The second to fourth digit ratio (2D:4D) is used as a potential marker for prenatal androgen exposure. It is associated with many behavioral and biological variables, including fertility and sexual behavior. However, direct association between 2D:4D and reproductive success--in populations where no contraceptives are used--has not been investigated. Here, we present a study conducted among the semi-nomad Himba population living in northern Namibia. 2D:4D ratios were calculated for a sample of this population (N=99; 60 women, 39 men), and the results were correlated with age, marital status, age at first marriage, number of children, and number of marriages. As found in the majority of previous studies, males had lower 2D:4D ratios than females. The 2D:4D ratio did not correlate with number of children. Females and males with a more masculine 2D:4D were married earlier and were more likely to have a husband or wife. We suggest that mating preferences for females with masculine 2D:4D are related to masculinity of phenotypic and personality traits of such women, which are beneficial in harsh environmental conditions and/or higher facial masculinity, which influences the perceived age of an individual. At the same time, masculine (physically strong, dominant, and hardworking) males might gather resources necessary to marry their first wife earlier.

A human immunodeficiency virus type (HIV-1)-based lentiviral vector pseudotyped with the vesicular stomatitis virus envelope glycoprotein and encoding the GFP reporter gene was used to evaluate different methods of lentiviral vector titration. GFP expression, viral DNA quantification and the efficiency of vector DNA integration were assayed after infection of conventional HIV-1-permissive cell lines and human primary adult fibroblasts with the vector. We found that vector titers based on GFP expression determined by flow cytometry may vary by more than 50-fold depending on the cell type and the promoter-cell combination used. Interestingly, we observed that the viral integration process in primary HDFa cells was significantly more efficient compared to that in SupT1 or 293T cells. We propose that determination of the amount of integrated viral DNA by quantitative PCR be used in combination with the reporter gene expression assay.

Regulation of inducible nitric oxide synthase (NOS2) expression is important given the role of this enzyme in inflammation, control of infections and immune regulation. In contrast to tumour necrosis factor-α (TNF-α) alone or CD40 stimulation alone, simultaneous stimulation of mouse macrophages through CD40 ligation and TNF-α led to up-regulation of NOS2 and nitric oxide production. This response was of functional relevance because CD40/TNF-α-stimulated macrophages acquired nitric oxide-dependent anti-Leishmania major activity. CD40 plus TNF-α up-regulated NOS2 independently of interferon-γ, interferon-α/β and interleukin-1. TNF receptor-associated factor 6 (TRAF6), an adapter protein downstream of CD40, appears to be required for NOS2 up-regulation because a CD40-TRAF6 blocking peptide inhibited up-regulation of NOS2 in CD40/TNF-α-stimulated macrophages. CCAAT/enhancer-binding protein-β (C/EBPβ), a transcription factor activated by TNF-α but not CD40, was required for NOS2 up-regulation because this enzyme was not up-regulated when C/EBPβ(-/-) macrophages received CD40 plus TNF-α stimulation. These results indicate that CD40 and TNF-α co-operate to up-regulate NOS2, probably via the effect of TRAF6 and C/EBPβ.

Cytomegalovirus (CMV) infection and reactivation pose a serious threat for patients after haematopoietic stem cell transplantation. We have previously shown that CD8(+) T cells targeting different CMV epitopes correlate with protection at different threshold frequencies in those patients. To investigate if this may relate to a different quality of these cells here we analyse the T-cell receptor diversity of pp50 (245-253)/HLA-A*0101 specific CD8(+) T cells with that of CD8(+) T cells targeting various pp65 peptides. The results from this pilot study show differences in the breadth of the T-cell receptor usage of the different cell populations. We observe for the first time that the T-cell receptor Vβ CDR3 spectratypes used by CMV pp50 (245-253)/HLA-A*0101-specific CD8(+) T cells can reach higher numbers than those used by CD8(+) T cells targeting various pp65 peptides in our patient cohort. This merits further investigation into the effectiveness of the different CMV-specific T cells and their impact on immunosenescence, which is important to eventually define the most useful source of adoptive therapy and monitoring protocols for cytomegalovirus-specific immune responses.

Dysferlin-deficient SJL mice are commonly used to study dysferlinopathy. We demonstrated that poloxamer 188 (P188), a membrane sealant, is effective in reducing the loss of muscle mass in SJL mice when administered using an osmotic pump for 6 weeks. We did not observe significant changes over a 2-week administration period, suggesting that longthier observation is necessary to determine the effectiveness of P188. We also examined exercise endurance in P188-administered SJL mice using a rolling cage. Phosphorylated p38 was found to be reduced in P188-administered SJL mice; additionally, using microarray analysis, we found diminished expression of atrogin-1, an E3 ubiquitin ligase, as the effector of muscular atrophy. Chronic infusion of P188 to dysferlin-deficient SJL mice reduced muscular atrophy, and administering p38 and atrogin-1 in the gastrocnemius muscle improved its motor function. These results provide a basis for potential treatments for dysferlin-deficient skeletal muscle fibers.

Inhibition of tumor suppressor p53 is cardioprotective against ischemic injury and provides resistance to subsequent cardiac remodeling. We investigated p53-mediated expansion of ischemic damage with a focus on mitochondrial integrity in association with autophagy and apoptosis. p53(-/-) heart showed that autophagic flux was promoted under ischemia without a change in cardiac tissue ATP content. Electron micrographs revealed that ischemic border zone in p53(-/-) mice had 5-fold greater numbers of autophagic vacuoles containing mitochondria, indicating the occurrence of mitophagy, with an apparent reduction of abnormal mitochondria compared with those in WT mice. Analysis of autophagic mediators acting downstream of p53 revealed that TIGAR (TP53-induced glycolysis and apoptosis regulator) was exclusively up-regulated in ischemic myocardium. TIGAR(-/-) mice exhibited the promotion of mitophagy followed by decrease of abnormal mitochondria and resistance to ischemic injury, consistent with the phenotype of p53(-/-) mice. In p53(-/-) and TIGAR(-/-) ischemic myocardium, ROS production was elevated and followed by Bnip3 activation which is an initiator of mitophagy. Furthermore, the activation of Bnip3 and mitophagy due to p53/TIGAR inhibition were reversed with antioxidant N-acetyl-cysteine, indicating that this adaptive response requires ROS signal. Inhibition of mitophagy using chloroquine in p53(-/-) or TIGAR(-/-) mice exacerbated accumulation of damaged mitochondria to the level of wild-type mice and attenuated cardioprotective action. These findings indicate that p53/TIGAR-mediated inhibition of myocyte mitophagy is responsible for impairment of mitochondrial integrity and subsequent apoptosis, the process of which is closely involved in p53-mediated ventricular remodeling after myocardial infarction.

We determined the fatty acid compositions of six species of freshwater microalgae belonging to the Chlorophyta, which were isolated from freshwater bodies in Assam, India. All six microalgae -Desmodesmus sp. DRLMA7, Desmodesmus elegans DRLMA13, Scenedesmus sp. DRLMA5, Scenedesmus sp. DRLMA9 Chlorella sp. DRLMA3 and Chlorococcum macrostigmatum DRLMA12-showed similar fatty acid profiles 16:0, 16:4, 18:1, 18:2, and 18:3 as major components. We also compared fatty acid compositions during the late exponential and stationary growth phases of D. elegans DRLMA13 and Scenedesmus sp. DRLMA9 in BG11 medium. We observed enhanced percentages of total saturated and monounsaturated fatty acids with a concomitant decrease in polyunsaturated fatty acid content upon the prolonged cultivation of both microalgae. Distinct morphological features of microalgal isolates were determined by scanning electron microscopic (SEM) studies. An ornamented cell wall was found in D. elegans DRLMA13, which is characteristic of small spineless species of Desmodesmus. The isolated microalgae were further distinguished through analysis of internal transcribed spacer 2 (ITS2) secondary structures and compensatory base changes (CBCs). Analysis of CBCs showed the relatedness of Chlorella sp. DRLMA3 with other Chlorella-like organisms, but it does not belong to the clade comprising Chlorella sensu stricto, which includes Chlorella vulgaris. The CBC count between Scenedesmus sp. DRLMA9 and other species of Scenedesmus provides evidence that this isolate represents a new species.

Functional magnetic resonance imaging (fMRI) has allowed insights into the spatiotemporal distribution of human brain networks. According to the neurophysiological property of the fetal brain to generate spontaneous activity, we aimed to determine the feasibility of investigating the maturation of intrinsic networks, beginning at gestational week 20 in healthy human fetuses by combining resting-state fMRI and an analytical approach, independent component analysis (ICA). In this study, functional images of 16 fetuses with morphologically normal brain development, from 20 to 36 gestational weeks of age, were acquired on a 1.5T unit (Philips Medical Systems, Best, The Netherlands) using single-shot, gradient-recalled echo-planar imaging. After preprocessing (motion correction, brain extraction), images were analyzed using single-subject ICA. We visualized a bilateral occipital network and medial and lateral prefrontal activity pattern that involved the future Brodmann areas 9-11. Furthermore, there was one either predominantly right (3/7 cases) or left (4/7 cases) hemispheric lateralized network that involved the superior temporal cortical regions (Brodmann areas 22 and 39). Frequency oscillations were in the range of 0.01-0.06Hz for all networks. This study shows that resting-state networks (RSNs) are shaped and are detectable in utero. Further investigations of resting-state measurements in the fetus may therefore allow developmental brain activity monitoring and may provide insights into early brain function.

Brazilin (7, 11b-dihydrobenz[b]indeno[1,2-d]pyran-3,6a,9,10 (6H)-tetrol), the major component of Caesalpinia sappan L., is a natural red pigment used for histological staining. Recent studies have shown that brazilin exhibits distinct biological effects, including anti-hepatotoxicity, antiplatelet activity, and anti-inflammatory activities. In the present study, we evaluated the effects of brazilin on MMP-1 and -3 expressions in human dermal fibroblasts exposed to ultraviolet B (UVB) irradiation. Brazilin showed protective effect on UVB-induced loss of cell viability of fibroblasts. Brazilin also blocked significantly UVB-induced Reactive Oxygen Species generation in fibroblasts. Brazilin inhibited UVB-induced MMP-1/3 expressions and secretions in a dose-dependent manner. Moreover, UVB-induced NF-κB activation was completely blocked by treatment with brazilin. These findings suggest that brazilin inhibits UVB-induced MMP-1/3 expressions and secretions by suppressing of NF-κB activation in human dermal fibroblasts. Thus, brazilin might be used as a potential agent for treatment of UV-induced skin photoaging.

Human immunodeficiency virus (HIV) particles that remain in the blood of patients are frequently ignored as targets for AIDS treatment. We therefore investigated the use of photodynamic therapy (PDT) with hematoporphyrin monomethyl ether (HMME) as a means of inactivating cell-free HIV in vitro. Virus particles including HIV-1(IIIB), resistant HIV-1 variants, HIV-1 clinical variants, and HIV-2 variants were incubated with HMME for 40 min, followed by irradiation with a 630-nm semiconductor laser at an energy density of 0.3 J/cm(2). The antiviral effects were evaluated by counting syncytium formation or measuring p24 antigen expression levels in supernatants by enzyme-linked immunosorbent assay. The relationships between photoinactivation and HMME concentrations, energy density, power density and antioxidants (NaN(3) and D: -mannitol) were also assessed using the above methods. All the tested virus particles were completely responsive to HMME-PDT. HMME concentration and energy density were positively correlated with photoinactivation of HIV, while power density was negatively correlated. Both sodium azide and D: -mannitol weakened the inhibitory effect of PDT on virus-induced membrane fusion, with D: -mannitol having a stronger effect. HMME-PDT can inactivate HIV particles, and may therefore represent a promising treatment for AIDS patients.

Although many low-penetrant genetic risk factors for breast cancer have been discovered, knowledge about the effect of multiple risk alleles is limited, especially in women <50 years. We therefore investigated the association between multiple risk alleles and breast cancer risk as well as individual effects according to age-approximated pre- and post-menopausal status.

Posttranscriptional gene silencing is mediated by RNA-induced silencing complexes (RISCs) that contain AGO proteins and single-stranded small RNAs. The assembly of plant AGO1-containing RISCs depends on the molecular chaperone HSP90. Here, we demonstrate that cyclophilin 40 (CYP40), protein phosphatase 5 (PP5), and several other proteins with the tetratricopeptide repeat (TPR) domain associates with AGO1 in an HSP90-dependent manner in extracts of evacuolated tobacco protoplasts (BYL). Intriguingly, CYP40, but not the other TPR proteins, could form a complex with small RNA duplex-bound AGO1. Moreover, CYP40 that was synthesized by in-vitro translation using BYL uniquely facilitated binding of small RNA duplexes to AGO1, and as a result, increased the amount of mature RISCs that could cleave target RNAs. CYP40 was not contained in mature RISCs, indicating that the association is transient. Addition of PP5 or cyclophilin-binding drug cyclosporine A prevented the association of endogenous CYP40 with HSP90-AGO1 complex and inhibited RISC assembly. These results suggest that a complex of AGO1, HSP90, CYP40, and a small RNA duplex is a key intermediate of RISC assembly in plants.

SlyD belongs to the FK506-binding protein (FKBP) family with both peptidylprolyl isomerase (PPIase) and chaperone activities, and is considered to be a ubiquitous cytosolic protein-folding facilitator in bacteria. It possesses a histidine- and cysteine-rich C-terminus binding to selected divalent metal ions (e.g., Ni(2+), Zn(2+)), which is important for its involvement in the maturation processes of metalloenzymes. We have determined the solution structure of C-terminus-truncated SlyD from Helicobacter pylori (HpSlyDΔC). HpSlyDΔC folds into two well-separated, orientation-independent domains: the PPIase-active FKBP domain and the chaperone-active insert-in-flap (IF) domain. The FKBP domain consists of a four-stranded antiparallel β-sheet with an α-helix on one side, whereas the IF domain folds into a four-stranded antiparallel β-sheet accompanied by a short α-helix. Intact H. pylori SlyD binds both Ni(2+) and Zn(2+), with dissociation constants of 2.74 and 3.79 μM respectively. Intriguingly, binding of Ni(2+) instead of Zn(2+) induces protein conformational changes around the active sites of the FKBP domain, implicating a regulatory role of nickel. The twin-arginine translocation (Tat) signal peptide from the small subunit of [NiFe] hydrogenase (HydA) binds the protein at the IF domain. Nickel binding and the recognition of the Tat signal peptide by the protein suggest that SlyD participates in [NiFe] hydrogenase maturation processes.

Neural tube defects (NTDs) are severe malformations of the central nervous system, affecting 1 of 1,000 live births. Mouse models were instrumental in defining the signaling pathways defective in NTDs, including the planar cell polarity (PCP), also called noncanonical Frizzled/Disheveled pathway. Based on the highly penetrant occurrence of NTDs in double Fzd3/Fzd6(-/-) mutant mice, we investigated the role of the human orthologues, FZD3 and FZD6, by resequencing a cohort of 473 NTDs patients and 639 ethnically matched controls. While we could not demonstrate a significant contribution of FZD3 gene, we identified five rare FZD6 variants that were absent in all controls and predicted to have a functional effect by computational analysis: one de novo frameshift mutation (c.1843_1844insA), three missense changes (p.Arg405Gln, p.Arg511Cys p.Arg511His), and one substitution (c.*20C>T) affecting the 3'-untranslated region (UTR) of the gene. The overall rate of predicted deleterious variants of FZD6 was 5.1-fold higher in cases compared to controls, resulting in a significantly increased NTDs mutation burden. This study demonstrates that rare nonsynonymous variants in FZD6 may contribute to NTDs in humans and enlarges the spectrum of mutations that link PCP pathway to NTDs.

Several studies report immunomodulatory effects of endogenous IL-10 after trauma. The present study investigates the effect of inhalative IL-10 administration on systemic and pulmonary inflammation in hemorrhagic shock. Male C57/BL6 mice (8 animals per group) were subjected to pressure-controlled hemorrhagic shock for 1.5 hrs followed by resuscitation and inhalative administration of either 50 μL PBS (Shock group) or 50 μg/kg recombinant mouse IL-10 dissolved in 50 μL PBS (Shock + IL-10 group). Animals were sacrificed after 4.5 hrs of recovery and serum IL-6, IL-10, KC, and MCP-1 concentrations were measured with ELISA kits. Acute pulmonary inflammation was assessed by pulmonary myeloperoxidase (MPO) activity and pulmonary H&E histopathology. Inhalative IL-10 administration decreased pulmonary inflammation without altering the systemic concentrations of IL-6, IL-10, and KC. Serum MCP-1 levels were significantly reduced following inhalative IL-10 administration. These findings suggest that inhalative IL-10 administration may modulate the pulmonary microenvironment without major alterations of the systemic inflammatory response, thus minimizing the potential susceptibility to infection and sepsis.

Heme oxygenase-1 (HO-1) is an anti-inflammatory enzyme that maintains homeostasis during cellular stress. Given previous findings that shorter length variants of a HO-1 promoter region GT(n) microsatellite polymorphism are associated with increased HO-1 expression in cell lines, we hypothesized that shorter variants would also be associated with increased levels of HO-1 expression, less inflammation and lower levels of inflammation-associated viral replication in human immunodeficiency virus (HIV)-infected subjects. Healthy donors (n = 20) with shorter GT(n) repeats had higher HO-1 mRNA transcript in peripheral blood mononuclear cells stimulated with lipopolysaccharide (r = -0.38, P = 0.05). The presence of fewer GT(n) repeats in subjects with untreated HIV disease was associated with higher HO-1 mRNA levels in peripheral blood (r = -0.41, P = 0.02); similar observations were made in CD14(+) monocytes from antiretroviral-treated subjects (r = -0.36, P = 0.04). In African-Americans, but not Caucasians, greater GT(n) repeats were correlated with higher soluble CD14 levels during highly active antiretroviral therapy (r = 0.38, P = 0.007) as well as higher mean viral load off-therapy (r = 0.24, P = 0.04). These data demonstrate that the HO-1 GT(n) microsatellite polymorphism is associated with higher levels of HO-1 expression and that this pathway may have important effects on the association between inflammation and HIV replication.

This paper presents cell culture substrates in the form of microcontainer arrays with overlaid surface topographies, and a technology for their fabrication. The new fabrication technology is based on microscale thermoforming of thin polymer films whose surfaces are topographically prepatterned on a micro- or nanoscale. For microthermoforming, we apply a new process on the basis of temporary back moulding of polymer films and use the novel concept of a perforated-sheet-like mould. Thermal micro- or nanoimprinting is applied for prepatterning. The novel cell container arrays are fabricated from polylactic acid (PLA) films. The thin-walled microcontainer structures have the shape of a spherical calotte merging into a hexagonal shape at their upper circumferential edges. In the arrays, the cell containers are arranged densely packed in honeycomb fashion. The inner surfaces of the highly curved container walls are provided with various topographical micro- and nanopatterns. For a first validation of the microcontainer arrays as in vitro cell culture substrates, C2C12 mouse premyoblasts are cultured in containers with microgrooved surfaces and shown to align along the grooves in the three-dimensional film substrates. In future stem-cell-biological and tissue engineering applications, microcontainers fabricated using the proposed technology may act as geometrically defined artificial microenvironments or niches.

Epigenetic regulation exerts a major influence on origins of DNA replication during development. The mechanisms for this regulation, however, are poorly defined. We showed previously that acetylation of nucleosomes regulates the origins that mediate developmental gene amplification during Drosophila oogenesis. Here we show that developmental activation of these origins is associated with acetylation of multiple histone lysines. Although these modifications are not unique to origin loci, we find that the level of acetylation is higher at the active origins and quantitatively correlated with the number of times these origins initiate replication. All of these acetylation marks were developmentally dynamic, rapidly increasing with origin activation and rapidly declining when the origins shut off and neighboring promoters turn on. Fine-scale analysis of the origins revealed that both hyperacetylation of nucleosomes and binding of the origin recognition complex (ORC) occur in a broad domain and that acetylation is highest on nucleosomes adjacent to one side of the major site of replication initiation. It was surprising to find that acetylation of some lysines depends on binding of ORC to the origin, suggesting that multiple histone acetyltransferases may be recruited during origin licensing. Our results reveal new insights into the origin epigenetic landscape and lead us to propose a chromatin switch model to explain the coordination of origin and promoter activity during development.

Normal rat kidney (NRK) fibroblasts exhibit growth-dependent changes in electrophysiological properties and intracellular calcium dynamics. The transition from a quiescent state to a density-arrested state results in altered calcium entry characteristics. This coincides with modulation of the expression of the genes encoding the calcium channels Trpc1, Trpc6 and Orai1, and of the intracellular calcium sensor Stim1. In the present study we have used gene selective short hairpin (sh) RNAs against these various genes to investigate their role in (a) capacitative store-operated calcium entry (SOCE); (b) non-capacitative OAG-induced receptor-operated calcium entry (ROCE); and (c) prostaglandin F(2α) (PGF(2α))-induced Ca(2+)-oscillations in NRK fibroblasts. Intracellular calcium measurements revealed that knockdown of the genes encoding Trpc1, Orai1 and Stim1 each caused a significant reduction of SOCE in NRK cells, whereas knockdown of the gene encoding Trpc6 reduced only the OAG-induced ROCE. Furthermore, our data show that knockdown of the genes encoding Trpc1, Orai1 and Stim1, but not Trpc6, substantially reduced the frequency (up to 60%) of PGF(2α)-induced Ca(2+) oscillations in NRK cells. These results indicate that in NRK cells distinct calcium channels control the processes of SOCE, ROCE and PGF(2α)-induced Ca(2+) oscillations.

CK2, a pleiotropic Ser/Thr kinase, is an important target for cancer therapy. We tested our novel tenfibgen-based nanocapsule for delivery of the inhibitor 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) and an siRNA directed against both CK2α and α' catalytic subunits to prostate cancer cells. We present data on the TBG nanocapsule itself and on CK2 inhibition or downregulation in treated cells, including effects on Nuclear Factor-kappa B (NF-κB) p65. By direct comparison of two CK2-directed cargos, our data provide proof that the TBG encapsulation design for delivery of drugs specifically to cancer cells has strong potential for small molecule- and nucleic acid-based cancer therapy.