surfaces, and interfaces) and on the electrode in order to design future intelligent and adaptive sensors. We present here an overview of MTFs applied to electrochemical sensing, in which we address their fabrication methods and the transport processes that are critical to the electrode response. We also summarize the current applications in biosensing and electroanalysis, as well as the challenges and opportunities brought by integrating MTF synthesis with electrode microfabrication, which is critical when moving from laboratory work to in situ sensing in the field of interest.

Safflower: A New Host of Cercospora beticola.

Safflower is an oilseed crop adapted to the small-grain production areas of the western Great Plains, including the Northern Plains Area (NPA). In the NPA, safflower production is being evaluated for potential rotation with sugar beet. Safflower is susceptible to Cercospora carthami, whereas sugar beet is susceptible to C. beticola C. carthami has not been observed on safflower in the NPA but C. beticola is ubiquitous on sugar beet. Observation of unusual leaf spots on irrigated safflower cv. Centennial at Sidney, MT prompted this investigation of safflower as a potential alternate host of C. beticola. Safflower plants were inoculated with four isolates of C. beticola (C1, C2, Sid1, and Sid2) and incubated in growth chambers; leaf spot symptoms appeared between 3 and 4 weeks later. Polymerase chain reaction (PCR) amplification of extracts from lesion leaf tissue with C. beticola-specific primers produced fragments comparable with amplified fragments from purified cultures of control C. beticola. PCR assay of cultures of single spores from diseased safflower leaf lesions also produced fragments comparable with fragments from C. beticola cultures. Antibody that was raised from isolate C2 also bound to antigens from the single-spore cultures of the four C. beticola isolates. Inoculum from single-spore cultures from infected safflower also infected sugar beet and produced typical Cercospora leaf spot symptoms. Assay of these leaf lesions by PCR resulted in amplification of target fragments with the C. beticola-specific primers. Our results demonstrate that safflower is a new host of C. beticola.

Possible interactions between the NS-1 protein and tumor necrosis factor alpha pathways in erythroid cell apoptosis induced by human parvovirus B19.

Human erythroid progenitor cells are the main target cells of the human parvovirus B19 (B19), and B19 infection induces a transient erythroid aplastic crisis. Several authors have reported that the nonstructural protein 1 (NS-1) encoded by this virus has a cytotoxic effect, but the underlying mechanism of NS-1-induced primary erythroid cell death is still not clear. In human erythroid progenitor cells, we investigated the molecular mechanisms leading to apoptosis after natural infection of these cells by the B19 virus. The cytotoxicity of NS-1 was concomitantly evaluated in transfected erythroid cells. B19 infection and NS-1 expression induced DNA fragmentation characteristic of apoptosis, and the commitment of erythroid cells to undergo apoptosis was combined with their accumulation in the G(2) phase of the cell cycle. Since B19- and NS-1-induced apoptosis was inhibited by caspase 3, 6, and 8 inhibitors, and substantial caspase 3, 6, and 8 activities were induced by NS-1 expression, there may have been interactions between NS-1 and the apoptotic pathways of the death receptors tumor necrosis factor receptor 1 and Fas. Our results suggest that Fas-FasL interaction was not involved in NS-1- or B19-induced apoptosis in erythroid cells. In contrast, these cells were sensitized to tumor necrosis factor alpha (TNF-alpha)-induced apoptosis. Moreover, the ceramide level was enhanced by B19 infection and NS-1 expression. Therefore, our results suggest that there may be a connection between the respective apoptotic pathways activated by TNF-alpha and NS-1 in human erythroid cells.

Determinants for sensitivity of human immunodeficiency virus coreceptor CXCR4 to the bicyclam AMD3100.

The bicyclam AMD3100 is a potent and selective inhibitor of the replication of human immunodeficiency virus type 1 and type 2 (HIV-1 and HIV-2). It was recently demonstrated that the compound inhibited HIV entry through CXCR4 but not through CCR5. Selectivity of AMD3100 for CXCR4 was further indicated by its lack of effect on HIV-1 and HIV-2 infection mediated by the CCR5, CCR3, Bonzo, BOB, and US28, coreceptors. AMD3100 completely blocked HIV-1 infection mediated by a mutant CXCR4 bearing a deletion of most of the amino-terminal extracellular domain. In contrast, relative resistance to AMD3100 was conferred by different single amino acid substitutions in the second extracellular loop (ECL2) or in the adjacent membrane-spanning domain, TM4. Only substitutions of a neutral residue for aspartic acid and of a nonaromatic residue for phenylalanine (Phe) were associated with drug resistance. This suggests a direct interaction of AMD3100 with these amino acids rather than indirect effects of their mutation on the CXCR4 structure. The interaction of aspartic acids of ECL2 and TM4 with AMD3100 is consistent with the positive charge of bicyclams, which might block HIV-1 entry by preventing electrostatic interactions between CXCR4 and the HIV-1 envelope protein gp120. Other features of AMD3100 must account for its high antiviral activity, in particular the presence of an aromatic linker between the cyclam units. This aromatic group might engage in hydrophobic interactions with the Phe-X-Phe motifs of ECL2 or TM4. These results confirm the importance of ECL2 for the HIV coreceptor activity of CXCR4.

The rhesus macaque CCR3 chemokine receptor is a cell entry cofactor for HIV-2, but not for HIV-1.

The eotaxin receptor (CCR3) is a CD4-associated coreceptor for human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2). By comparison with other chemokine receptors, such as CCR5 and CXCR4, the primary sequences of human CCR3 and its rhesus macaque homolog were markedly different in their extracellular domains. Human CD4+ cells expressing CCR3 from either human or macaque origin could be infected by HIV-2, with apparently similar efficiency, but only cells expressing human CCR3 could be infected by HIV-1. It suggests that HIV-1 and HIV-2 envelope proteins interact differently with the CCR3 coreceptor HIV-1 could infect cells expressing chimeric human/macaque CCR3 bearing either the first and second, or the third and fourth extracellular domains of human CCR3. As previously observed for CCR5, there seems to be a certain functional redundancy between domains supporting the coreceptor activity of CCR3. In spite of their close genetic relationship to HIV-2, two macaque simian immunodeficiency virus strains were apparently unable to use the CCR3 coreceptor from either human or simian origin.

A biometric study on the relationships between the deep palmar arch and the superficial palmar arch, the distal wrist and palmar creases.

The topographical localisation of the palmar arches is important in hand surgery. The aim of this study was to contribute with biometric data on their positions and to correlate this with the size od the palm. We studied 60 hands of 30 adult fixed cadavers, of Brazilian origin, from both sexes and between the ages of 21 and 70 years. The arteries of 54 hands were injected with latex Neoprene. Before dissection the distance between distal wrist crease (DWC) and the proximal palmar digital crease of the middle finger (PDMC) was measured. Also, we recorded the distance between the DWC and the proximal and distal palmar creases (PPC, DPC). After dissecting the superficial palmar region, the distance between the superficial palmar arch (SPA) and the DWC was recorder. We then dissected the deep palmar arch (DPA). The average distance between the DPA and DWC was always measured in the midline of the palm. The average distance DWC-DPA was 33.7 +/- 2.6 mm in the female and 36 +/- 4.0 mm in the male. The difference was statistically significant. The average distance between DPA and the PPC was 24.2 +/- 3.0 mm in the female and 27.1 +/- 4.1 mm in the male; this difference was significant. In 83% of cases the DPA was proximal to the SPA and in 14.9% was distal to it. The linear regression test for the relation between DWC-PDMC and DWC-DPA was significant in the male and this fact allowed us to obtain the linear equation to predict the distance DWC-DPA. Other parameters were also considered. The results may be useful as a reference to radiologists as well as to surgeons.

Resistance to a drug blocking human immunodeficiency virus type 1 entry (RPR103611) is conferred by mutations in gp41.

A triterpene derived from betulinic acid (RPR103611) blocks human immunodeficiency virus type 1 (HIV-1) infection and fusion of CD4+ cells with cells expressing HIV-1 envelope proteins (gp120 and gp41), suggesting an effect on virus entry. This compound did not block infection by a subtype D HIV-1 strain (NDK) or cell-cell fusion mediated by the NDK envelope proteins. The genetic basis of drug resistance was therefore addressed by testing envelope chimeras derived from NDK and a drug-sensitive HIV-1 strain (LAI, subtype B). A drug-resistant phenotype was observed for all chimeras bearing the ectodomain of NDK gp41, while the origins of gp120 and of the membrane anchor and cytoplasmic domains of gp41 had no apparent role. The envelope gene of a LAI variant, fully resistant to the antiviral effect of RPR103611, was cloned and sequenced. Its product differed from the parental sequence at two positions in gp41, with changes of arginine 22 to alanine (R22A) and isoleucine 84 to serine (I84S), the gp120 being identical. In the context of LAI gp41, the I84S substitution was sufficient for drug resistance. Therefore, in two different systems, differences in gp41 were associated with sensitivity or resistance to RPR103611. Modifications of gp41 can affect the quaternary structure of gp120 and gp41 and the accessibility of gp120 to antiviral agents such as neutralizing antibodies. However, a direct effect of RPR103611 on a gp41 target must also be envisioned, in agreement with the blocking of apparently late steps of HIV-1 entry. This compound could be a valuable tool for structure-function studies of gp41.

Usage of the coreceptors CCR-5, CCR-3, and CXCR-4 by primary and cell line-adapted human immunodeficiency virus type 2.

The chemokine receptors CCR-5 and CXCR-4, and possibly CCR-3, are the principal human immunodeficiency virus type 1 (HIV-1) coreceptors, apparently interacting with HIV-1 envelope, in association with CD4. Cell lines coexpressing CD4 and these chemokine receptors were infected with a panel of seven primary HIV-2 isolates passaged in peripheral blood mononuclear cells (PBMC) and three laboratory HIV-2 strains passaged in T-cell lines. The CCR-5, CCR-3, and CXCR-4 coreceptors could all be used by HIV-2. The ability to use CXCR-4 represents a major difference between HIV-2 and the closely related simian immunodeficiency viruses. Most HIV-2 strains using CCR-5 could also use CCR-3, sometimes with similar efficiencies. As observed for HIV-1, the usage of CCR-5 or CCR-3 was observed principally for HIV-2 strains derived from asymptomatic individuals, while HIV-2 strains derived from AIDS patients used CXCR-4. However, there were several exceptions, and the patterns of coreceptor usage seemed more complex for HIV-2 than for HIV-1. The two T-tropic HIV-2 strains tested used CXCR-4 and not CCR-5, while T-tropic HIV-1 can generally use both. Moreover, among five primary HIV-2 strains all unable to use CXCR-4, three could replicate in CCR-5-negative PBMC, which has not been reported for HIV-1. These observations suggest that the CCR-5 coreceptor is less important for HIV-2 than for HIV-1 and indicate that HIV-2 can use other cell entry pathways and probably other coreceptors. One HIV-2 isolate replicating in normal or CCR-5-negative PBMC failed to infect CXCR-4+ cells or the U87MG-CD4 and sMAGI cell lines, which are permissive to infection by HIV-2 but not by HIV-1. This suggests the existence of several HIV-2-specific coreceptors, which are differentially expressed in cell lines and PBMC.

Role of the first and third extracellular domains of CXCR-4 in human immunodeficiency virus coreceptor activity.

The CXCR-4 chemokine receptor and CD4 behave as coreceptors for cell line-adapted human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and for dual-tropic HIV strains, which also use the CCR-5 coreceptor. The cell line-adapted HIV-1 strains LAI and NDK and the dual-tropic HIV-2 strain ROD were able to infect CD4+ cells expressing human CXCR-4, while only LAI was able to infect cells expressing the rat homolog of CXCR-4. This strain selectivity was addressed by using human-rat CXCR-4 chimeras. All chimeras tested mediated LAI infection, but only those containing the third extracellular domain (e3) of human CXCR-4 mediated NDK and ROD infection. The e3 domain might be required for the functional interaction of NDK and ROD, but not LAI, with CXCR-4. Alternatively, LAI might also interact with e3 but in a different way. Monoclonal antibody 12G5, raised against human CXCR-4, did not stain cells expressing rat CXCR-4. Chimeric human-rat CXCR-4 allowed us to map the 12G5 epitope in the e3 domain. The ability of 12G5 to neutralize infection by certain HIV-1 and HIV-2 strains is also consistent with the role of e3 in the coreceptor activity of CXCR-4. The deletion of most of the amino-terminal extracellular domain (e1) abolished the coreceptor activity of human CXCR-4 for ROD and NDK but not for LAI. These results indicate that HIV strains have different requirements for their interaction with CXCR-4. They also suggest differences in the interaction of dual-tropic HIV with CCR-5 and CXCR-4.

Human immunodeficiency virus strains differ in their ability to infect CD4+ cells expressing the rat homolog of CXCR-4 (fusin).

A clade B strain of human immunodeficiency virus type 1 (HIV-1(LAI)) could infect CD4+ cells expressing human CXCR-4 (fusin) or its rat homolog with similar efficacy. By contrast, cells expressing rat CXCR-4 were not permissive to HIV-1(NDK) (clade D), HIV-2(ROD), or HIV-1(LAI) with chimeric envelope protein gp120 bearing the V3 domain from HIV-1(NDK). The reciprocal chimeric gp120 (HIV-1(NDK) with V3 from HIV-1(LAI)) could mediate infection of cells expressing either human or rat CXCR-4. Genetically divergent HIV strains have different requirements for interaction with the CXCR-4 coreceptor, and the gp120 V3 domain seems to be involved in this interaction.

Possible role of the V3 domain of gp120 in resistance to an amphotericin B derivative (MS8209) blocking human immunodeficiency virus entry.

MS8209, an amphotericin B derivative blocking human immunodeficiency virus type 1 (HIV-1) entry after CD4 binding, neutralized the HIV-2 strains EHO and ROD10 but not ROD(CEM). In the V3 domain of gp120, ROD(CEM) differed from ROD10 at two positions (a threonine instead of an isoleucine at position 312 and an arginine instead of a glutamine at position 329), and drug resistance was conferred to HIV-1 by substitution of the ROD(CEM) V3 but not the ROD10 V3. V3 mutations may prevent the interaction of gp120 with MS8209 or modify the mechanism of virus entry, rendering it less accessible to neutralization.

Trans-activation of the long terminal repeat of human immunodeficiency virus type 1 by the parvovirus B19 NS1 gene product.

Persistent parvovirus B19 infections in human immunodeficiency virus type 1 (HIV-1)-infected patients have been reported. The two viruses could share common target cells. The NS1 protein of B19 regulates B19 expression and we have investigated its possible effect on the long terminal repeat (LTR) of HIV-1. In transient transfection experiments, NS1 trans-activated the expression of reporter genes under the control of the HIV-1 LTR. The effect of NS1 was apparent only in the presence of the HIV-1 Tat protein, and required intact TAR and TATA box sequences.

Comparative Training Responses to Rope Skipping and Jogging.

In brief: It has been suggested that ten minutes of rope skipping is equal to 30 minutes of jogging for improved cardiovascular efficiency. This study compared physiological adaptations to six-week programs of jogging and rope skipping. Twenty-six sedentary volunteers (17 women and nine men) aged 18 to 35 years were assigned to a jogging, rope-skipping, or control group. Training frequency was five times per week; each session was 30 minutes for the jogging group and ten minutes for the rope-skipping groups. Significant differences (p <.05) in o2 max were observed in each group. o2 max increased 5.1 ml· kg(-1)· min(-1) for the jogging group (13%) and 2.8 ml· kg(-1)· min(-1) for the rope-skipping group (7%). The rope-skipping group had higher injury and drop-out rates. It was concluded that ten minutes of rope skipping does not elicit a training response comparable to 30 minutes of jogging.

The effect of rope skipping rate on energy expenditure of males and females.

The purpose was to study the effects of skipping rate on energy expenditure and sex differences in response to rope skipping. Responses of 19 males and 11 females were measured while skipping for 5 min at 125, 135 and 145 skips . min-1. Expired air was routed through a hollow handle to collection bags to provide uninterrupted exercise. Values at the respective rates for the total sample were: VO2 (l . min-1) 2.79, 2.83, 2.85; VO2 (ml . kg-1 . min-1) 41.1, 42.0 42.5; HR (beats . min-1) 176, 177, 177; VE (l . min) 102.2, 103.5, 106.3; R 1.09, 1.07, 1.05; energy expenditure (kj . min-1) 58.6, 59.4, 60.3. Sex differences were found in that females had significantly lower VO2 both in l . min-1 and ml . kg-1 . min-1 but higher HR values than males. Comparison of VO2 values of the females to VO2max values reported for females in the literature suggested that they may have been exercising close to their maximum. There were no differences in any of the values due to skipping rate nor was there interaction between sex and rate. Retrospective cinematographic analysis on two subjects suggested that the failure to find significant differences due to rate may be due to a decrease in vertical displacement resulting in a relatively constant work output as skipping rate increased. Average MET values at the different rates ranged from 11.7 to 12.5, which supported findings from other studies that rope skipping is very strenuous exercise.