ENMD-2076 is a unique orally bioavailable Aurora kinase and VEGFR inhibitor. The purpose of this phase 1 study of ENMD-2076 was to determine the MTD, pharmacokinetic, and pharmacodynamic profiles and preliminary antitumor activity.
Patients with refractory advanced solid malignancies were treated with ENMD-2076 orally with continuous once daily dosing. Doses from 60 to 200 mg/m2 were evaluated using a standard 3 (to 4) +3 design. Pharmacokinetic parameters were studied on days 1, 28, and 30 to 35 of cycle 1. Expanded MTD cohorts included patients with ovarian cancer, colorectal cancer, and refractory solid tumors.
A total of 67 patients (46 F, 21M; ages 30–76) entered the study. Dose levels of 60, 80, 120, 200, and 160 mg/m2 were evaluated. Two patients experienced grade 3 hypertension at 200 mg/m2, and additional grade 3 neutropenia events limited tolerability at this dose. An intermediate dose of 160 mg/m2 was determined to be the MTD. The most common drug-related adverse events included hypertension, nausea/vomiting, and fatigue. The pharmacokinetics of ENMD-2076 were characterized by a rapid absorption phase (Tmax 3–7.8 hours), a t1/2 of 27.3 to 38.3 hours after a single dose, and dose proportional exposure. Decreased plasma sVEGFR2 was observed posttreatment. Two patients with platinum refractory/resistant ovarian cancer had RECIST partial responses.
ENMD-2076 was well tolerated, had a linear pharmacokinetic profile, and showed promising antitumor activity, particularly in ovarian cancer. The recommended phase 2 dose of ENMD-2076 is 160 mg/m2 administered orally once daily with continuous dosing.
Attachment of glutathione (GSH) to cysteine residues in proteins (S-glutathionylation) is a reversible post-translational modification that can profoundly alter protein structure and function. Often serving in a protective role, e.g., by temporarily saving protein thiols from irreversible oxidation and inactivation, glutathionylation can be identified and semi-quantitatively assessed using anti-GSH antibodies, thought to be specific for recognition of the S-glutathionylation modification. Here we describe an alternate mechanism of protein glutathionylation in which the sulfur atoms of the GSH and the protein’s thiol group are covalently bound via a crosslinking agent, rather than through a disulfide bond. This form of thiol crosslinking has been shown to occur and confirmed by mass spectrometry at the solution chemistry level, as well as in experiments documenting the potent antiproliferative activity of the bis-diazeniumdiolate Double JS-K in H1703 cells in vitro and in vivo. The modification is recognized by the anti-GSH antibody as if it were authentic S-glutathionylation, requiring mass spectrometry to distinguish between them.
We previously demonstrated that the histone deacetylase (HDAC) inhibitor, 4b, which preferentially targets HDAC1 and HDAC3, ameliorates Huntington's disease (HD)-related phenotypes in different HD model systems. In the current study, we investigated extensive behavioral and biological effects of 4b in N171-82Q transgenic mice and further explored potential molecular mechanisms of 4b action. We found that 4b significantly prevented body weight loss, improved several parameters of motor function and ameliorated Huntingtin (Htt)-elicited cognitive decline in N171-82Q transgenic mice. Pathways analysis of microarray data from the mouse brain revealed gene networks involving post-translational modification, including protein phosphorylation and ubiquitination pathways, associated with 4b drug treatment. Using real-time qPCR analysis, we validated differential regulation of several genes in these pathways by 4b, including Ube2K, Ubqln, Ube2e3, Usp28 and Sumo2, as well as several other related genes. Additionally, 4b elicited increases in the expression of genes encoding components of the inhibitor of kappaB kinase (IKK) complex. IKK activation has been linked to phosphorylation, acetylation and clearance of the Htt protein by the proteasome and the lysosome, and accordingly, we found elevated levels of phosphorylated endogenous wild-type (wt) Htt protein at serine 16 and threonine 3, and increased AcK9/pS13/pS16 immunoreactivity in cortical samples from 4b-treated mice. We further show that HDAC inhibitors prevent the formation of nuclear Htt aggregates in the brains of N171-82Q mice. Our findings suggest that one mechanism of 4b action is associated with the modulation of the ubiquitin–proteasomal and autophagy pathways, which could affect accumulation, stability and/or clearance of important disease-related proteins, such as Htt.
To develop stem/progenitor cell-based therapy for cystic fibrosis (CF) lung disease, it is first necessary to identify markers of human lung epithelial progenitor/stem cells and to better understand the potential for differentiation into distinct lineages. Here we investigated integrin α6β4 as an epithelial progenitor cell marker in the human distal lung. We identified a subpopulation of α6β4+ cells that localized in distal small airways and alveolar walls and were devoid of pro-surfactant protein C expression. The α6β4+ epithelial cells demonstrated key properties of stem cells ex vivo as compared to α6β4- epithelial cells, including higher colony forming efficiency, expression of stem cell-specific transcription factor Nanog, and the potential to differentiate into multiple distinct lineages including basal and Clara cells. Co-culture of α6β4+ epithelial cells with endothelial cells enhanced proliferation. We identified a subset of adeno-associated virus (AAVs) serotypes, AAV2 and AAV8, capable of transducing α6β4+ cells. In addition, reconstitution of bronchi epithelial cells from CF patients with only 5% normal α6β4+ epithelial cells significantly rescued defects in Cl- transport. Therefore, targeting the α6β4+ epithelial population via either gene delivery or progenitor cell-based reconstitution represents a potential new strategy to treat CF lung disease.
Expression of isotopically labeled peptide standards as artificial concatamers (QconCATs) allows for the multiplex quantification of proteins in unlabeled samples by mass spectrometry. We have developed a generalizable QconCAT design strategy, which we term IQcat, wherein concatenated peptides are binned by isoelectric point to facilitate MS-sample enrichment by isoelectric focusing. Our method utilizes a rapid (~2 week), inexpensive and scalable purification of arg/lys labeled IQcat standards in the E. coli auxotroph AT713. With this pipeline we assess the fidelity of IQcat-based absolute quantification for 10 yeast proteins over a broad concentration range in a single information-rich isoelectric fraction. The technique is further employed for a quantitative study of androgen-dependent protein expression in cultured prostate cancer cells.
absolute quantification; LNCaP; OFFGEL; prostate cancer; QconCAT
The long-term cardiovascular outcomes of a population-based cohort presenting to the emergency department (ED) with chest pain and classified with a clinical risk stratification algorithm are not well documented. The Olmsted County Chest Pain Study is a community-based study that included all consecutive patients presenting with chest pain consistent with unstable angina presenting to all Olmsted County EDs. Patients were classified according to the Agency for Health Care Policy and Research (AHCPR) criteria. Patients with ST elevation myocardial infarction and chest pain of non-cardiac origin were excluded. Main outcome measures were major adverse cardiovascular and cerebrovascular events (MACCE) at 30 days and a median follow up of 7.3 years, and mortality through a median of 16.6 years. The 2271 patients were classified: 436 (19.2%) as high-, 1,557 (68.6%) as intermediate- and 278 (12.2%) as low-risk. Thirty-day MACCE occurred in 11.5% in high-risk, 6.2% in intermediate-risk, and 2.5% in the lowrisk group (p<0.001). At 7.3 years, significantly more MACCE were recorded in the intermediate (HR 1.91; 95% CI 1.33–2.75) and high-risk groups (HR 2.45; 95% CI 1.67–3.58). Intermediate- and high-risk patients demonstrated a 1.38 fold (95% CI 0.95–2.01, p=0.09) and a 1.68 fold (95% CI 1.13–2.50, p = 0.011) higher mortality compared to lowrisk patients at 16.6 years. At 7.3 and 16.6 years of follow-up, biomarkers were not incrementally predictive of cardiovascular risk. In conclusion, a widely-applicable rapid clinical algorithm using AHCPR criteria can reliably predict long-term mortality and cardiovascular outcomes. This algorithm, when applied in the ED, affords an excellent opportunity to identify patients who might benefit from optimization of their cardiovascular risk management.
Although the risk factor for apolipoprotein E (apoE) polymorphism in Alzheimer's disease (AD) has been well described, the role that apoE plays in other neurodegenerative diseases, including Pick's disease, is not well established. To examine a possible role of apoE in Pick's disease, an immunohistochemical analysis was performed utilizing a novel site-directed antibody that is specific for an amino-terminal fragment of apoE. Application of this antibody, termed the amino-terminal apoE cleavage fragment (nApoECF) antibody, consistently labeled Pick bodies within area CA1 of the hippocampus in 4 of the 5 cases examined. Co-localization of the nApoECF antibody with PHF-1, a general marker for Pick bodies, as well as with an antibody to caspase-cleaved tau (TauC3) was evident within the hippocampus. While staining of the nApoECF antibody was robust in area CA1, little co-localization with PHF-1 in Pick bodies within the dentate gyrus was observed. A quantitative analysis indicated that approximately 86% of the Pick bodies identified in area CA1 labeled with the nApoECF antibody. The presence of truncated apoE within Pick bodies suggests a broader role of apoE beyond AD and raises the question as to whether this protein contributes to pathogenesis associated with Pick's disease.
Alterations in cost–benefit decision making accompany numerous neuropsychiatric conditions, including schizophrenia, attention deficit hyperactivity disorder, and addiction. Central cholinergic systems have been linked to the etiology and/or treatment of many of these conditions, but little is known about the role of cholinergic signaling in cost–benefit decision making.
The goal of these experiments was to determine how cholinergic signaling is involved in cost–benefit decision making, using a behavioral pharmacological approach.
Male Long-Evans rats were trained in either “probability discounting” or “delay discounting” tasks, in which rats made discrete-trial choices between a small food reward and a large food reward associated with either varying probabilities of omission or varying delays to delivery, respectively. The effects of acute administration of different doses of nicotinic and muscarinic acetylcholine receptor agonists and antagonists were assessed in each task.
In the probability discounting task, acute nicotine administration (1.0 mg/kg) significantly increased choice of the large risky reward, and control experiments suggested that this was due to robust nicotine-induced impairments in behavioral flexibility. In the delay discounting task, the muscarinic antagonists scopolamine (0.03, 0.1, and 0.3 mg/kg) and atropine (0.3 mg/kg) both significantly increased choice of the small immediate reward. Neither mecamylamine nor oxotremorine produced reliable effects on either of the decision making tasks.
These data suggest that cholinergic receptors play multiple roles in decision making contexts which include consideration of reward delay or probability. These roles should be considered when targeting these receptors for therapeutic purposes.
Cholinergic; Nicotinic; Muscarinic; Delay discounting; Probability discounting; Impulsivity; Risk; Perseveration
Expansions of the non-coding GGGGCC hexanucleotide repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene were recently identified as the long sought-after cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) on chromosome 9p. In this study we aimed to determine whether the length of the normal - unexpanded - allele of the GGGGCC repeat in C9ORF72 plays a role in the presentation of disease or affects age at onset in C9ORF72 mutation carriers. We also studied whether the GGGGCC repeat length confers risk or affects age at onset in FTD and ALS patients without C9ORF72 repeat expansions. C9ORF72 genotyping was performed in 580 FTD, 995 ALS and 160 FTD-ALS patients and 1444 controls, leading to the identification of 211 patients with pathogenic C9ORF72 repeat expansions and an accurate quantification of the length of the normal alleles in all patients and controls. No meaningful association between the repeat length of the normal alleles of the GGGGCC repeat in C9ORF72 and disease phenotype or age at onset was observed in C9ORF72 mutation carriers or non-mutation carriers.
Amyotrophic lateral sclerosis; Frontotemporal Dementia; C9ORF72; Repeat-expansion disease; Association study
Toxoplasma gondii transmission between intermediate hosts is dependent on the ingestion of walled cysts formed during the chronic phase of infection. Immediately following consumption, the parasite must ensure survival of the host by preventing adverse inflammatory responses and/or by limiting its own replication. Since the Toxoplasma secreted effectors rhoptry 16 kinase (ROP16) and dense granule 15 (GRA15) activate the JAK-STAT3/6 and NF-κB signaling pathways, respectively, we explored whether a particular combination of these effectors impacted intestinal inflammation and parasite survival in vivo. Here we report that expression of the STAT-activating version of ROP16 in the type II strain (strain II+ROP16I) promotes host resistance to oral infection only in the context of endogenous GRA15 expression. Protection was characterized by a lower intestinal parasite burden and dampened inflammation. Host resistance to the II+ROP16I strain occurred independently of STAT6 and the T cell coinhibitory receptors B7-DC and B7-H1, two receptors that are upregulated by ROP16. In addition, coexpression of ROP16 and GRA15 enhanced parasite susceptibility within tumor necrosis factor alpha/gamma interferon-stimulated macrophages in a STAT3/6-independent manner. Transcriptional profiling of infected STAT3- and STAT6-deficient macrophages and parasitized Peyer's patches from mice orally challenged with strain II+ROP16I suggested that ROP16 activated STAT5 to modulate host gene expression. Consistent with this supposition, the ROP16 kinase induced the sustained phosphorylation and nuclear localization of STAT5 in Toxoplasma-infected cells. In summary, only the combined expression of both GRA15 and ROP16 promoted host resistance to acute oral infection, and Toxoplasma may possibly target the STAT5 signaling pathway to generate protective immunity in the gut.
The imatinib paradigm in chronic myeloid leukemia (CML) established continuous BCR-ABL inhibition as a design principle for ABL tyrosine kinase inhibitors (TKIs). However, clinical responses seen in patients treated with the ABL TKI dasatinib despite its much shorter plasma half-life and the apparent rapid restoration of BCR-ABL signaling activity following once-daily dosing suggested acute, potent inhibition of kinase activity may be sufficient to irrevocably commit CML cells to apoptosis. To determine the specific requirements for ABL TKI-induced CML cell death for a panel of clinically important ABL TKIs (imatinib, nilotinib, dasatinib, ponatinib, and DCC-2036), we interrogated response of CML cell lines and primary CML cells following acute drug exposure using intracellular FACS and immunoblot analyses of BCR-ABL signaling, apoptosis measurements, liquid chromatography/tandem mass spectrometry of intracellular drug levels, and biochemical TKI dissociation studies. Importantly, significant intracellular TKI stores were detected following drug washout, levels of which tracked with onset of apoptosis and incomplete return of BCR-ABL signaling, particularly pSTAT5, to baseline. Among TKIs tested, ponatinib demonstrated the most robust capacity for apoptotic commitment showing sustained suppression of BCR-ABL signaling even at low intracellular levels following extensive washout, consistent with high-affinity binding and slow dissociation from ABL kinase. Together, our findings suggest commitment of CML cells to apoptosis requires protracted incomplete restoration of BCR-ABL signaling mediated by intracellular retention of TKIs above a quantifiable threshold. These studies refine our understanding of apoptotic commitment in CML cells and highlight parameters important to design of therapeutic kinase inhibitors for CML and other malignancies.
Parkinson’s disease (PD) is a multifactorial movement disorder characterized by progressive neurodegeneration. Genome-wide association studies (GWAS) have nominated over fifteen distinct loci associated with risk of PD, however the biological mechanisms by which these loci influence disease risk are mostly unknown. GWAS are only the first step in the identification of disease genes: the specific causal variants responsible for the risk within the associated loci and the interactions between them must be identified to fully comprehend their impact on the development of PD. In the present study, we first attempted to replicate the association signals of 17 PD GWAS loci in our series of 1381 patients with PD and 1328 controls. BST1, SNCA, HLA-DRA, CCDC62/HIP1R and MAPT all showed a significant association with PD under different models of inheritance and LRRK2 showed a suggestive association. We then examined the role of coding LRRK2 variants in the GWAS association signal for that gene. The previously identified LRRK2 risk mutant p.M1646T and protective haplotype p.N551K-R1398H-K1423K did not explain the association signal of LRRK2 in our series. Finally, we investigated the gene-gene interaction between PARK16 and LRRK2 that has previously been proposed. We observed no interaction between PARK16 and LRRK2 GWAS variants, but did observe a non-significant trend toward interaction between PARK16 and LRRK2 variants within the protective haplotype. Identification of causal variants and the interactions between them is the crucial next step in making biological sense of the massive amount of data generated by GWAS studies. Future studies combining larger sample sizes will undoubtedly shed light on the complex molecular interplay leading to the development of PD.
Association studies in genetics; Parkinson’s disease/Parkinsonism
This study examines the neural mechanisms through which younger and older adults ignore irrelevant information, a process that is necessary to effectively encode new memories. Some age-related memory deficits have been linked to a diminished ability to dynamically gate sensory input, resulting in problems inhibiting the processing of distracting stimuli. Whereas oscillatory power in the alpha band (8–12Hz) over visual cortical areas is thought to dynamically gate sensory input in younger adults, it is not known whether older adults use the same mechanism to gate out sensory input. Here we identified a task in which both older and younger adults could suppress the processing of irrelevant sensory stimuli, allowing us to use electroencephalography (EEG) to explore the neural activity associated with suppression of visual processing. As expected, we found that the younger adults’ suppression of visual processing was correlated with robust modulation of alpha oscillatory power. However, older adults did not modulate alpha power to suppress processing of visual information. These results demonstrate that suppression of alpha power is not necessary to inhibit the processing of distracting stimuli in older adults, suggesting the existence of alternative strategies for suppressing irrelevant, potentially distracting information.
Aging; Attention; EEG; Oscillations; Sensory gating; Visual working memory
To understand the online health information-seeking behaviors of people with diabetes, determine whether they utilize online social media, and to see if they would be willing to use these sites to discuss health information.
Design and sample
57 participants were recruited from the South-Eastern US between June and October 2009 and asked to take an online survey. Participants were asked demographic data, information about their diabetes, and Internet and online social networking use.
The majority of participants utilize popular online social networking sites and many would be willing to discuss health information online.
Popular online social networks and online social media have the potential to serve as important platforms for nursing and public health interventions. In particular, these venues may serve as appropriate tools to reach minority populations and people in rural areas. Further research is needed to understand how we can use these Internet sites to reach people directly where they are and for delivering diabetes education and support.
The use of tyrosine kinase inhibitors (TKIs) against EGFR/c-Met in non-small cell lung cancer (NSCLC) has been shown to be effective in increasing patient progression free survival (PFS), but their efficacy is limited due to the development of resistance and tumor recurrence. Therefore, understanding the molecular mechanisms underlying development of drug resistance in NSCLC is necessary for developing novel and effective therapeutic approaches to improve patient outcome. This study aims to understand the mechanism of EGFR/c-Met tyrosine kinase inhibitor (TKI) resistance in NSCLC. H2170 and H358 cell lines were made resistant to SU11274, a c-Met inhibitor, and erlotinib, an EGFR inhibitor, through step-wise increases in TKI exposure. The IC50 concentrations of resistant lines exhibited a 4–5 and 11–22-fold increase for SU11274 and erlotinib, respectively, when compared to parental lines. Furthermore, mTOR and Wnt signaling was studied in both cell lines to determine their roles in mediating TKI resistance. We observed a 2–4-fold upregulation of mTOR signaling proteins and a 2- to 8-fold upregulation of Wnt signaling proteins in H2170 erlotinib and SU11274 resistant cells. H2170 and H358 cells were further treated with the mTOR inhibitor everolimus and the Wnt inhibitor XAV939. H358 resistant cells were inhibited by 95% by a triple combination of everolimus, erlotinib and SU11274 in comparison to 34% by a double combination of these drugs. Parental H2170 cells displayed no sensitivity to XAV939, while resistant cells were significantly inhibited (39%) by XAV939 as a single agent, as well as in combination with SU11274 and erlotinib. Similar results were obtained with H358 resistant cells. This study suggests a novel molecular mechanism of drug resistance in lung cancer.
In the event of a deliberate or accidental radiological emergency, the skin would likely receive substantial ionizing radiation (IR) poisoning which could negatively impact cellular proliferation, communication, and immune-regulation within the cutaneous microenvironment. Indeed, as we have previously shown, local IR exposure to the murine ear causes a reduction of two types of cutaneous dendritic cells (cDC), including interstitial DC (iDC) of the dermis and Langerhans cells (LC) of the epidermis, in a dose and time dependent manner. These APCs are critical regulators of skin homeostasis, immuno-surveillance, and the induction of T and B cell-mediated immunity as previously demonstrated using conditional cDC knockout mice. To mimic a radiological emergency, we developed a murine model of sub-lethal total body irradiation (TBI). Our data would suggest that TBI results in the reduction of cDC from the murine ear that was not due to a systemic response to IR as a loss was not observed in shielded ears. We further determined that this reduction was due, in part, to the up-regulation of the chemoattractant CCL21 on lymphatic vessels as well as CCR7 expressed on cDC. Migration as a potential mechanism was confirmed using CCR7−/− mice where cDC were not depleted following TBI. Finally, we demonstrated that the loss of cDC following TBI results in an impaired contact hypersensitivity (CHS) response to hapten by using a modified CHS protocol. Taken together, these data suggest that IR exposure may result in diminished immuno-surveillance in the skin, which could render the host more susceptible to pathogens.
Cellular prion protein (PrPC) has attracted considerable attention for its role in transmissible spongiform encephalopathies (TSEs). In spite of being a point of intense research effort critical questions still remain regarding the physiological function of PrPC and how these functions may change with the conversion of the protein into the infectious and pathological conformation (PrPSc). While emerging evidence suggests PrPC/Sc are involved in signal transduction there is little consensus on the signaling pathways associated with the normal and diseased states. The purported involvement of PrPC in signal transduction, and the association of TSEs with neural pathology, makes kinome analysis of human neurons an interesting and appropriate model to characterize patterns of signal transduction following activation of PrPC by two commonly employed experimental ligands; antibody-induced dimerization by 6H4 and the amino acids 106-126 PrP peptide fragment (PrP 106–126). Analysis of the induced kinome responses reveals distinct patterns of signaling activity following each treatment. Specifically, stimulation of human neurons with the 6H4 antibody results in alterations in mitogen activated protein kinase (MAPK) signaling pathways while the 106-126 peptide activates growth factor related signaling pathways including vascular endothelial growth factor (VEGF) signaling and the phosphoinositide-3 kinase (PI3K) pathway. These pathways were validated through independent functional assays. Collectively these results indicate that stimulation of PrPC with distinct ligands, even within the same cell type, results in unique patterns of signaling. While this investigation highlights the apparent functional versatility of PrPC as a signaling molecule and may offer insight into cellular mechanisms of TSE pathology it also emphasizes the potential dangers associated with attributing activation of specific intracellular events to particular receptors through artificial models of receptor activation.
kinome; PrP; cell signaling; peptide array; transmissible spongiform encephalopathy
Microbial sewage communities consist of a combination of human fecal microorganisms and nonfecal microorganisms, which may be residents of urban sewer infrastructure or flowthrough originating from gray water or rainwater inputs. Together, these different microorganism sources form an identifiable community structure that may serve as a signature for sewage discharges and as candidates for alternative indicators specific for human fecal pollution. However, the structure and variability of this community across geographic space remains uncharacterized. We used massively parallel 454 pyrosequencing of the V6 region in 16S rRNA genes to profile microbial communities from 13 untreated sewage influent samples collected from a wide range of geographic locations in the United States. We obtained a total of 380,175 high-quality sequences for sequence-based clustering, taxonomic analyses, and profile comparisons. The sewage profile included a discernible core human fecal signature made up of several abundant taxonomic groups within Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria. DNA sequences were also classified into fecal, sewage infrastructure (i.e., nonfecal), and transient groups based on data comparisons with fecal samples. Across all sewage samples, an estimated 12.1% of sequences were fecal in origin, while 81.4% were consistently associated with the sewage infrastructure. The composition of feces-derived operational taxonomic units remained congruent across all sewage samples regardless of geographic locale; however, the sewage infrastructure community composition varied among cities, with city latitude best explaining this variation. Together, these results suggest that untreated sewage microbial communities harbor a core group of fecal bacteria across geographically dispersed wastewater sewage lines and that ambient water quality indicators targeting these select core microorganisms may perform well across the United States.
Influenza viruses often evade host immunity via antigenic drift and shift despite previous influenza virus infection and/or vaccination. Vaccines that match circulating virus strains are needed for optimal protection. Development of a universal influenza virus vaccine providing broadly cross-protective immunity will be of great importance. The nucleoprotein (NP) of influenza A virus is highly conserved among all strains of influenza A viruses and has been explored as an antigen for developing a universal influenza virus vaccine. In this work, we generated a recombinant parainfluenza virus 5 (PIV5) containing NP from H5N1 (A/Vietnam/1203/2004), a highly pathogenic avian influenza (HPAI) virus, between HN and L (PIV5-NP-HN/L) and tested its efficacy. PIV5-NP-HN/L induced humoral and T cell responses in mice. A single inoculation of PIV5-NP-HN/L provided complete protection against lethal heterosubtypic H1N1 challenge and 50% protection against lethal H5N1 HPAI virus challenge. To improve efficacy, NP was inserted into different locations within the PIV5 genome. Recombinant PIV5 containing NP between F and SH (PIV5-NP-F/SH) or between SH and HN (PIV5-NP-SH/HN) provided better protection against H5N1 HPAI virus challenge than did PIV5-NP-HN/L. These results suggest that PIV5 expressing NP from H5N1 has the potential to be utilized as a universal influenza virus vaccine.
Outer retinal injury has been well described in glaucoma. To better understand the source of this injury, we wanted to develop a reliable model of partial retinal ganglion cell (RGC) axotomy.
Endodiathermy spots were placed along the inferior 180° adjacent to the optic nerve margin in the right eyes of four cynomolgus monkeys. Fluorescein angiography, spectral domain optical coherence tomography (SD-OCT), and multifocal electroretinography (mfERG) were performed at various intervals. Two animals were sacrificed at 3 months. Two animals were sacrificed at 4 months, at which time they underwent an injection of fluorescent microspheres to measure regional choroidal blood flow. Retinal immunohistochemistry for glial fibrillary acidic protein (GFAP), rhodopsin, S-cone opsin, and M/L-cone opsin were performed, as were axon counts of the optic nerves.
At 3 months, there was marked thinning of the inferior nerve fiber layer on SD-OCT. The mfERG waveforms were consistent with inner but not outer retinal injury. Greater than 95% reduction in axons was seen in the inferior optic nerves but no secondary degeneration superiorly. There was marked thinning of the nerve fiber and ganglion cell layers in the inferior retinas. However, the photoreceptor histology was similar in the axotomized and nonaxotomized areas. Regional choroidal blood flow was not affected by the axotomy.
Unlike experimental glaucoma, hemiretinal endodiathermy axotomy (HEA) of the RGCs produces no apparent anatomic, functional, or blood flow effects on the outer retina and choroid.
Outer retinal injury has been well described in glaucoma. In this study, we characterize a new model of partial retinal ganglion cell axotomy and compare the functional, anatomic, and vascular effects to those previously described in experimental glaucoma in the nonhuman primate.
endodiathermy axotomy; choroidal blood flow; photoreceptor morphology; multifocal electroretinography; optical coherence tomography
Angiotensin-converting enzyme (ACE) is assumed to influence the activity of the hypothalamic–pituitary–adrenocortical (HPA) axis, which shows hyperactivity in depressed patients. ACE could thus be a promising candidate gene for late-life depression but this has not been examined previously. Depression was assessed in 1005 persons aged at least 65 years, at baseline and over the 10-year follow-up. A clinical level of depression (DEP) was defined as having a score of ⩾16 on the Centre for Epidemiology Studies-Depression scale or a diagnosis of current major depression based on the Mini International Neuropsychiatric Interview and according to DSM-IV criteria. Seven single-nucleotide polymorphisms (SNPs) in the ACE gene were genotyped and diurnal cortisol secretion, as an index of HPA axis activity, was measured. Multivariable analyses were adjusted for socio-demographic and vascular factors, cognitive impairment, and apolipoprotein E. Strong significant associations were found between all seven SNPs and DEP and, in particular, first-onset DEP in persons without a past history of depression (P-values ranging from 0.005 to 0.0004). These associations remained significant after correction for multiple testing. The genotypes that were associated with an increased risk of DEP were also significantly associated with an increase in cortisol secretion under stress conditions. Variants of the ACE gene influence cortisol secretion and appear as susceptibility factors for late-life depression in the elderly population. Whether this could represent a common pathophysiological mechanism linking HPA axis and late-life depression remains to be explored.
Angiotensin-converting enzyme; cortisol; elderly; HPA axis; late-life depression; prospective study
Conopeptides, often generically referred to as conotoxins, are small neurotoxins found in the venom of predatory marine cone snails. These molecules are highly stable and are able to efficiently and selectively interact with a wide variety of heterologous receptors and channels, making them valuable pharmacological probes and potential drug leads. Recent advances in next-generation RNA sequencing and high-throughput proteomics have led to the generation of large data sets that require purpose-built and dedicated bioinformatics tools for efficient data mining.
Here we describe ConoSorter, an algorithm that categorizes cDNA or protein sequences into conopeptide superfamilies and classes based on their signal, pro- and mature region sequence composition. ConoSorter also catalogues key sequence characteristics (including relative sequence frequency, length, number of cysteines, N-terminal hydrophobicity, sequence similarity score) and automatically searches the ConoServer database for known precursor sequences, facilitating identification of known and novel conopeptides. When applied to ConoServer and UniProtKB/Swiss-Prot databases, ConoSorter is able to recognize 100% of known conotoxin superfamilies and classes with a minimum species specificity of 99%. As a proof of concept, we performed a reanalysis of Conus marmoreus venom duct transcriptome and (i) correctly classified all sequences previously annotated, (ii) identified 158 novel precursor conopeptide transcripts, 106 of which were confirmed by protein mass spectrometry, and (iii) identified another 13 novel conotoxin gene superfamilies.
Taken together, these findings indicate that ConoSorter is not only capable of robust classification of known conopeptides from large RNA data sets, but can also facilitate de novo identification of conopeptides which may have pharmaceutical importance.
ConoSorter; Cone snail; Venomics; Transcriptome; Proteome; Conopeptides; Conotoxins
Amyloid fibrils are self-assembled protein aggregates implicated in a number of human diseases. Fragmentation-dominated models for the self-assembly of amyloid fibrils have had important successes in explaining the kinetics of amyloid fibril formation but predict fibril length distributions that do not match experiments. Here we resolve this inconsistency using a combination of experimental kinetic measurements and computer simulations. We provide evidence for a structural transition that occurs at a critical fibril mass concentration, or ‘CFC’, above which fragmentation of fibrils is suppressed. Our simulations predict the formation of distinct fibril length distributions above and below the CFC, which we confirm by electron microscopy. These results point to a new picture of amyloid fibril growth in which structural transitions that occur during self-assembly have strong effects on the final population of aggregate species with small, and potentially cytotoxic, oligomers dominating for long periods of time at protein concentrations below the CFC.
There have been significant advances with regard to BRAF-targeted therapies against metastatic melanoma. However, the majority of patients receiving BRAF inhibitors (BRAFi) manifest disease progression within a year. We have recently shown that melanoma patients treated with BRAFi exhibit an increase in melanoma-associated antigens and in CD8+ tumor-infiltrating lymphocytes in response to therapy. To characterize such a T-cell infiltrate, we analyzed the complementarity-determining region 3 (CDR3) of rearranged T-cell receptor (TCR) β chain-coding genes in tumor biopsies obtained before the initiation of BRAFi and 10–14 d later. We observed an increase in the clonality of tumor-infiltrating lymphocytes in 7 of 8 patients receiving BRAFi, with a statistically significant 21% aggregate increase in clonality. Over 80% of individual T-cell clones detected after initiation of BRAFi treatment were new clones. Interestingly, the comparison of tumor infiltrates with clinical responses revealed that patients who had a high proportion of pre-existing dominant clones after the administration of BRAFi responded better to therapy than patients who had a low proportion of such pre-existing dominant clones following BRAFi. These data suggest that although the inhibition of BRAF in melanoma patients results in tumor infiltration by new lymphocytes, the response to treatment appears to be related to the presence of a pre-existing population of tumor-infiltrating T-cell clones.
BRAF; T cells; TILs; melanoma; vemurafenib
Brain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome.
High-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM.
We generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype.
These studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention.