Alisporivir (ALV), a cyclophilin inhibitor, is a host-targeting antiviral (HTA) with multigenotypic anti-hepatitis C virus (HCV) activity and a high barrier to resistance. Recent advances have supported the concept of interferon (IFN)-free regimens to treat chronic hepatitis C. As the most advanced oral HTA, ALV with direct-acting antivirals (DAAs) represents an attractive drug combination for IFN-free therapy. In this study, we investigated whether particular DAAs exhibit additive, synergistic, or antagonistic effects when combined with ALV. Drug combinations of ALV with NS3 protease, NS5B polymerase, and NS5A inhibitors were investigated in HCV replicons from genotypes 1a, 1b, 2a, 3, and 4a (GT1a to -4a). Combinations of ALV with DAAs exerted an additive effect on GT1 and -4. A significant and specific synergistic effect was observed with ALV-NS5A inhibitor combination on GT2 and -3. Furthermore, ALV was fully active against DAA-resistant variants, and ALV-resistant variants were fully susceptible to DAAs. ALV blocks the contact between cyclophilin A and domain II of NS5A, and NS5A inhibitors target domain I of NS5A; our data suggest a molecular basis for the use of these two classes of inhibitors acting on two distinct domains of NS5A. These results provide in vitro evidence that ALV with NS5A inhibitor combination represents an attractive strategy and a potentially effective IFN-free regimen for treatment of patients with chronic hepatitis C. Due to its high barrier and lack of cross-resistance, ALV could be a cornerstone drug partner for DAAs.
We report the development of single, locally crystallized nanopores in HfO2 membranes for biosensing applications. HfO2 is chosen for its isoelectric point of 7.0, mechanical and chemical stability in solution, and for its potential as a high-k material for nanopore ionic field effect transistor applications. The HfO2 membrane is deposited on a graphene layer suspended over a 300 nm FIB hole, where graphene is used as the mechanical support. Exposure of the membrane to a focused electron beam causes crystallization in the vicinity of the nanopore during pore formation. We investigate the effects of crystallization on the electrical and surface properties of HfO2 films. Our surface analysis of HfO2 reveals improved hydrophilicity of crystallized HfO2, a notable advantage over the hydrophobicity of as-deposited HfO2. We also demonstrate detection of dsDNA translocation through HfO2 nanopores under various applied bias levels. In addition, our device architecture also presents a promising first step toward the realization of high-k HfO2 nanopore transistors.
The development of two distinct classes of hepatitis C antiviral agents, direct-acting antivirals (DAAs) and host-targeting antivirals (HTAs), have distinctly impacted the hepatitis C virus (HCV) field by generating higher sustained virological response (SVR) rates within infected patients, via reductions in both adverse side effects and duration of treatment when compared to the old standard of care. Today DAAs are actively incorporated into the standard of care and continue to receive the most advanced clinical trial analysis. With a multitude of innovative and potent second-generation DAA compounds currently being tested in clinical trials, it is clear that the future of DAAs looks very bright. In comparison to the other class of compounds, HTAs have been slightly less impactful, despite the fact that primary treatment regimens for HCV began with the use of an HTA - interferon alpha (IFNα). The compound was advantageous in that it provided a broad-reaching antiviral response; however deleterious side effects and viral/patient resistance has since made the compound outdated. HTA research has since moved onward to target a number of cellular host factors that are required for HCV viral entry and replication such as scavenger receptor-BI (SR-BI), 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCoA reductase), cyclophilin A (CypA), fatty acid synthase (FASN) and miRNA-122. The rationale behind pursuing these HTAs is based upon the extremely low mutational rate that occurs within eukaryotic cells, thereby creating a high genetic barrier to drug resistance for anti-HCV compounds, as well as pan-genotypic coverage to all HCV genotypes and serotypes. As the end appears near for HCV, it becomes important to ask if the development of novel HTAs should also be analyzed in combination with other DAAs, in order to address potential hard-to-treat HCV patient populations. Since the treatment regimens for HCV began with the use of a global HTA, could one end the field as well?
host-targeting antivirals; alisporivir; HCV; cyclophilin A
Selective drug delivery to hypoxic tumor niches remains a significant therapeutic challenge that calls for new conceptual approaches. Sickle red blood cells (SSRBCs) have shown an ability to target such hypoxic niches and induce tumoricidal properties when used together with exogenous pro-oxidants. Here we determine whether the delivery of a model therapeutic encapsulated in murine SSRBCs can be enhanced by ex vivo photosensitization under conditions that delay autohemolysis to a time that coincides with maximal localization of SSRBCs in a hypoxic tumor. Hyperspectral imaging of 4T1 carcinomas shows oxygen saturation levels <10% in a large fraction (commonly 50% or more) of the tumor. Using video microscopy of dorsal skin window chambers implanted with 4T1 tumors, we demonstrate that allogeneic SSRBCs, but not normal RBCs (nRBCs), selectively accumulate in hypoxic 4T1 tumors between 12-24 hours after systemic administration. We further show that ex vivo photo-oxidation can program SSRBCs to postpone hemolysis/release of a model therapeutic to a point that coincides with their maximum sequestration in hypoxic tumor microvessels. Under these conditions, drug-loaded photosensitized SSRBCs show a 3-4 fold greater drug delivery to tumors compared to non-photosensitized SSRBCs, drug-loaded photosensitized nRBCs, and free drug. These results demonstrate that photo-oxidized SSRBCs, but not photo-oxidized nRBCs, sequester and hemolyze in hypoxic tumors and release substantially more drug than photo-oxidized nRBCs and non-photo-oxidized SSRBCs. Photo-oxidation of drug-loaded SSRBCs thus appears to exploit the unique tumor targeting and carrier properties of SSRBCs to optimize drug delivery to hypoxic tumors. Such programmed and drug-loaded SSRBCs therefore represent a novel and useful tool for augmenting drug delivery to hypoxic solid tumors.
blood; cancer; carrier; chemotherapy; particle; photosensitizer
Chediak-Higashi syndrome (CHS) is a rare, autosomal recessive congenital immunodeficiency caused by mutations in CHS1, a gene encoding a putative lysosomal trafficking protein. In the majority of patients, this disorder is typically characterized by infantile-onset hemophagocytic lymphohistiocytosis (HLH), which is lethal unless allogeneic transplantation is performed. A small number of individuals have the attenuated form of the disease and do not benefit from transplant. Improved outcomes of transplantation have been reported when performed before the development of HLH, thus it is important to quickly differentiate patients that present with the childhood form of disease and to prematurely enroll them into a transplantation protocol. In addition, this would also preclude those that exhibit clinical phenotypes of adolescent and adult CHS from this treatment. Patients with an absence of cytotoxic T lymphocyte (CTL) function have a high risk for developing HLH, and could therefore benefit the most from early hematopoietic stem cell transplantation (HSCT). However, although normal CTL cytotoxicity or bi-allelic missense mutations do not exclude the occurrence of HLH in childhood, a more conservative approach is justified. This article summarizes recent advances in the clinical characterization of CHS patients, provides updates on promising new testing methods, and focuses on specific therapeutic approaches.
Chediak-Higashi syndrome; LYST/CHS1; Phenotype-genotype correlation; CTL cytotoxicity; Hemophagocytic lymphohistiocytosis
The highly tunable properties of poly(ethylene glycol) (PEG)-based hydrogel systems permit their use in a wide array of regenerative medicine and drug delivery applications. One of the most valuable properties of PEG hydrogels is their intrinsic resistance to protein adsorption and cell adhesion, as it allows for a controlled introduction of desired bioactive factors including proteins, peptides, and drugs. Acrylate-PEG-N-hydroxysuccinimide (Acr-PEG-NHS) is widely utilized as a PEG linker to functionalize bioactive factors with photocrosslinkable groups. This enables their facile incorporation into PEG hydrogel networks or the use of PEGylation strategies for drug delivery. However, PEG linkers can sterically block integrin binding sites on functionalized proteins and reduce cell-material interactions. In this study we demonstrate that reducing the density of PEG linkers on protein backbones during functionalization results in significantly improved cell adhesion and spreading to bioactive hydrogels. However, this reduction in functionalization density also increases protein loss from the matrix over time due to ester hydrolysis of the Acr-PEG-NHS linkers. To address this, a novel PEG linker, acrylamide-PEG-isocyanate (Aam-PEG-I), with enhanced hydrolytic stability was synthesized. It was found that decreasing functionalization density with Aam-PEG-I resulted in comparable increases in cell adhesion and spreading to Acr-PEG-NHS systems while maintaining protein and bioactivity levels within the hydrogel network over a significantly longer time frame. Thus, Aam-PEG-I provides a new option for protein functionalization for use in a wide range of applications that improves initial and sustained cell-material interactions to enhance control of bioactivity.
Poly(ethylene glycol); hydrogels; protein functionalization; biostability; cell-material interactions
Acute ethanol administration is associated with sedation and analgesia as well as behavioral disinhibition and memory loss but the mechanisms underlying these effects remain to be elucidated. During the past decade, insects have emerged as important model systems to understand the neural and genetic bases of alcohol effects. However, novel assays to assess ethanol's effects on complex behaviors in social or isolated contexts are necessary. Here we used the honey bee as an especially relevant model system since bees are typically exposed to ethanol in nature when collecting standing nectar crop of flowers, and there is recent evidence for independent biological significance of this exposure for social behavior. Bee's inhibitory control of the sting extension response (SER) and a conditioned-place aversion assay were used to study ethanol effects on analgesia, behavioral disinhibition, and associative learning. Our findings indicate that although ethanol, in a dose-dependent manner, increases SER thresholds (analgesic effects), it disrupts the ability of honey bees to inhibit SER and to associate aversive stimuli with their environment. These results suggest that ethanol's effects on analgesia, behavioral disinhibition and associative learning are common across vertebrates and invertebrates. These results add to the use of honey bees as an ethanol model to understand ethanol's effects on complex, socially relevant behaviors.
Obstructive sleep apnea (OSA) is a common disorder characterized by the reduction or complete cessation in airflow resulting from an obstruction of the upper airway. Several studies have observed an increased risk for cardiovascular morbidity and mortality among OSA patients. Metabolic syndrome (MetS), a cluster of cardiovascular risk factors characterized by the presence of insulin resistance, is often found in patients with OSA, but the complex interplay between these two syndromes is not well understood. In this study, we present the results of a genetic association analysis of 373 candidate SNPs for MetS selected in a previous genome wide association analysis (GWAS). The 384 selected SNPs were genotyped using the Illumina VeraCode Technology in 387 subjects retrospectively assessed at the Internal Medicine Unit of the “Virgen de Valme” University Hospital (Seville, Spain). In order to increase the power of this study and to validate our findings in an independent population, we used data from the Framingham Sleep study which comprises 368 individuals. Only the rs11211631 polymorphism was associated with OSA in both populations, with an estimated OR=0.57 (0.42-0.79) in the joint analysis (p=7.21 × 10-4). This SNP was selected in the previous GWAS for MetS components using a digenic approach, but was not significant in the monogenic study. We have also identified two SNPs (rs2687855 and rs4299396) with a protective effect from OSA only in the abdominal obese subpopulation. As a whole, our study does not support that OSA and MetS share major genetic determinants, although both syndromes share common epidemiological and clinical features.
Obstructive sleep apnea; Metabolic syndrome; Polymorphisms; Genome wide association analysis
We introduce a two-loop power control that allows an efficient use of the overall power resources for commercial wireless networks based on cross-layer optimization. This approach maximizes the network's utility in the outer-loop as a function of the averaged signal to interference-plus-noise ratio (SINR) by considering adaptively the changes in the network characteristics. For this purpose, the concavity property of the utility function was verified with respect to the SINR, and an iterative search was proposed with guaranteed convergence. In addition,
the outer-loop is in charge of selecting the detector that minimizes the overall power consumption (transmission and detection). Next the inner-loop implements a feedback power control in order to achieve the optimal SINR in the transmissions despite channel variations and roundtrip delays. In our proposal, the utility maximization process and detector selection and feedback power control are decoupled problems, and as a result, these strategies are implemented at two different time scales in the two-loop framework. Simulation results show that substantial utility gains may be achieved by improving the power management in the wireless network.
We study the electrochemistry of single layer graphene edges using a nanopore-based structure consisting of stacked graphene and Al2O3 dielectric layers. Nanopores, with diameters ranging from 5 to 20 nm, are formed by an electron beam sculpting process on the stacked layers. This leads to unique edge structure which, along with the atomically thin nature of the embedded graphene electrode, demonstrates electrochemical current densities as high as 1.2 × 104 A/cm2. The graphene edge embedded structure offers a unique capability to study the electrochemical exchange at an individual graphene edge, isolated from the basal plane electrochemical activity. We also report ionic current modulation in the nanopore by biasing the embedded graphene terminal with respect to the electrodes in the fluid. The high electrochemical specific current density for a graphene nanopore-based device can have many applications in sensitive chemical and biological sensing, and energy storage devices.
Nanopores; graphene; graphene electrochemistry; nano-bio sensors; stacked graphene
Alisporivir is the most advanced host-targeting antiviral cyclophilin (Cyp) inhibitor in phase III studies and has demonstrated a great deal of promise in decreasing hepatitis C virus (HCV) viremia in infected patients. In an attempt to further elucidate the mechanism of action of alisporivir, HCV replicons resistant to the drug were selected. Interestingly, mutations constantly arose in domain II of NS5A. To demonstrate that these mutations are responsible for drug resistance, they were reintroduced into the parental HCV genome, and the resulting mutant viruses were tested for replication in the presence of alisporivir or in the absence of the alisporivir target, CypA. We also examined the effect of the mutations on NS5A binding to itself (oligomerization), CypA, RNA, and NS5B. Importantly, the mutations did not affect any of these interactions. Moreover, the mutations did not preserve NS5A-CypA interactions from alisporivir rupture. NS5A mutations alone render HCV only slightly resistant to alisporivir. In sharp contrast, when multiple NS5A mutations are combined, significant resistance was observed. The introduction of multiple mutations in NS5A significantly restored viral replication in CypA knockdown cells. Interestingly, the combination of NS5A mutations renders HCV resistant to all classes of Cyp inhibitors. This study suggests that a combination of multiple mutations in domain II of NS5A rather than a single mutation is required to render HCV significantly and universally resistant to Cyp inhibitors. This in accordance with in vivo data that suggest that alisporivir is associated with a low potential for development of viral resistance.
The nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication both in vitro and in vivo and represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arose in vitro under SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA
We found a highly divergent circovirus in serum samples from several dogs. Phylogenetic analysis indicates that canine circovirus genotype 1 (CaCV-1) represents the first circovirus reported in dogs and is genetically most closely related to the only known mammalian circovirus, porcine circovirus. Here we report the complete genome sequence of the CaCV-1 strain NY214, which will help toward understanding the evolutionary and pathogenic characteristics of mammalian circoviruses.
Humanized Bone marrow/Liver/Thymus (BLT) mice recapitulate the mucosal transmission of HIV, permitting study of early events in HIV pathogenesis and evaluation of preexposure prophylaxis methods to inhibit HIV transmission. Human hematopoiesis is reconstituted in NOD-scid mice by implantation of human fetal liver and thymus tissue to generate human T cells plus intravenous injection of autologous liver-derived CD34+ hematopoietic stem cells to engraft the mouse bone marrow. In side-by-side comparisons, we show that NOD-scid mice homozygous for a deletion of the IL-2Rγ-chain (NOD-scid IL-2Rγ−/−) are far superior to NOD-scid mice in both their peripheral blood reconstitution with multiple classes of human leukocytes (e.g., a mean of 182 versus 14 CD4+ T cells per μl 12 weeks after CD34+ injection) and their susceptibility to intravaginal HIV exposure (84% versus 11% viremic mice at 4 weeks). These results should speed efforts to obtain preclinical animal efficacy data for new HIV drugs and microbicides.
HIV/AIDS pathogenesis; HIV vaginal transmission; Humanized mice; HIV animal models; Hematopoietic stem cells; BLT mice; NOD-scid mice; NOD-scid IL-2Rγ−/− mice
The objective of this study was to compare quality of life measures in patients with neurocysticercosis (NCC) to those of a matched control group. The NCC outpatients and their controls were recruited from two neurology referral hospitals in Mexico City, Mexico during 2007–2008. The quality of life of 224 NCC patients was compared with 224 age-sex-hospital-day matched controls using the short form 12 v2 (SF-12 v2) quality of life survey. Medical chart reviews were also conducted for the NCC outpatients to evaluate presenting clinical manifestations. Compared with the controls, NCC patients had a significantly lower score for each of the eight domains of health evaluated and significantly lower Physical and Mental Component Summary scores. Chart reviews indicated that hydrocephalus (48%), severe headaches (47%), and epilepsy (31%) were the most common clinical manifestations in these NCC outpatients.
Neurocysticercosis (NCC) is a major public health problem in many developing countries where health education, sanitation, and meat inspection infrastructure are insufficient. The condition occurs when humans ingest eggs of the pork tapeworm Taenia solium, which then develop into larvae in the central nervous system. Although NCC is endemic in many areas of the world and is associated with considerable socio-economic losses, the burden of NCC remains largely unknown. This study provides the first estimate of disability adjusted life years (DALYs) associated with NCC in Mexico.
DALYs lost for symptomatic cases of NCC in Mexico were estimated by incorporating morbidity and mortality due to NCC-associated epilepsy, and morbidity due to NCC-associated severe chronic headaches. Latin hypercube sampling methods were employed to sample the distributions of uncertain parameters and to estimate 95% credible regions (95% CRs).
In Mexico, 144,433 and 98,520 individuals are estimated to suffer from NCC-associated epilepsy and NCC-associated severe chronic headaches, respectively. A total of 25,341 (95% CR: 12,569–46,640) DALYs were estimated to be lost due to these clinical manifestations, with 0.25 (95% CR: 0.12–0.46) DALY lost per 1,000 person-years of which 90% was due to NCC-associated epilepsy.
This is the first estimate of DALYs associated with NCC in Mexico. However, this value is likely to be underestimated since only the clinical manifestations of epilepsy and severe chronic headaches were included. In addition, due to limited country specific data, some parameters used in the analysis were based on systematic reviews of the literature or primary research from other geographic locations. Even with these limitations, our estimates suggest that healthy years of life are being lost due to NCC in Mexico.
Neurocysticercosis (NCC) is a major public health problem caused by the larvae of the parasite Taenia solium. The condition occurs when humans ingest eggs of the pork tapeworm Taenia solium, which then develop into larvae in the central nervous system. The disease is predominantly found and considered important in Latin American, Asian, and African countries and is associated with a large social and economic burden. Very few studies have been conducted to evaluate the burden of NCC and there are no estimates from Mexico. We estimated the disability adjusted life years (DALYs) lost due to NCC in Mexico incorporating morbidity and mortality due to NCC-associated epilepsy, and morbidity due to NCC-associated severe chronic headaches. NCC-associated epilepsy and severe chronic headaches were estimated to cause a loss of approximately 0.25 healthy year of life per 1,000 persons annually in Mexico. This is the first estimate of DALYs associated with NCC in Mexico. However, this value is likely to be underestimated since only the clinical manifestations of epilepsy and severe chronic headaches were included.
Although the mammalian immune system is generally thought to develop in a linear fashion, findings in avian and murine species argue instead for the developmentally ordered appearance (or “layering”) of unique hematopoietic stem cells (HSC) that give rise to distinct lymphocyte lineages at different stages of development. Here, we provide evidence of an analogous “layered” immune system in humans. Our results suggest that fetal and adult T cells are distinct populations that arise from different populations of HSC present at different stages of development. We also provide evidence that the fetal T cell lineage is biased towards immune tolerance. These observations offer a mechanistic explanation for the tolerogenic properties of the developing fetus and for variable degrees of immune responsiveness at birth.
In this study, we established a flow cytometry live cell-based assay that permits the screening of hepatitis C
virus (HCV) inhibitors. Specifically, we created a stable cell line, which harbors a subgenomic replicon encoding an
NS5A-YFP fusion protein. This system allows direct measurement of YFP fluorescence in live hepatoma cells in which
the HCV replicon replicates. We demonstrated that this stable fluorescent system permits the rapid and sensitive
quantification of HCV replication inhibition by direct-acting antiviral agents (DAA) including protease and NS5A
inhibitors and host-targeting antiviral agents (HTA) including cyclophilin inhibitors. This flow cytometry-based live cell
assay is well suited for multiple applications such as the evaluation of HCV replication as well as antiviral drug screening.
Direct-acting antiviral; FACS assay; HCV; hepatoma cells; host-targeting antiviral; inhibitors.
We report inelastic neutron scattering experiments on single crystals of
(Tc = 38 K). In addition to confirming the
resonance previously found in powder samples, we find that spin excitations in the
normal state form longitudinally elongated ellipses along the QAFM
direction in momentum space, consistent with density functional theory predictions.
On cooling below Tc, while the resonance preserves its momentum
anisotropy as expected, spin excitations at energies below the resonance become
essentially isotropic in the in-plane momentum space and dramatically increase their
correlation length. These results suggest that the superconducting gap structures in
Ba0.67Ka0.33Fe2As2 are more
complicated than those suggested from angle resolved photoemission experiments.
Immobilization of lipase on appropriate solid supports is one way to improve their stability and activity, and can be reused for large scale applications. A sample, cost- effective and high loading capacity method is still challenging.
A facile method of lipase immobilization was developed in this study, by the use of polydopamine coated magnetic nanoparticles (PD-MNPs). Under optimal conditions, 73.9% of the available lipase was immobilized on PD-MNPs, yielding a lipase loading capacity as high as 429 mg/g. Enzyme assays revealed that lipase immobilized on PD-MNPs displayed enhanced pH and thermal stability compared to free lipase. Furthermore, lipase immobilized on PD-MNPs was easily isolated from the reaction medium by magnetic separation and retained more than 70% of initial activity after 21 repeated cycles of enzyme reaction followed by magnetic separation.
Immobilization of enzyme onto magnetic iron oxide nanoparticles via poly-dopamine film is economical, facile and efficient.
Chromatin binding plays a central role in the molecular mechanism of LEDGF/p75 in HIV-1 DNA integration. Conflicting results have been reported in regards to the relevance of the LEDGF/p75 chromatin binding element PWWP domain in its HIV-1 cofactor activity.
Here we present evidence that re-expression of a LEDGF/p75 mutant lacking the PWWP domain (ΔPWWP) rescued HIV-1 infection in cells verified to express background levels of endogenous LEDGF/p75 that do not support efficient HIV-1 infection. The HIV-1 cofactor activity of LEDGF/p75 ΔPWWP was similar to that of LEDGF/p75 wild type (WT). A possible molecular explanation for the nonessential role of PWWP domain in the HIV-1 cofactor activity of LEDGF/p75 comes from the fact that coexpression of HIV-1 integrase significantly restored the impaired chromatin binding activity of LEDGF/p75 ΔPWWP. However, integrase failed to promote chromatin binding of a non-chromatin bound LEDGF/p75 mutant that lacks both the PWWP domain and the AT hook motifs (ΔPWWP/AT) and that exhibits negligible HIV-1 cofactor activity. The effect of integrase on the chromatin binding of LEDGF/p75 requires the direct interaction of these two proteins. An HIV-1 integrase mutant, unable to interact with LEDGF/p75, failed to enhance chromatin binding, whereas integrase wild type did not increase the chromatin binding strength of a LEDGF/p75 mutant lacking the integrase binding domain (ΔIBD).
Our data reveal that the PWWP domain of LEDGF/p75 is not essential for its HIV-1 cofactor activity, possibly due to an integrase-mediated increase of the chromatin binding strength of this LEDGF/p75 mutant.
To measure vitreous, aqueous, subretinal fluid and plasma levels of vascular endothelial growth factor in late stages of retinopathy of prematurity.
Interventional study. We enrolled patients with clinical diagnoses of bilateral stage V retinopathy of prematurity, confirmed by b-scan ultrasound and programmed for vitrectomy. During surgery we took samples from blood, aqueous, vitreous, and subretinal fluids. The vascular endothelial growth factor concentration in each sample was measured by ELISA reaction. A control sample of aqueous, vitreous and blood was taken from patients with congenital cataract programmed for phacoemulsification. For statistical analysis, a Mann–Whitney and a Wilcoxon W test was done with a significant P value of 0.05.
We took samples of 16 consecutive patients who met the inclusion criteria. The vascular endothelial growth factor levels in the study group were: aqueous, 76.81 ± 61.89 pg/mL; vitreous, 118.53 ± 65.87 pg/mL; subretinal fluid, 1636.58 ± 356.47 pg/mL; and plasma, 74.64 ± 43.94 pg/mL. There was a statistical difference between the study and the control group (P < 0.001) in the aqueous and vitreous samples.
Stage 5 retinopathy of prematurity has elevated intraocular levels of vascular endothelial growth factor, which remains high despite severe retinal lesion. There was no statistical difference in plasma levels of the molecule between the control and study group.
VEGF; late stages; ROP
Lens epithelium-derived growth factor (LEDGF)/p75 is a cellular cofactor for HIV-1 DNA integration. It is well established that the simultaneous binding of LEDGF/p75 to chromatin and to HIV-1 integrase is required for its cofactor activity. However, the exact molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet completely understood. Our hypothesis is that evolutionarily conserved regions in LEDGF/p75 exposed to solvent and harboring posttranslational modifications may be involved in its HIV-1 cofactor activity. Therefore, a panel of LEDGF/p75 deletion mutants targeting these protein regions were evaluated for their HIV-1 cofactor activity, chromatin binding, integrase interaction, and integrase-to-chromatin-tethering activity by using different cellular and biochemical approaches. The deletion of amino acids 267 to 281 reduced the cofactor activity of LEDGF/p75 to levels observed for chromatin-binding-defective mutants. This region contains a serine cluster (residues 271, 273, and 275) recurrently found to be phosphorylated in both human and mouse cells. Importantly, the conversion of these Ser residues to Ala was sufficient to impair the ability of LEDGF/p75 to mediate HIV-1 DNA integration, although these mutations did not alter chromatin binding, integrase binding, or the integrase-to-chromatin-tethering capability of LEDGF/p75. These results clearly indicated that serine residues 271, 273, and 275 influence the HIV-1 cofactor activity of integrase-to-chromatin-tethering-competent LEDGF/p75.
LEDGF/p75 can tether over-expressed lentiviral integrase proteins to chromatin but how this underlies its integration cofactor role for these retroviruses is unclear. While a single integrase binding domain (IBD) binds integrase, a complex N-terminal domain ensemble (NDE) interacts with unknown chromatin ligands. Whether integration requires chromatin tethering per se, specific NDE-chromatin ligand interactions or other emergent properties of LEDGF/p75 has been elusive. Here we replaced the NDE with strongly divergent chromatin-binding modules. The chimeras rescued integrase tethering and HIV-1 integration in LEDGF/p75-deficient cells. Furthermore, chromatin ligands could reside inside or outside the nucleosome core, and could be protein or DNA. Remarkably, a short Kaposi's sarcoma virus peptide that binds the histone 2A/B dimer converted GFP-IBD from an integration blocker to an integration cofactor that rescues over two logs of infectivity. NDE mutants were corroborative. Chromatin tethering per se is a basic HIV-1 requirement and this rather than engagement of particular chromatin ligands is important for the LEDGF/p75 cofactor mechanism.
Like other retroviruses, HIV-1 integrates a DNA copy of its genome into a host cell chromosome in each replication cycle. The resulting integrated proviruses are the basis for two important clinical problems: the inability to eradicate HIV-1 from the body and the permanent archiving of drug-resistant viruses. The DNA recombining steps catalyzed by the viral integrase are known, but the process as it occurs between the incoming virus and chromatin in cells is incompletely understood. LEDGF/p75 has been identified as an HIV-1 integration cofactor. If integrase is over-expressed in the absence of other viral components, it becomes linked to chromatin via LEDGF/p75. Whether this chromatin attachment function is the necessary and sufficient basis for its cofactor role in the viral life cycle has been unclear. The current work shows that HIV-1 cofactor function is preserved if the chromatin binding modules of LEDGF/p75 are replaced with widely disparate chromatin linkages. These results are evidence that chromatin tethering per se rather than connections to specific chromatin ligands is central to the LEDGF/p75 mechanism. They also have implications for targeting of lentiviral vectors within the human genome.
The fibrinogen (Fg) binding MSCRAMM Clumping factor A (ClfA) from Staphylococcus aureus interacts with the C-terminal region of the fibrinogen (Fg) γ-chain. ClfA is the major virulence factor responsible for the observed clumping of S. aureus in blood plasma and has been implicated as a virulence factor in a mouse model of septic arthritis and in rabbit and rat models of infective endocarditis. We report here a high-resolution crystal structure of the ClfA ligand binding segment in complex with a synthetic peptide mimicking the binding site in Fg. The residues in Fg required for binding to ClfA are identified from this structure and from complementing biochemical studies. Furthermore, the platelet integrin αIIbβ3 and ClfA bind to the same segment in the Fg γ-chain but the two cellular binding proteins recognize different residues in the common targeted Fg segment. Based on these differences, we have identified peptides that selectively antagonize the ClfA-Fg interaction. The ClfA-Fg binding mechanism is a variant of the “Dock, Lock and Latch” mechanism previously described for the Staphylococcus epidermidis SdrG–Fg interaction. The structural insights gained from analyzing the ClfANFg peptide complex and identifications of peptides that selectively recognize ClfA but not αIIbβ3 may allow the design of novel anti-staphylococcal agents. Our results also suggest that different MSCRAMMs with similar structural organization may have originated from a common ancestor but have evolved to accommodate specific ligand structures.
Staphylococcus aureus (S. aureus) is a common pathogen that can cause a range of diseases from mild skin infections to life-threatening sepsis in humans. Some surface proteins on S. aureus play important roles in the S. aureus disease process. One of these bacterial surface proteins is clumping factor A (ClfA) that binds to the C-terminal region of one of the three chains of fibrinogen (Fg), a blood protein that plays a key role in coagulation. We carried out biochemical and structural studies to understand the binding mechanism of ClfA to Fg and to define the residues in Fg that interact with ClfA. Interestingly, the platelet integrin, which is important for platelet aggregation and thrombi formation, also binds to the same region of Fg as ClfA. Despite the fact that the two proteins bind at the same region, the mode of recognition is significantly different. Exploiting this difference in recognition, we have demonstrated that agents could be designed that inhibit the ClfA–Fg interaction but do not interfere with the interaction of Fg with the platelet integrin. This opens the field for the design of a novel class of anti-staph therapeutics.