Mutations in the TP53 tumor suppressor gene are common in HNSCC and correlate with radioresistance. Currently, there are no clinically available therapeutic approaches targeting p53 in HNSCC. Here we propose a strategy which uses TP53 mutational status to individualize anti-metabolic strategies for potentiation of radiation toxicity in HNSCC cells.
Glycolytic flux and mitochondrial respiration were evaluated in wild-type (wt) and mutant (mut) TP53 HNSCC cell lines. Sensitivity to external beam radiation (XRT) was measured using a clonogenic assay.
HNSCC cells expressing mutTP53 demonstrated radioresistance compared to HNSCC cells expressing wtTP53. Glycolytic inhibition potentiated radiation toxicity in mutTP53, but not wtTP53 expressing HNSCC cells. The relative sensitivity of mutp53 HNSCC cells to glycolytic inhibition is due to a glycolytic dependence associated with decreased mitochondrial complex II and IV activity. Wild-typeTP53 expressing cells maintain mitochondrial reserves and are relatively insensitive to glycolytic inhibition. Inhibition of respiration using metformin increases glycolytic dependence in wtTP53 expressing cells and potentiates the effects of glycolyic inhibition on radiation toxicity.
TP53 mutation in HNSCC cells correlates with a metabolic shift away from mitochondrial respiration toward glycolysis resulting in increased sensitivity to the potentiating effects of glycolytic inhibition on radiation toxicity. In contrast, wtTP53 expressing cells require inhibition of both mitochondrial respiration and glycolysis to become sensitized to radiation. One can therefore, use TP53 mutational status as a marker of altered tumor cell metabolism to individualize HNSCC treatment selection of specific targeted metabolic agents that can overcome cellular resistance to radiation therapy.
p53; 2-deoxyglucose; metformin; mitochondria; radiation
The purpose of this study is to investigate whether children with congenital esotropia are more likely than controls to develop mental illness by early adulthood.
Retrospective, population-based cohort.
Children (<19 years) diagnosed with congenital esotropia while residing in Olmsted County, Minnesota, from January 1, 1965, through December 31, 1994, and their one-to-one non-strabismic birth- and gender-matched controls.
The medical records of patients with esotropia and their controls were retrospectively reviewed for the subsequent development of psychiatric disease.
Main Outcome Measures
The development of mental illness and associated co-morbidities among patients with congenital esotropia and their controls.
A mental health disorder was diagnosed in 42 (33%) of the 127 patients with congenital esotropia followed to a mean age of 20.4 years compared to 16% of controls (p=0.002). Congenital esotropia increased the odds of developing a psychiatric illness 2.6 times (Confidence interval: 1.5- 4.8) compared to controls. The number of mental health diagnoses (p=0.019) and the use of psychotropic medications (p= 0.015) were significantly more common among esotropic patients compared to non-strabismic controls.
Congenital esotropia, similar to those with intermittent exotropia or convergence insufficiency, increases the odds of devloping mental illness by early adulthood 2.6 times compared to controls. The etiology of this association does not appear to be associated with premature birth.
Planktonic microbial activity and community structure is dynamic, and can change dramatically on time scales of hours to days. Yet for logistical reasons, this temporal scale is typically under-sampled in the marine environment. In order to facilitate higher-resolution, long-term observation of microbial diversity and activity, we developed a protocol for automated collection and fixation of marine microbes using the Environmental Sample Processor (ESP) platform. The protocol applies a preservative (RNALater) to cells collected on filters, for long-term storage and preservation of total cellular RNA. Microbial samples preserved using this protocol yielded high-quality RNA after 30 days of storage at room temperature, or onboard the ESP at in situ temperatures. Pyrosequencing of complementary DNA libraries generated from ESP-collected and preserved samples yielded transcript abundance profiles nearly indistinguishable from those derived from conventionally treated replicate samples. To demonstrate the utility of the method, we used a moored ESP to remotely and autonomously collect Monterey Bay seawater for metatranscriptomic analysis. Community RNA was extracted and pyrosequenced from samples collected at four time points over the course of a single day. In all four samples, the oxygenic photoautotrophs were predominantly eukaryotic, while the bacterial community was dominated by Polaribacter-like Flavobacteria and a Rhodobacterales bacterium sharing high similarity with Rhodobacterales sp. HTCC2255. However, each time point was associated with distinct species abundance and gene transcript profiles. These laboratory and field tests confirmed that autonomous collection and preservation is a feasible and useful approach for characterizing the expressed genes and environmental responses of marine microbial communities.
metatranscriptomics; gene expression; automated sampling; marine bacterioplankton; RNA preservation; Monterey Bay
Human cell lines are useful for studying cancer biology and pre-clinically modeling cancer therapy, but can be misidentified and cross contamination is unfortunately common. The purpose of this study was to develop a panel of validated head and neck cell lines representing the spectrum of tissue sites and histologies that could be used for studying the molecular, genetic, and phenotypic diversity of head and neck cancer.
A panel of 122 clinically and phenotypically diverse head and neck cell lines from head and neck squamous cell carcinoma (HNSCC), thyroid cancer, cutaneous squamous cell carcinoma, adenoid cystic carcinoma, oral leukoplakia, immortalized primary keratinocytes, and normal epithelium, was assembled from the collections of several individuals and institutions. Authenticity was verified by performing short tandem repeat (STR) analysis. Human papillomavirus (HPV) status and cell morphology were also determined.
Eighty-five of the 122 cell lines had unique genetic profiles. HPV-16 DNA was detected in 2 cell lines. These 85 cell lines included cell lines from the major head and neck primary tumor sites, and close examination demonstrates a wide range of in vitro phenotypes.
This panel of 85 genomically validated head and neck cell lines represents a valuable resource for the head and neck cancer research community that can help advance understanding of the disease by providing a standard reference for cell lines that can be utilized for biological as well as preclinical studies.
head and neck cancer; short tandem repeat (STR) profiling; head and neck squamous cell carcinoma; thyroid cancer; human papillomavirus
The Cheiruridae are a diverse group of trilobites and several subfamilies within the clade have been the focus of recent phylogenetic studies. This paper focuses on the relationships of one of those subfamilies, the Ordovician Eccoptochilinae. We analyze sixteen species from six genera within the traditionally defined group, using the pilekiid Anacheirurus frederici as an outgroup. To assess the monophyly of the Eccoptochilinae seven sphaerexochine species, Kawina arnoldi, Sphaerexochus arenosus, S. atacius, S. latifrons, S. mirus, S. parvus, and S. scabridus were included in the analysis as well. The results of this analysis show that the genus Eccoptochile represents a paraphyletic grade and species traditionally assigned to Parasphaerexochus and Skelipyx plot within Pseudosphaerexochus. Also, representative species of Sphaerexochinae plot within the traditionally defined Eccoptochilinae, suggesting Eccoptochilinae itself is paraphyletic. To resolve this, we propose all species of Pseudosphaerexochus be placed within Sphaerexochinae and Eccoptochilinae be restricted to a monotypic Eccoptochile clavigera.
To characterize tumor growth and metastatic potential in head and neck squamous cell carcinoma (HNSCC) cell lines in an orthotopic murine model of oral tongue cancer, and to correlate TP53 mutation status with these findings.
Cells from each of 48 HNSCC cell lines were orthotopically injected into the oral tongues of nude mice. Tumor volume, cervical lymph node metastasis, and mouse survival were recorded. Direct sequencing of the TP53 gene and western blot analysis for the p53 protein after induction with 5-fluorouracil was performed. Cell lines were categorized as either mutant TP53 or wild-type TP53, and lines with TP53 mutation were further categorized on the basis of type of mutation (disruptive or non-disruptive), and level of p53 protein expression. The behavior of tumors in these different groups was compared.
The 48 HNSCC cell lines showed a wide range of behavior from highly aggressive and metastatic to no tumor formation. Mice injected with cells harboring disruptive TP53 mutations had faster tumor growth, greater incidence of cervical lymph node metastasis, and shorter survival than mice injected with cells lacking these mutations.
HNSCC cell lines display a wide spectrum of behavior in an orthotopic model of oral cancer. Cell lines with disruptive TP53 mutations are more aggressive in this system, corroborating clinical reports that have linked these mutations to poor patient outcome.
head and neck squamous cell carcinoma; TP53; disruptive TP53 mutation; cervical lymph node metastasis; orthotopic nude mouse model
Given the poor immunogenicity of current H5N1 influenza vaccines, additives and adjuvants remain a viable solution for increasing efficacy. Here, we demonstrate that a 20-amino acid peptide (EB) possessing influenza antiviral activity also enhances the immune response to H5N1 vaccination in mice. The addition of EB to formalin-inactivated whole-virus vaccine induced virion aggregation and these aggregates were readily engulfed by phagocytic cells in vitro. In vivo, mice vaccinated with a suboptimal dose of inactivated vaccine containing EB peptide had reduced morbidity, improved viral clearance, and faster recovery than mice receiving vaccine alone. This phenomenon was not accompanied by an increase in virus-specific antibodies. Instead, cell-mediated immunity was enhanced as demonstrated by increased interferon-γ production from splenocytes. This data demonstrates that the EB peptide may a useful adjuvant for boosting the efficacy of poorly immunogenic influenza vaccines.
H5N1 influenza virus; inactivated influenza vaccine; adjuvant; cell-mediated immunity
Proteorhodopsin (PR) is a photoprotein that functions as a light-driven proton pump in diverse marine Bacteria and Archaea. Recent studies have suggested that PR may enhance both growth rate and yield in some flavobacteria when grown under nutrient-limiting conditions in the light. The direct involvement of PR, and the metabolic details enabling light-stimulated growth, however, remain uncertain. Here, we surveyed transcriptional and growth responses of a PR-containing marine flavobacterium during carbon-limited growth in the light and the dark. As previously reported (Gómez-Consarnau et al., 2007), Dokdonia strain MED134 exhibited light-enhanced growth rates and cell yields under low carbon growth conditions. Inhibition of retinal biosynthesis abolished the light-stimulated growth response, supporting a direct role for retinal-bound PR in light-enhanced growth. Among protein-coding transcripts, both PR and retinal biosynthetic enzymes showed significant upregulation in the light. Other light-associated proteins, including bacterial cryptochrome and DNA photolyase, were also expressed at significantly higher levels in the light. Membrane transporters for Na+/phosphate and Na+/alanine symporters, and the Na+-translocating NADH-quinone oxidoreductase (NQR) linked electron transport chain, were also significantly upregulated in the light. Culture experiments using a specific inhibitor of Na+-translocating NQR indicated that sodium pumping via NQR is a critical metabolic process in the light-stimulated growth of MED134. In total, the results suggested the importance of both the PR-enabled, light-driven proton gradient, as well as the generation of a Na+ ion gradient, as essential components for light-enhanced growth in these flavobacteria.
flavobacteria; marine; photoheterotrophy; proteorhodopsin; transcriptomics
Variants of unknown significance (VUS) complicate the assignment of risk to new DNA sequence variants found in at-risk populations. This study focused on the poorly studied linker region of the cancer-associated BRCA2 protein encoded by exons twelve through fourteen of BRCA2. To develop a new method to characterize VUS in this region of BRCA2, we first chose to study 4 reported VUS occurring on evolutionarily conserved residues within the linker region. To determine if these VUS represent neutral changes or if they impact the function of the BRCA2 protein, we stably transfected expression plasmids encoding wild-type or each mutant peptide into T47D breast cancer cells, which are wild-type for BRCA2. Four mutant peptide expressing cell lines and a wild-type linker region expressing cell line next were studied by challenging transfected cell lines with the DNA crosslinking compound cisplatin (10 μM) for 5 days. Expression of the wild-type linker region and certain mutant linker peptides (N2452D and I2285V) decreased apoptosis (as demonstrated by cell death detection assay) in transfected cell lines, indicating that the linker region peptide directly or indirectly affects the DNA damage repair pathway. By determining the cell survival and assaying the apoptotic index of treated cell lines, one could potentially use this screen to determine that a particular VUS has a functional impact on BRCA2 function, and hence is of functional significance. We conclude that this method is useful for screening the effect of linker region VUS on BRCA2 function, and to identify mutations for further testing. We also conclude that mutations in the linker region may have heretofore unappreciated roles in BRCA2 function.
BRCA2; breast cancer; cisplatin; variants of unknown significance; mutation screen
Herpes simplex ocular infection is a major cause of corneal blindness. Local antiviral treatments exist but are associated with corneal toxicity, and resistance has become an issue. We evaluated the biodistribution and efficacy of a humanized anti-herpes simplex virus (anti-HSV) IgG FAb fragment (AC-8; 53 kDa) following repeated topical administration. AC-8 was found in the corneal epithelium, anterior stroma, subepithelial stromal cells, and retinal glial cells, with preferential entry through the ocular limbus. AC-8 was active against 13 different strains of HSV-1, with 50% and 90% mean effective concentrations (MEC50 and MEC90, respectively) ranging from 0.03 to 0.13 μg/ml, indicating broad-spectrum activity. The in vivo efficacy of AC-8 was evaluated in a mouse model of herpes-induced ocular disease. Treatment with low-dose AC-8 (1 mg/ml) slightly reduced the ocular disease scores. A greater reduction of the disease scores was observed in the 10-mg/ml AC-8-treated group, but not as much as with trifluridine (TFT). AC-8 treatment reduced viral titers but less than trifluridine. AC-8 did not display any toxicity to the cornea or other structures in the eye. In summary, topical instillation of an anti-HSV FAb can be used on both intact and ulcerated corneas. It is well tolerated and does not alter reepithelialization. Further studies to improve the antiviral effect are needed for AC-8 to be considered for therapeutic use.
Tumor metabolism is an essential contributor to disease progression and response to treatment. An understanding of the metabolic phenotype of head and neck squamous cell carcinoma (HNSCC) will allow development of appropriate anti-metabolic strategies for this tumor type.
A panel of 15 HNSCC cell lines was assayed for glucose and glutamine dependence and sensitivity to metabolic inhibitors. Additionally, broad-spectrum metabolomic analysis using mass spectrometry/liquid chromatography was combined with individual measurements of reducing potential, adenosine triphosphate (ATP) and lactate production to characterize cellular metabolic phenotypes.
HNSCC energy and reducing potential levels closely mirrored extra-cellular glucose concentrations. Glucose starvation induced cell death despite activation of secondary energetic pathways. Conversely, glutamine was not required for HNSCC survival and did not serve as a significant source of energy. 2-deoxyglucose (2-DG) and its fluorinated derivative decreased glycolytic and Krebs cycle activity, cellular energy and reducing potential and inhibited HNSCC cell proliferation. 2-DG effects were potentiated by the addition of metformin, but not inhibitors of the pentose phosphate pathway or glutaminolysis. Despite dependence on glucose catabolism, we identified a subset of cell lines relatively resistant to starvation. Exploration of one such cell line (HN30) suggests that the presence of wild-type p53 can partially protect tumor cells from glucose starvation.
HNSCC tumor cells are dependent on glucose, not glutamine for energy production and survival, providing a rationale for treatment strategies targeting glucose catabolism. However, anti-metabolic strategies may need to be tailored to the tumor background, more specifically, p53 status.
glycolysis; glutamine; reducing potential; 2-deoxyglucose; metabolism
Identification of novel indications for commonly prescribed drugs could accelerate translation of therapies. We investigated whether any clinically-used drugs might have utility for treating prostate cancer by coupling an efficient, high-throughput laboratory-based screen and a large, prospective cohort study. In stage 1, we conducted an in vitro prostate cancer cell cytotoxicity screen of 3,187 compounds. Digoxin emerged as the leading candidate given its potency in inhibiting proliferation in vitro (mean IC50=163 nM) and common use. In stage 2, we evaluated the association between the leading candidate drug from stage 1 and prostate cancer risk in 47,884 men followed 1986–2006. Regular digoxin users (versus nonusers: RR=0.76, 95% CI 0.61–0.95), especially users for ≥10 years (RR=0.54, 95% CI 0.37–0.79, P-trend<0.001), had a lower prostate cancer risk. Digoxin was highly potent in inhibiting prostate cancer cell growth in vitro and its use was associated with a 25% lower prostate cancer risk.
Digoxin; prostate cancer; risk; cohort; transdisciplinary; cytotoxicity
Sequencing of microbial community RNA (metatranscriptome) is a useful approach for assessing gene expression in microorganisms from the natural environment. This method has revealed transcriptional patterns in situ, but can also be used to detect transcriptional cascades in microcosms following experimental perturbation. Unambiguously identifying differential transcription between control and experimental treatments requires constraining effects that are simply due to sampling and bottle enclosure. These effects remain largely uncharacterized for “challenging” microbial samples, such as those from anoxic regions that require special handling to maintain in situ conditions. Here, we demonstrate substantial changes in microbial transcription induced by sample collection and incubation in experimental bioreactors. Microbial communities were sampled from the water column of a marine oxygen minimum zone by a pump system that introduced minimal oxygen contamination and subsequently incubated in bioreactors under near in situ oxygen and temperature conditions. Relative to the source water, experimental samples became dominated by transcripts suggestive of cell stress, including chaperone, protease, and RNA degradation genes from diverse taxa, with strong representation from SAR11-like alphaproteobacteria. In tandem, transcripts matching facultative anaerobic gammaproteobacteria of the Alteromonadales (e.g., Colwellia) increased 4–13 fold up to 43% of coding transcripts, and encoded a diverse gene set suggestive of protein synthesis and cell growth. We interpret these patterns as taxon-specific responses to combined environmental changes in the bioreactors, including shifts in substrate or oxygen availability, and minor temperature and pressure changes during sampling with the pump system. Whether such changes confound analysis of transcriptional patterns may vary based on the design of the experiment, the taxonomic composition of the source community, and on the metabolic linkages between community members. These data highlight the impressive capacity for transcriptional changes within complex microbial communities, underscoring the need for caution when inferring in situ metabolism based on transcript abundances in experimental incubations.
The partial genomes of seven ocular Herpes simplex type-1 virus isolates were sequenced in a multiplex format. The data will be useful for studies of virulence and population structure and for the design of mutagenesis strategies for structure–function studies of each of the proteins.
Little is known about the role of sequence variation in the pathology of HSV-1 keratitis virus. The goal was to show that a multiplex, high-throughput genome-sequencing approach is feasible for simultaneously sequencing seven HSV-1 ocular strains.
A genome sequencer was used to sequence the HSV-1 ocular isolates TFT401, 134, CJ311, CJ360, CJ394, CJ970, and OD4, in a single lane. Reads were mapped to the HSV-1 strain 17 reference genome by high-speed sequencing. ClustalW was used for alignment, and the Mega 4 package was used for phylogenetic analysis (www.megasoftware.net). Simplot was used to compare genetic variability and high-speed sequencing was used to identify SNPs (developed by Stuart Ray, Johns Hopkins University School of Medicine, Baltimore, MD, http://sray.med.som.jhml.edu/SCRoftware/simplot).
Approximately 95% to 99% of the seven genomes were sequenced in a single lane with average coverage ranging from 224 to 1345. Phylogenetic analysis of the sequenced genome regions revealed at least three clades. Each strain had approximately 200 coding SNPs compared to strain 17, and these were evenly spaced along the genomes. Four genes were highly conserved, and six were more variable. Reduced coverage was obtained in the highly GC-rich terminal repeat regions.
Multiplex sequencing is a cost-effective way to obtain the genomic sequences of ocular HSV-1 isolates with sufficient coverage of the unique regions for genomic analysis. The number of SNPs and their distribution will be useful for analyzing the genetics of virulence, and the sequence data will be useful for studying HSV-1 evolution and for the design of structure–function studies.
Background. Vaccinia virus keratitis (VACVK) is a complication of smallpox vaccination that can result in blindness. There are no Food and Drug Administration–approved treatments for VACVK, and vaccinia immunoglobulin (VIG) is contraindicated in isolated VACVK. We used a rabbit model of infection to compare several therapeutic options for VACVK.
Methods. Rabbit eyes were infected with 105 plaque-forming units of the Dryvax strain of vaccinia virus and scored daily for 28 days using a modified MacDonald-Shadduck scoring system. Animals were treated for 10 days after the onset of keratitis with albumin, VIG, prednisolone acetate, trifluridine, or combinations thereof. Ocular viral titers and vaccinia-specific antibody titers were determined by plaque assay and enzyme-linked immunosorbent assay, respectively.
Results. Treatment with intravenous VIG neither exacerbated nor ameliorated VACVK. Topical prednisolone acetate interfered with viral clearance, and ocular disease rebounded in prednisolone-treated groups. The most effective treatment was topical trifluridine alone.
Conclusions. We conclude that (1) VIG did not negatively affect the treatment of isolated keratitis, (2) topical corticosteroids should not be used for treating VACVK, and (3) treatment with topical trifluridine, with or without intravenous VIG, is the preferred therapeutic regimen for treating VACVK.
The EB peptide is a 20-mer that was previously shown to have broad spectrum in vitro activity against several unrelated viruses, including highly pathogenic avian influenza, herpes simplex virus type I, and vaccinia, the prototypic orthopoxvirus. To expand on this work, we evaluated EB for in vitro activity against the zoonotic orthopoxviruses cowpox and monkeypox and for in vivo activity in mice against vaccinia and cowpox.
In yield reduction assays, EB had an EC50 of 26.7 μM against cowpox and 4.4 μM against monkeypox. The EC50 for plaque reduction was 26.3 μM against cowpox and 48.6 μM against monkeypox. A scrambled peptide had no inhibitory activity against either virus. EB inhibited cowpox in vitro by disrupting virus entry, as evidenced by a reduction of the release of virus cores into the cytoplasm. Monkeypox was also inhibited in vitro by EB, but at the attachment stage of infection. EB showed protective activity in mice infected intranasally with vaccinia when co-administered with the virus, but had no effect when administered prophylactically one day prior to infection or therapeutically one day post-infection. EB had no in vivo activity against cowpox in mice.
While EB did demonstrate some in vivo efficacy against vaccinia in mice, the limited conditions under which it was effective against vaccinia and lack of activity against cowpox suggest EB may be more useful for studying orthopoxvirus entry and attachment in vitro than as a therapeutic against orthopoxviruses in vivo.
EB peptide; vaccinia; cowpox; monkeypox; poxvirus entry; poxvirus attachment
Angiogenesis plays an important role in tumor growth and metastasis; therefore, inhibition of angiogenesis is a promising strategy for developing new anticancer drugs. Type 2 methionine aminopeptidase (MetAP2) protein is likely a molecular target of angiogenesis inhibitors.
Nitroxoline, an antibiotic used to treat urinary tract infections, was identified from a high-throughput screen of a library of 175 000 compounds for MetAP2 inhibitors and from a parallel screen using the Johns Hopkins Drug Library to identify currently used clinical drugs that can also inhibit human umbilical vein endothelial cells (HUVEC) proliferation. To investigate the mechanism of action of nitroxoline, inhibition of MetAP2 activity and induction of senescence were assessed in HUVEC. To test the antiangiogenic activity of nitroxoline, endothelial tube formation in Matrigel and microvessel formation in Matrigel plugs in vivo were assessed. Antitumor efficacy of nitroxoline was evaluated in mouse models of human breast cancer xenograft (n = 10) and bladder cancer orthotopic xenograft (n = 11). Furthermore, the mechanism of action of nitroxoline was investigated in vivo.
Nitroxoline inhibited MetAP2 activity in vitro (half maximal inhibitory concentration [IC50] = 54.8 nM, 95% confidence interval [CI] = 22.6 to 132.8 nM) and HUVEC proliferation (IC50 = 1.9 μM, 95% CI = 1.54 to 2.39 μM). Nitroxoline inhibited MetAP2 activity in HUVEC in a dose-dependent manner and induced premature senescence in a biphasic manner. Nitroxoline inhibited endothelial tube formation in Matrigel and reduced microvessel density in vivo. Mice (five per group) treated with nitroxoline showed a 60% reduction in tumor volume in breast cancer xenografts (tumor volume on day 30, vehicle vs nitroxoline, mean = 215.4 vs 86.5 mm3, difference = 128.9 mm3, 95% CI = 32.9 to 225.0 mm3, P = .012) and statistically significantly inhibited growth of bladder cancer in an orthotopic mouse model (tumor bioluminescence intensities of vehicle [n = 5] vs nitroxoline [n = 6], P = .045).
Nitroxoline shows promise as a potential therapeutic antiangiogenic agent.
To determine if a peptide, TAT-Cd0, inhibits Herpes simplex virus type 1 infection of human corneal epithelial cells.
TAT-Cd0 and a control peptide, E50,51TAT-Cd0, were added at various times throughout infection with the lacz-expressing hrR3 virus, and viral replication was measured by β-galactosidase activity. Toxicity was assessed using a dye reduction assay.
The CC50 value for TAT-Cd0 was ∼100 μM. In assays with peptide present at all times, TAT-Cd0 was 150-fold more active than E50,51TAT-Cd0 (EC50 0.2 vs. 30.0 μM). The EC50 values of TAT-Cd0 for entry inhibition, cell protection, virus inactivation, and inhibition of attachment were 0.1, 0.4, 9.5, and 3.0 μM, respectively. TAT-Cd0 was less effective when added 1 h postinfection (EC50 = 30.0 μM).
TAT-Cd0 is an effective inhibitor of Herpes simplex virus type 1 infection in human corneal epithelial cells and affects multiple steps before, or very early, in infection. The peptide has potential as an antiviral and further studies are warranted.
The HSV-1 US1 gene encodes an ocular virulence determinant that is highly modified after translation. To better determine sequence variability in the protein, the gene was sequenced in six ocular isolates, and bioinformatics analysis was carried out. The data will be useful for designing structure-function studies.
The herpes simplex virus type 1 (HSV-1) US1 gene encodes host-range and ocular virulence determinants. Mutations in US1 affecting virulence are known in strain OD4, but the genomic variation across several strains is not known. The goal was to determine the degree of sequence variation in the gene from several ocular HSV isolates.
The US1 gene from six ocular HSV-1 isolates, as well as strains KOS and F, were sequenced, and bioinformatics analyses were applied to the data.
Strains 17, F, CJ394, and CJ311 had identical amino acid sequences. With the other strains, most of the variability was concentrated in the amino-terminal third of the protein. MEME analysis identified a 63-residue core sequence (motif 1) present in all α-herpesvirus US1 homologs that were located in a region identified as structured. Ten amino acids were absolutely conserved in all the α-herpesvirus US1 homologs and were all located in the central core. Consensus-binding motifs for cyclin-dependent kinases and pocket proteins were also identified.
These results suggest that significant sequence variation exists in the US1 gene, that the α22 protein contains a conserved central core region with structurally variable regions at the amino- and carboxyl termini, that 10 amino acids are conserved in α-herpes US1 homologs, and that additional host proteins may interact with the HSV-1 US1 and US1.5 proteins. This information will be valuable in designing further studies on structure-function relationships and on the role these play in host-range determination and keratitis.
The antiviral peptide, entry blocker (EB), inhibits influenza virus replication by preventing attachment to cells. Here, we identified the minimal and optimal EB sequence that retained antiviral activity with a 50% inhibitory concentration (IC50) and 50% effective concentration (EC50) similar to those of the full-length EB peptide and several truncated variants that possessed up to 10-fold lower IC50s. These data have implications for improving the antiviral efficacy of EB-derived peptides while decreasing production costs and easing synthesis.
It is unknown whether breast cancer (BC) characteristics among young women treated with radiotherapy (RT) for Hodgkin's lymphoma (HL) differ from sporadic BC.
Using population-based data, we calculated BC risk following HL according to clinicopathologic features.
Compared with BC in the general population, risks of oestrogen receptor (ER)-positive/progesterone receptor (PR)-positive and ER-negative/PR-negative BC in young, irradiated HL survivors were increased five-fold (95% confidence interval (CI)=3.81–6.35) and nine-fold (95% CI=6.93–12.25), respectively. Among 15-year survivors, relative risk of ER-negative/PR-negative BC exceeded by two-fold (P=0.002) than that of ER-positive/PR-positive BC.
Radiotherapy may disproportionately contribute to the development of BC with adverse prognostic features among young HL survivors.
Hodgkin's lymphoma; second breast cancer; radiation-related breast cancer
Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide. To explore the genetic origins of this cancer, we used whole exome sequencing and gene copy number analyses to study 32 primary tumors. Tumors from patients with a history of tobacco use had more mutations than did tumors from patients who did not use tobacco, and tumors that were negative for human papilloma virus (HPV) had more mutations than did HPV-positive tumors. Six of the genes that were mutated in multiple tumors were assessed in up to 88 additional HNSCCs. In addition to previously described mutations in TP53, CDKN2A, PIK3CA and HRAS, we identified mutations in FBXW7 and NOTCH1. Interestingly, nearly 40% of the 28 mutations identified in NOTCH1 were predicted to truncate the gene product, suggesting that NOTCH1 may function as a tumor suppressor gene rather than an oncogene in this tumor type.
Preimplantation embryos from cattle, sheep, and goats may be cryopreserved for short- or long-term storage. Preimplantation embryos consist predominantly of water, and the avoidance of intracellular ice crystal formation during the cryopreservation process is of paramount importance to maintain embryo viability. Embryos are placed into a hypertonic solution (1.4 – 1.5 M) of a cryoprotective agent (CPA) such as ethylene glycol (EG) or glycerol (GLYC) to create an osmotic gradient that facilitates cellular dehydration. After embryos reach osmotic equilibrium in the CPA solution, they are individually loaded in the hypertonic CPA solution into 0.25 ml plastic straws for freezing. Embryos are placed into a controlled rate freezer at a temperature of -6°C. Ice crystal formation is induced in the CPA solution surrounding the embryo, and crystallization causes an increase in the concentration of CPA outside of the embryo, causing further cellular dehydration. Embryos are cooled at a rate of 0.5°C/min, enabling further dehydration, to a temperature of -34°C before being plunged into liquid nitrogen (-196°C). Cryopreserved embryos must be thawed prior to transfer to a recipient (surrogate) female. Straws containing the embryos are removed from the liquid nitrogen dewar, held in room temperature air for 3 to 5 sec, and placed into a 37°C water bath for 25 to 30 sec. Embryos cryopreserved in GLYC are placed into a 1 M solution of sucrose for 10 min for removal of the CPA before transfer to a recipient (surrogate) female. Embryos cryopreserved in EG, however, may be directly transferred to the uterus of a recipient.
We demonstrate an integrated FPGA solution to project highly stabilized, aberration-corrected stimuli directly onto the retina by means of real-time retinal image motion signals in combination with high speed modulation of a scanning laser. By reducing the latency between target location prediction and stimulus delivery, the stimulus location accuracy, in a subject with good fixation, is improved to 0.15 arcminutes from 0.26 arcminutes in our earlier solution. We also demonstrate the new FPGA solution is capable of delivering stabilized large stimulus pattern (up to 256x256 pixels) to the retina.
(170.0170) Medical optics and biotechnology; (170.4460) Ophthalmic optics