New Methodology for Accurate Determination of Molecular Co-localization at the Nanoscale.

A new methodology using nanoparticle projectile secondary ion mass spectrometry was developed to identify statistically significant co-localization of tagged proteins versus random aggregations at the nanoscale. The custom instrument was run in the unique event-by-event bombardment detection mode with 1040 keV Au28008+ individual projectiles each probing an area with a diameter of ∼20 nm. In a model experiment, antibodies tagged with fluorine, iodine, and bromine were attached on a silicon wafer in a 1:1:1 ratio. To determine whether the three different antibodies were homogeneously distributed at the nanoscale or if there were fluctuations due to the slightly different physical properties of the tags, a "co-localization factor" was introduced. It is shown for the first time that the differences in the hydrophobicity of the tags induced fluctuations, causing differential attachment of the tags at the nanoscale. When tags with the same physical and chemical properties were used, the analysis of co-localization factors shows that the attachment became random.

Trigeminal autonomic cephalalgias.

The trigeminal autonomic cephalalgias (TACs) are a group of primary headache disorders characterised by lateralized symptoms: prominent headache and ipsilateral cranial autonomic features, such as conjunctival injection, lacrimation and rhinorrhea. The TACs are: cluster headache (CH), paroxysmal hemicrania (PH), short-lasting unilateral neuralgiform headache attacks with conjunctival injection and tearing (SUNCT)/short-lasting neuralgiform headache attacks with cranial autonomic features (SUNA) and hemicrania continua (HC). Their diagnostic criteria are outlined in the International Classification of Headache Disorders, third edition-beta (ICHD-IIIb). These conditions are distinguished by their attack duration and frequency, as well as response to treatment. HC is continuous and by definition responsive to indomethacin. The main differential when considering this headache is chronic migraine. Other TACs are remarkable for their short duration and must be distinguished from other short-lasting painful conditions, such as trigeminal neuralgia and primary stabbing headache. Cluster headache is characterised by exquisitely painful attacks that occur in discrete episodes lasting 15-180 min a few times a day. In comparison, PH occurs more frequently and is of shorter duration, and like HC is responsive to indomethacin. SUNCT/SUNA is the shortest duration and highest frequency TAC; attacks can occur over a hundred times every day.

The relationship between parental genetic or phenotypic divergence and progeny variation in the maize nested association mapping population.

Appropriate selection of parents for the development of mapping populations is pivotal to maximizing the power of quantitative trait loci detection. Trait genotypic variation within a family is indicative of the family's informativeness for genetic studies. Accurate prediction of the most useful parental combinations within a species would help guide quantitative genetics studies. We tested the reliability of genotypic and phenotypic distance estimators between pairs of maize inbred lines to predict genotypic variation for quantitative traits within families derived from biparental crosses. We developed 25 families composed of ~200 random recombinant inbred lines each from crosses between a common reference parent inbred, B73, and 25 diverse maize inbreds. Parents and families were evaluated for 19 quantitative traits across up to 11 environments. Genetic distances (GDs) among parents were estimated with 44 simple sequence repeat and 2303 single-nucleotide polymorphism markers. GDs among parents had no predictive value for progeny variation, which is most likely due to the choice of neutral markers. In contrast, we observed for about half of the traits measured a positive correlation between phenotypic parental distances and within-family genetic variance estimates. Consequently, the choice of promising segregating populations can be based on selecting phenotypically diverse parents. These results are congruent with models of genetic architecture that posit numerous genes affecting quantitative traits, each segregating for allelic series, with dispersal of allelic effects across diverse genetic material. This architecture, common to many quantitative traits in maize, limits the predictive value of parental genotypic or phenotypic values on progeny variance.

Molecular characterization of a new lytic bacteriophage isolated from cheese whey.

In this study, we isolated and characterized a lytic Lactococcus lactis bacteriophage from the sera of a failed fermentation. The phage was isolated and cultured in L. lactis subsp. cremoris in M17 medium. The isolated bacteriophage was characterized by multiplex PCR, pulsed-field electrophoresis, DNA restriction digestion, analysis of the N-terminal sequence of the phage major structural protein, transmission electron microscopy and sequencing and analysis of a conserved fragment of its genome. Analysis of the viral genome indicates that its genome is composed of a DNA strand of approximately 48 kb in length, and PCR and microscopy confirmed that IL-P1 belongs to the group of 936-type phages in the family Siphoviridae, which is the most abundant type of lactococcal virus in dairy products worldwide. To our knowledge, this is the first report of a virus within this family that has a presumptive genome larger than 40 kb.

On the Surface Mapping using Individual Cluster Impacts.

This paper describes the advantages of using single impacts of large cluster projectiles (e.g. C(60) and Au(400)) for surface mapping and characterization. The analysis of co-emitted time-resolved photon spectra, electron distributions and characteristic secondary ions shows that they can be used as surface fingerprints for target composition, morphology and structure. Photon, electron and secondary ion emission increases with the projectile cluster size and energy. The observed, high abundant secondary ion emission makes cluster projectiles good candidates for surface mapping of atomic and fragment ions (e.g., yield >1 per nominal mass) and molecular ions (e.g., few tens of percent in the 500 < m/z < 1500 range).

Incidence and risk factors of surgical wound infection in children: a prospective study.

BACKGROUND AND AIMS: to establish the incidence and risk factors of surgical site infection (SSI) in children operated in the Department of Paediatric Surgery of the Clinic of Surgery of Tartu University Hospital. MATERIAL AND METHODS: the data of wound healing were obtained for 589 children operated between 15 March 2003 and 31 March 2005. The operations were divided into general surgical (451), orthopaedic (70) and urological (68). The surgical wounds were classified as clean (442), clean-contaminated (96), contaminated (36) and dirty-infected (15). Univariate and multivariate analyses were performed to identify risk factors. RESULTS: There were 7 SSI cases, overall rate being 1.2%. Superficial wound infection occurred in 5 cases and deep wound infection occurred in 2 cases. There was no organ/space infection. SSI was significantly more frequent in the case of contaminated and dirty-infected compared with clean or clean-contaminated operations, 7.8% and 0.6%, respectively (p = 0.0008). Wound infection endangered more children who had operation related complications compared with non-complicated cases, 11.1% and 0.4%, respectively (p < 0.0001).

High-throughput genotyping of KIR2DL2/L3, KIR3DL1/S1, and their HLA class I ligands using real-time PCR.

Killer immunoglobulin-like receptors (KIRs) expressed on natural killer cells are critical components of innate immunity. Interactions between KIRs and their human leukocyte antigen (HLA) ligands have been shown to influence autoimmune and infectious disease course in defined populations. However, the low throughput and high cost of current methods impede confirmation of the universality of these findings. To support large epidemiology surveys, we developed a high-throughput real-time polymerase chain reaction-based assay to identify carriers of KIR3DL1, KIR3DS1, KIR2DL2, and KIR2DL3 and their HLA ligands. The platform performed with 100% sensitivity and specificity in detection of carrier and non-carrier on reference samples. The application of this platform will further clarify the nature and impact of the KIR-HLA epistatic interaction on disease course in large global population-based studies.

HLA class I allele and haplotype diversity in Ugandans supports the presence of a major east African genetic cluster.

The objective of this study was to characterize the class I human leukocyte antigen (HLA) genetic composition of the Ugandan population to better define its relationship with other African groups. Samples from 175 individuals from Kampala (Uganda) were subjected to class I HLA-A, -B, and -C sequence-based typing. The high concordance between the major alleles and haplotypes found in the current and Kenyan populations and interpopulation genetic distance analysis strongly supported the presence of an East African cluster that contained the current Ugandan population along with Kenyan Luo and Nandi populations. The congruence of major alleles in different populations would permit consideration of East Africa as an integrated setting when designing and evaluating much needed malaria, tuberculosis, and AIDS vaccines.

Evidence for nerve growth factor-mediated paracrine effects in human epidermis.

Nerve growth factor (NGF) is critical to the development and maintenance of the peripheral nervous system, but its possible roles in other organ systems are less well characterized. We have recently shown that human epidermal melanocytes, pigment cells derived from the neural crest, express the NGF receptor (p75 NGF-R) in vitro (Peacocke, M., M. Yaar, C. P. Mansur, M. V. Chao, and B. A. Gilchrest. 1988. Proc. Natl. Acad. Sci. USA. 85:5282-5286). Using cultured human skin-derived cells we now demonstrate that the melanocyte p75 NGF-R is functional, in that NGF stimulation modulates melanocyte gene expression; that exposure to an NGF gradient is chemotactic for melanocytes and enhances their dendricity; and that keratinocytes, the dominant epidermal cell type, express NGF messenger RNA and hence are a possible local source of NGF for epidermal melanocytes in the skin. These combined data suggest a paracrine role for NGF in human epidermis.

Rats with high or low sociability are differently affected by chronic variable stress.

Depression is strongly related to social behavior. We have previously shown that social behavior of rats is individually stable. The purpose of the present study was to compare the sensitivity of animals with different sociability to chronic variable stress (CVS). Four social interaction tests were performed with 60 single-housed male Sprague-Dawley rats. Twenty rats with the lowest and 20 with the highest average social activity time were selected as low sociability (LS) and high sociability (HS) rats, respectively. Both groups were further divided into control and stress groups with equal average body weight. The CVS procedure lasted for 3 weeks. The stressors applied were cold water and wet bedding, imitation of injection, stroboscopic light, movement restriction in a small cage, tail pinch with a clothespin, and strong illumination during the predicted dark phase. In HS-rats, but not in LS-rats, CVS reduced sucrose intake compared with baseline after 3 weeks, suggesting that HS-rats are more vulnerable to anhedonia elicited by CVS. LS-animals were more anxious in the social interaction and open field tests, but stress eliminated differences with HS-animals in the social interaction test and increased their activity in the forced swimming test. In LS-rats stress increased ex vivo dopamine levels and reduced 5-HT levels in the frontal cortex, suggesting that the increased behavioral activity after stress may be related to increased impulsivity. This study thus revealed that animals with high sociability trait are more vulnerable to anhedonia elicited by chronic stress in conditions of single housing.

The trk family of receptors mediates nerve growth factor and neurotrophin-3 effects in melanocytes.

We have recently shown that (a) human melanocytes express the p75 nerve growth factor (NGF) receptor in vitro; (b) that melanocyte dendricity and migration, among other behaviors, are regulated at least in part by NGF; and (c) that cultured human epidermal keratinocytes produce NGF. We now report that melanocyte stimulation with phorbol 12-tetra decanoate 13-acetate (TPA), previously reported to induce p75 NGF receptor, also induces trk in melanocytes, and TPA effect is further potentiated by the presence of keratinocytes in culture. Moreover, trk in melanocytes becomes phosphorylated within minutes after NGF stimulation. As well, cultures of dermal fibroblasts express neurotrophin-3 (NT-3) mRNA; NT-3 mRNA levels in cultured fibroblasts are modulated by mitogenic stimulation, UV irradiation, and exposure to melanocyte-conditioned medium. Moreover, melanocytes constitutively express low levels of trk-C, and its expression is downregulated after TPA stimulation. NT-3 supplementation to cultured melanocytes maintained in Medium 199 alone prevents cell death. These combined data suggest that melanocyte behavior in human skin may be influenced by neurotrophic factors, possibly of keratinocyte and fibroblast origin, which act through high affinity receptors.

Evaluation of aldrithiol-2-inactivated preparations of HIV type 1 subtypes A, B, and D as reagents to monitor T cell responses.

The development of HIV vaccines is an urgent priority and there is need to generate reagents representing multiple subtypes that can be used to screen HIV-1-specific responses. We used Aldrithiol-2 (AT-2), a mild oxidizing reagent, to eliminate the infectivity of HIV while maintaining its structure and ability to be processed for presentation to T cells. Inactivated subtype A, B, and D viruses were evaluated for their ability to stimulate T cell responses in PBMC samples from 18 U.S. subjects infected with HIV-1 subtype B and 32 Ugandan subjects infected with subtypes A and D or recombinants AC and AD. Five HIV-1-negative samples were also analyzed. T cell responses to AT-2-inactivated viral isolates were monitored by interferon-gamma (IFN-gamma) intracellular cytokine secretion (ICS) analysis; matched microvesicle preparations served as negative controls. Among the 18 subtype B infected subjects, 39% had CD3(+) CD4 (+) IFN-gamma responses and 67% had CD3(+) CD8(+) IFN-gamma responses. Of the 32 Ugandan subjects, 34% demonstrated CD3(+) CD4(+) IFN-gamma responses and 78% demonstrated CD3(+) CD8(+) IFN-gamma responses. Both subtype-specific and cross-reactive responses were observed. Responses to the AT-2 viruses tended to be lower in magnitude than those detected by a set of overlapping gag peptides. Robust lymphoproliferative responses to AT-2 viruses were seen in a subset of subjects. In conclusion, AT-2-inactivated HIV-1 virions stimulated both CD4 and CD8 HIV-1-specific responses and may provide an additional reagent for screening HIV-1-specific responses in HIV seropositives and vaccinees.

Relevance of group Milleri streptococci in thoracic surgery: a clinical update.

Group Milleri streptococci (GMS), a heterogeneous group of streptococci, are associated with purulent infections. This study was a retrospective analysis of all consecutive thoracic infections of GMS between 2001 and 2004. Of 246 surgical GMS infections, thoracic infections accounted for 4.5 per cent, including 10 pleural infections (eight empyemas and two infected pleural effusions) and one mediastinal infection. The etiology of pleural infection was parapneumonic (7), second to esophageal perforation (1), liver transplantation (1), and liver resection (1). Polymicrobial infections were present in 64 per cent. All patients underwent removal of the infected masses, including drainage (3), thoracoscopic decortication (5), thoracotomy with debridement (2), and incision with drainage (1). The case fatality rate was 9 per cent (there was one patient with congestive heart disease unfit to undergo surgical empyema evacuation) and the recurrence rate was 27.3 per cent (three patients). Combined antibiotic/surgical treatment was successful in all other cases. GMS isolates were susceptible to clindamycin and all beta-lactam antibiotics except ceftazidime, but were resistant to aminoglycosides. If found intrathoracically, GMS frequently progress to severe empyema. Therefore, timely removal of pleural collection by percutaneous drainage or surgical intervention seems indicated. If surgery is required, thoracoscopic decortication may be the preferred approach.

HIVIS-DNA or HIVISopt-DNA priming followed by CMDR vaccinia-based boosts induce both humoral and cellular murine immune responses to HIV.

BACKGROUND: In order to develop a more effective prophylactic HIV-1 vaccine it is important optimize the components, improve Envelope glycoprotein immunogenicity as well as to explore prime-boost immunization schedules. It is also valuable to include several HIV-1 subtype antigens representing the world-wide epidemic. METHODS: HIVIS-DNA plasmids which include Env genes of subtypes A, B and C together with Gag subtypes A and B and RTmut/Rev of subtype B were modified as follows: the Envelope sequences were shortened, codon optimized, provided with an FT4 sequence and an immunodominant region mutated. The reverse transcriptase (RT) gene was shortened to contain the most immunogenic N-terminal fragment and fused with an inactivated viral protease vPR gene. HIVISopt-DNA thus contains fewer plasmids but additional PR epitopes compared to the native HIVIS-DNA. DNA components were delivered intradermally to young Balb/c mice once, using a needle-free Biojector® immediately followed by dermal electroporation. Vaccinia-based MVA-CMDR boosts including Env gene E and Gag-RT genes A were delivered intramuscularly by needle, once or twice. RESULTS: Both HIVIS-DNA and HIVISopt-DNA primed humoral and cell mediated responses well. When boosted with heterologous MVA-CMDR (subtypes A and E) virus inhibitory neutralizing antibodies were obtained to HIV-1 subtypes A, B, C and AE. Both plasmid compositions boosted with MVA-CMDR generated HIV-1 specific cellular responses directed against HIV-1 Env, Gag and Pol, as measured by IFNγ ELISpot. It was shown that DNA priming augmented the vector MVA immunological boosting effects, the HIVISopt-DNA with a trend to improved (Env) neutralization, the HIVIS-DNA with a trend to better (Gag) cell mediated immune reponses. CONCLUSIONS: HIVIS-DNA was modified to obtain HIVISopt-DNA that had fewer plasmids, and additional epitopes. Even with one DNA prime followed by two MVA-CMDR boosts, humoral and cell-mediated immune responses were readily induced by priming with either DNA construct composition. Priming by HIV-DNA augmented neutralizing antibody responses revealed by boosting with the vaccinia-based heterologous sequences. Cellular and antibody responses covered selected strains representing HIV-1 subtypes A, B, C and CRF01_AE. We assume this is related to the inclusion of heterologous full genes in the vaccine schedule.

Effects of low dose N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine administration on exploratory and amphetamine-induced behavior and dopamine D2 receptor function in rats with high or low exploratory activity.

Individual differences in behavioral traits are associated with sensitivity to various neurochemical and psychopharmacological manipulations. In this study exploratory and amphetamine-induced behavior in rats with persistently high or low exploratory activity (HE and LE, respectively) was examined before and after a partial denervation of the locus coeruleus (LC) projections with the selective neurotoxin DSP-4 (N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine; 10 mg/kg). Partial LC denervation prevented the increase in exploratory activity over repeated test sessions in the LE animals, but had no effect in HE-rats. Amphetamine- (0.5 mg/kg) induced locomotor activity was attenuated by DSP-4 pretreatment only in HE-rats. These results suggest differential involvement of LC noradrenergic transmission in novelty- and amphetamine-induced behavior in animals with persistent differences in novelty-related behavior. In addition to partial noradrenaline depletion in the frontal cortex and hippocampus, which occurred in both HE- and LE-rats, DSP-4 treatment also decreased the content of dopamine and its metabolites in the nucleus accumbens, and the metabolite levels in striatum, but only in the LE-animals. 5-HIAA levels were also reduced in the nucleus accumbens and striatum in LE-rats by the neurotoxin. D(2) receptor function, as determined by dopamine-stimulated [(35)S]GTPgammaS binding, was increased by DSP-4 treatment in the striatum of LE-rats, but reduced in HE-rats. No effect of partial LC denervation was found on dopamine-stimulated [(35)S]GTPgammaS binding in the nucleus accumbens. Together these findings suggest that LC noradrenergic neurotransmission is differently involved in dopaminergic mechanisms which mediate novelty-related vs amphetamine-induced behavior.

Mass Spectrometry of Nanoparticles is Different.

Secondary ion mass spectrometry, SIMS, is a method of choice for the characterization of nanoparticles, NPs. For NPs with large surface-to-volume ratios, heterogeneity is a concern. Assays should thus be on individual nano-objects rather than an ensemble of NPs; however, this may be difficult or impossible. This limitation can be side-stepped by probing a large number of dispersed NPs one-by-one and recording the emission from each NP separately. A large collection of NPs will likely contain subsets of like-NPs. The experimental approach is to disperse the NPs and hit an individual NP with a single massive cluster (e.g., C-60, Au-400). At impact energies of ~1 keV/atom, they generate notable secondary ion (SI) emission. Examination of small NPs (≤20 nm in diameter) shows that the SI emission is size-dependent and impacts are not all equivalent. Accurate identification of the type of impact is key for qualitative assays of core or outer shell composition. For quantitative assays, the concept of effective impacts is introduced. Selection of co-emitted ejecta combined with rejection (anticoincidence) of substrate ions allows refining chemical information within the projectile interaction volume. Last, to maximize the SI signal, small NPs (≤5 nm in diameter) can be examined in the transmission mode where the SI yields are enhanced ~10-fold over those in the (conventional) reflection direction. Future endeavors should focus on schemes acquiring SIs, electrons, and photons concurrently.

Retinoids and state of differentiation modulate CRABP II gene expression in a skin equivalent.

Cellular retinoic acid-binding proteins (CRABPs) are a family of proteins that specifically bind retinoic acid (RA) and have been implicated in mediating its action, although their exact function is still unknown. Two CRABPs have been identified and cloned. CRABP I is present in many tissues and cultured cells; CRABP II, first detected in embryonic and neonatal skin of rats and chicks, is now recognized as the predominant form in human epidermis. Using a human living skin equivalent model composed of a dermis and an epidermis and human cDNAs recently cloned in our laboratory, we have studied the effects of 10(-6) M RA and etretin (ET) on the expression of CRABPs under different culture conditions intended to favor greater or lesser degrees of epidermal differentiation. Total cellular RNA was isolated separately from the dermis and epidermis and processed for northern blot analysis. At a presumptive physiologic RA concentration, only the gene for CRABP II, and not for CRABP I, was expressed. CRABP II transcripts were far more abundant on a per cell basis in epidermal keratinocytes than in dermal fibroblasts under all conditions studied. Epidermal differentiation, stimulated by air exposure of the cultures, tended to enhance CRABP II expression, and treatment with presumptive therapeutic concentrations of the two retinoid compounds tended to decrease CRABP II expression. Opposite effects of air exposure and retinoid treatment were observed on steady state levels of mRNA for selected markers of epidermal differentiation: involucrin, transglutaminase, and spr I. These results are consistent with earlier work at the protein level examining the effect of retinoids on CRABP activity and state of differentiation both in vivo and in vitro. Thus, the skin equivalent appears to be an excellent model system for investigating the role of CRABPs in mediating retinoid effects at the cellular and molecular levels.

Epidermal differentiation enhances CRABP II expression in human skin.

The cellular retinoic acid-binding proteins (CRABP I and II) are thought to mediate the effects of retinoic acid on target cells. We have used riboprobes complementary to CRABP I and II mRNAs to study the expression of these messages in normal and abnormal human skin. CRABP II was expressed predominantly in the suprabasal layers of the epidermis, with stronger expression in newborn than in sun-protected adult skin. Interestingly, the epidermis adjacent to or overlying squamous cell or basal cell carcinomas also showed strong expression, whereas the tumor cells were negative, with the exception of more differentiated cells surrounding the "keratin pearls" within squamous cell carcinomas. CRABP II mRNA was also found in the more differentiated cells of the hair follicles, in the outer root sheath. CRABP I message was undetectable in the epidermis or in the dermis of normal skin but was detected in the cells of the papillary dermis surrounding basal and squamous cell carcinomas. These data suggest that increased levels of CRABP II mRNA accompany keratinocyte differentiation in vivo.

Thymidine dinucleotide mimics the effect of solar simulated irradiation on p53 and p53-regulated proteins.

The tumor suppressor protein p53 participates in DNA repair and cell cycle regulation in response to injuries like ultraviolet (UV) irradiation. We have previously reported that the thymidine dinucleotide (pTpT), a common target for DNA photoproduct formation by UV light, mimics many effects of UV irradiation in cultured skin-derived cells, at least in part through the activation of p53. In this report we compare the effects of solar-simulated irradiation and pTpT on p53 and p53-regulated proteins involved in cellular growth arrest and DNA repair in cultured human dermal fibroblasts. We find that, like UV irradiation, pTpT increases the levels of p53, p21, and proliferating-cell nuclear antigen. The magnitude and time course of the inductions are UV dose dependent and consistent with known regulatory interactions among these nuclear proteins. These data confirm and expand previous studies of UV effects on nuclear proteins involved in cell cycle regulation and DNA repair. Our observations suggest that such protective effects can also be induced by pTpT in the absence of initial DNA damage, rendering cells more capable of responding to subsequent DNA damage.

Treatment of human melanocytes and S91 melanoma cells with the DNA repair enzyme T4 endonuclease V enhances melanogenesis after ultraviolet irradiation.

Tanning is a protective response of ultraviolet (UV)-irradiated skin that decreases damage from subsequent sun exposures by increasing the epidermal content of melanin, a brown-black pigment that absorbs light energy throughout the UV and visible portions of the electromagnetic spectrum. The melanin pigment is made by epidermal melanocytes and transferred to surrounding keratinocytes. The action spectrum, time course, and histologic features of tanning are well studied, but the initiating molecular events are unknown. Previous work has shown that T4 endonuclease V, a prokaryotic DNA repair enzyme that catalyzes the first and rate-limiting step in repair of UV-induced pyrimidine dimers, delivered in carrier liposomes (T4N5), enhances repair of UV-induced DNA damage in cultured human cells and protects against photocarcinogenesis in an animal model. We now report that T4N5 treatment enhances UV-induced melanogenesis, as measured by melanin content, tyrosinase activity, 14C-dopa incorporation, and visual assessment in both S91 murine melanoma cells and human melanocytes. T4N5 treatment also increases cell yields following UV irradiation. These data suggest that tanning can be stimulated through enhanced DNA repair.